Safety, Immunogenicity and Efficacy of Pfs230D1M-EPA/AS01 Vaccine, a Transmission Blocking Vaccine Against Plasmodium Falciparum, in an Age De-Escalation Trial of Children and a Family Compound Trial in Mali
Phase: PHASE2
Conditions: Malaria
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

Safety, Immunogenicity and Efficacy of Pfs230D1M-EPA/AS01 Vaccine, a 
Transmission Blocking Vaccine against Plasmodium falciparum, in an Age De-
Escalation Trial of Children and a Family Compound Trial in Mali 
 
 
NIH Protocol Number: 19-I-N086 
Project Federalwide Assurance (FWA): #00005897 
 
FMPOS Protocol Number: N°2019/10/CE/FMPOS 
Project FWA: #00001769 
 
 
Conducted by: 
Laboratory of Malaria Immunology and Vaccinology (LMIV) 
National Institute of Allergy and Infectious Diseases (NIAID) 
National Institutes of Health (NIH) 
and 
Faculté de Médecine Pharmacie d’Odonto Stomatologie (FMPOS) 
University of Sciences, Techniques, & Technologies of Bamako (USTTB) 
 
 
Investigational New Drug (IND) Sponsor: 
Office of Clinical Research Policy and Regulatory Operations (OCRPRO) 
Division of Clinical Research, NIAID, NIH 
 
IND Number: 17130 
 
 
Version 7.0 
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CONFIDENTIAL 
 
 

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Team Roster 
NIH Staff  
 
NIAID Principal Investigator (PI): 
Patrick Duffy, MD 
LMIV/NIAID 
BG 29B RM 4NN06 
9000 Rockville Pike 
Bethesda, MD 20814 
Tel: 301-761-5089 
Email: patrick.duffy@nih.gov  
 
Associate Investigators: 
David Cook, MD 
Sara Healy, MD, MPH 
Jen Hume, DPhil 
Jennifer Kwan, PhD 
Aye Diallo Sousa, PA-C 
 
Biostatisticians: 
Jonathan Fintzi, PhD 
Biostatistics Research Branch/NIAID/NIH 
Tel: 301-761-5662 
Email: jon.fintzi@nih.gov 
 
Bruce Swihart, PhD 
Biostatistics Research Branch/NIAID/NIH 
Tel: 240-669-5256 
Email: bruce.swihart@nih.gov 
 
 
USTTB Staff  
 
FMPOS PI: 
 
Issaka Sagara, MD, MSPH, PhD 
Malaria Research and Training Center (MRTC) 
Epidemiology Department of Parasitic Diseases (DEAP)/FMPOS/USTTB 
BP 1805, Point G 
Bamako, Mali 
Tel: +223-2022-8109 
Email: isagara@icermali.org 
 
 

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Associate Investigators: 
Mahamadou S. Sissoko, MD, MSPH, PhD    
Abdoulaye Katile, MD, MSPH 
Mahamadoun Hamady Assadou, MD, MPH 
Mamady Kone, MD 
Bourama Kamate, MD 
Kourane Sissoko, MD 
Bayaya Haidara, MD 
Amatigue Zeguimé, PharmD  
Kadidia Baba Cisse, BSc 
Kalifa Diarra, PharmD 
Daman Sylla, MD 
Mamadou B. Coulibaly, PharmD, PhD 
Amagana Dolo, PharmD, PhD 
Cheick F. Traore, PhD 
Amadou Niangaly, PharmD, PhD 
 
 
  
 

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Safety Oversight 
 
Data and Safety Monitoring Board (DSMB): 
NIAID DSMB 
 
Sponsor Medical Monitor: 
Shirley Jankelevich, MD 
Clinical Monitoring Research Program 
Frederick National Laboratory for Cancer Research  
Leidos Biomedical Research, Inc.  
5705 Industry Lane, Room 248  
Frederick, Maryland 21704 USA 
Tel: 301-846-7322 
Fax: 301-846-6224 
Cell: 301-401-3426 
Email: shirley.jankelevich@nih.gov 
 
Independent Safety Monitor: 
Mamadou Dembele, MD 
Service Medicine Interne 
Centre Hospitalo-Universitaire du Point G 
Tel: + 223 2022 5003  
Mobile: +223 7604 9387 
Email: hassiramadydembele@yahoo.fr 
 
 
Institutional Review Boards (IRBs) 
 
NIH IRB 
 
FMPOS Ethics Committee (EC) 
Point G 
Bamako, Mali 
 
 
Clinical Trial Site 
 
Doneguebougou Mali, West Africa and surrounding villages 
Malaria Research and Training Center (MRTC), University of Sciences, Techniques & 
Technologies of Bamako (USTTB)  
Mali, West Africa 
 
 

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Laboratories 
 
Clinical Laboratories: 
MRTC/DEAP Clinical Laboratory 
FMPOS/USTTB 
BP 1805, Point G 
Bamako, Mali 
 
Rodolphe Merieux Laboratory 
Bamako, Mali 
 
Entomological Laboratory: 
MRTC Medical Entomological Laboratory 
FMPOS, USTTB 
BP 1805, Point G 
Bamako, Mali 
 
Immunology Laboratories: 
LMIV/NIAID/NIH 
BG 29B  
9000 Rockville Pike 
Bethesda, MD 20814, USA 
 
MRTC/DEAP Immunology Laboratory 
FMPOS/USTTB 
BP 1805, Point G 
Bamako, Mali 
 
Collaborating Laboratories: 
Center for Human Immunology, Autoimmunity, and Inflammation 
National Heart, Lung and Blood Institute (NHLBI) 
Building 10 - Magnuson CC, 7N110A 
10 Center Dr 
Bethesda, MD 20892-1655 USA 
 
Laboratory of Malaria and Vector Research (LMVR) 
12735 Twinbrook Parkway, Twinbrook 3 
Rockville, Maryland 20852 USA 
 
Laboratory of Parasitic Diseases 
NIAID/NIH 
Building 4, Room B1-03 
4 Memorial Drive 
Bethesda, MD 20892 USA 
 

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NIH Intramural Sequencing Center 
National Human Genome Research Institute (NHGRI) 
5625 Fishers Lane  
Room 5S-16C MSC9400 
Bethesda, MD 20892 USA 
 
NIH Research Technologies Branch 
Genomic Technologies Section 
Building 50, Room 5509 
50 South Drive, MSC 8005 
Bethesda, MD 20892 USA 
 
PATH Malaria Vaccine Initiative (MVI) 
455 Massachusetts Avenue NW, Suite 1000 
Washington, DC 20001 USA 
 
 
 

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Table of Contents 
Team Roster .................................................................................................................................... 2 
List of Tables ................................................................................................................................ 11 
List of Figures ............................................................................................................................... 13 
List of Appendices ........................................................................................................................ 15 
List of Abbreviations .................................................................................................................... 16 
Protocol Summary ........................................................................................................................ 19 
Précis ............................................................................................................................................. 26 
1 
Background Information and Scientific Rationale ............................................................... 28 
 
Background Information............................................................................................. 28 
 
Development of the Study Agent, Pfs230D1M-EPA/AS01 ................................. 28 
 
Rationale for Study ..................................................................................................... 30 
 
Rationale for Study Design: Vaccination of School-Aged Children .................... 30 
 
Rationale for Study Design: Family Compound and Requirement of Broad 
Vaccine Coverage ................................................................................................................. 34 
 
Rationale for Study Design: Vaccine Dose and Regimen .................................... 37 
 
Rationale for Study Design: Inclusion of Children Under 5 Years of Age .......... 43 
2 
Previous Preclinical Experience with Pfs230D1M-EPA/AS01 ............................................ 44 
3 
Previous Human Experience with Pfs230-EPA .................................................................... 45 
 
Pfs230D1M-EPA/Alhydrogel in Healthy Adults (#15-I-0044) .................................. 45 
 
Safety of Pfs230D1M-EPA/Alhydrogel in Healthy Adults .................................. 46 
 
Immunogenicity and Functional Activity of Pfs230D1M-EPA/Alhydrogel in 
Healthy Adults ...................................................................................................................... 47 
 
Pfs230D1M-EPA/AS01 in Healthy Malian Adults (#17-I-N006) ............................. 51 
 
Safety of Pfs230D1M-EPA/AS01 in Healthy Adults (Primary Series) ................ 52 
 
Safety of Pfs230D1M-EPA/AS01 in Healthy Adults (Year 2, 4th Dose) ............. 57 
 
Immunogenicity and Functional Activity (SMFA) of Pfs230D1M-EPA/AS01 in 
Healthy Adults (Primary Series + 4th Dose; Year 1 + 2) ...................................................... 58 
 
Functional Activity by Direct Skin Feeds ............................................................. 65 
 
Pfs230D1M-EPA/AS01 in Healthy Malian Children and Adults (19-I-N086) ......... 66 
 
Safety in Healthy Malian Children and Adults ..................................................... 67 
 
Immunogenicity in Healthy Malian Children ....................................................... 69 
 
Functional Activity by DSF in Healthy Malian Children ..................................... 70 
4 
Malaria Transmission Dynamics in Mali .............................................................................. 70 
5 
Experience in Children with AS01 Adjuvant ....................................................................... 77 
 
RTS,S/AS01E Pediatric Safety Data ........................................................................... 78 
 
Clinical Development Plan ......................................................................................... 81 
6 
Study Objectives ................................................................................................................... 82 
 
Pilot Objectives .......................................................................................................... 82 
 
Primary Objectives ................................................................................................ 82 
 
Secondary Objectives ............................................................................................ 82 
 
Exploratory Objectives ......................................................................................... 82 
 
Main Objectives .......................................................................................................... 82 
 
Primary Objectives ................................................................................................ 82 
 
Secondary Objectives ............................................................................................ 83 
 
Exploratory Objectives ......................................................................................... 83 

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Study Design ......................................................................................................................... 83 
 
Description of the Study Design ................................................................................. 83 
 
Study Groups .............................................................................................................. 84 
 
Pilot Safety Group................................................................................................. 84 
 
Main Study Group................................................................................................. 86 
 
Year 2: Pilot and Main Study Groups ................................................................... 88 
8 
Study Endpoints .................................................................................................................... 89 
 
Pilot Endpoints ........................................................................................................... 89 
 
Primary Endpoints ................................................................................................ 89 
 
Secondary Endpoints ............................................................................................ 89 
 
Exploratory Endpoints .......................................................................................... 90 
 
Main Endpoints........................................................................................................... 90 
 
Primary Endpoints ................................................................................................ 90 
 
Secondary Endpoints ............................................................................................ 90 
 
Exploratory Endpoints .......................................................................................... 90 
 
Sample Size and Estimated Duration of Study ........................................................... 90 
 
Study Definitions ........................................................................................................ 91 
9 
Study Population ................................................................................................................... 93 
 
Study Site .................................................................................................................... 93 
 
Rationale for Subject Selection .................................................................................. 95 
 
Recruitment Plan ........................................................................................................ 96 
 
Inclusion Criteria for Groups 1, 2, 3 ........................................................................... 96 
 
Inclusion Criteria for Group 4 .................................................................................... 97 
 
Inclusion Criteria for Groups 1, 2, 3, (Year 2) ........................................................... 97 
 
Inclusion Criteria for Group 4 (Year 2) ...................................................................... 98 
 
Exclusion Criteria for Groups 1, 2, 3.......................................................................... 98 
 
Exclusion Criteria for Group 4 ................................................................................. 100 
 
Exclusion Criteria for Groups 1, 2, 3 (Year 2) ......................................................... 100 
 
Exclusion Criteria for Group 4 (Year 2) ................................................................... 102 
 
Justification for Exclusion of Special Populations ................................................... 103 
 
Justification of Exclusion of Pregnant Women .................................................. 103 
 
Justification for Exclusion of Children Under the Age of Five (Vaccination) ... 103 
10 Study Agents ....................................................................................................................... 103 
 
Pfs230D1M-EPA/AS01 ........................................................................................... 103 
 
Manufacturing ..................................................................................................... 103 
 
Disposition and Dispensation ............................................................................. 104 
 
Formulation, Packaging, and Labeling ............................................................... 104 
 
Storage, Shipping, and Stability ......................................................................... 104 
 
Preparation, Dosage, and Administration of Study Agents ................................ 105 
 
Administration .................................................................................................... 105 
 
Comparator vaccine .................................................................................................. 106 
 
HAVRIX® (Hepatitis A Vaccine) ....................................................................... 106 
 
TYPHIM Vi (Salmonella typhi vaccine) ............................................................ 106 
 
Menactra® ........................................................................................................... 107 
 
Contraindications to Vaccination ............................................................................. 107 
 
Indications for Deferral of Vaccination .................................................................... 108 

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Concomitant Medications and Procedures ............................................................... 108 
 
Prohibited Medications and Procedures ................................................................... 108 
 
Vaccine Accountability ............................................................................................ 109 
11 Study Schedule .................................................................................................................... 109 
 
Screening .................................................................................................................. 109 
 
Screening for Arms 1, 2, 3 .................................................................................. 110 
 
Screening for Arm 4 ............................................................................................ 111 
 
Assignment to Groups .............................................................................................. 112 
 
Pilot Safety Study ............................................................................................... 112 
 
Main Study .......................................................................................................... 112 
 
Early Termination Visit ............................................................................................ 112 
12 Study Procedures/Evaluations ............................................................................................ 112 
 
Photographs of Rash or Injection Site Reactions ..................................................... 112 
 
Blood Draw............................................................................................................... 113 
 
Clinical Laboratory Testing ...................................................................................... 113 
 
Electrocardiogram .................................................................................................... 113 
 
Malaria Diagnostics .................................................................................................. 113 
 
Blood Smears (BS) ............................................................................................. 113 
 
Rapid Diagnostics ............................................................................................... 114 
 
Malaria Polymerase Chain Reaction ................................................................... 114 
 
Immunologic Laboratory Testing ............................................................................. 115 
 
ELISA ................................................................................................................. 115 
 
Transmission Assays ........................................................................................... 115 
 
Standard Membrane Feeding Assays .................................................................. 116 
 
Direct Skin Feeds ................................................................................................ 116 
 
Entomological Procedures and Insectary Used in DSF ...................................... 118 
 
Schistosomiasis and Helminthes Infection Testing .................................................. 119 
 
Other Laboratory Assays .......................................................................................... 119 
 
Collection of Malaria Prevention Measures During the Transmission Season ........ 120 
 
Exploration of Human Movement During Transmission Season (Optional) ........... 120 
 
Human Movement (2019-2020) ............................................................................... 121 
13 Research Use of Stored Human Samples, Specimens, or Data .......................................... 122 
14 Data Sharing Plan ............................................................................................................... 123 
15 Assessment of Safety .......................................................................................................... 124 
 
Definitions ................................................................................................................ 124 
 
Documenting, Recording, and Reporting Adverse Events ....................................... 125 
 
Investigator Assessment of Adverse Events ............................................................. 126 
 
Severity ............................................................................................................... 128 
 
Causality ............................................................................................................. 128 
 
Follow-up of Adverse Events and Serious Adverse Events ..................................... 130 
 
Investigator Reporting Responsibilities to the Sponsor ........................................... 130 
 
Adverse Events ................................................................................................... 130 
 
Serious Adverse Events ...................................................................................... 130 
 
Unanticipated Problems ...................................................................................... 131 
 
Pregnancy ............................................................................................................ 131 
 
Investigator Reporting Procedures to the NIH IRB .................................................. 132 

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Definitions........................................................................................................... 132 
 
Investigator Reporting Responsibilities to the Local IRB ........................................ 134 
 
Sponsor’s Reporting Responsibilities ....................................................................... 134 
 
Pausing Rules for the Protocol ................................................................................. 134 
 
Pausing Rules for an Individual Subject ............................................................. 134 
 
Pausing Rules for a Particular Age Group .......................................................... 135 
 
Reporting a Pause ............................................................................................... 135 
 
Resumption of a Paused Study ........................................................................... 135 
 
Halting Rules for the Protocol .................................................................................. 135 
 
Reporting a Study Halt........................................................................................ 136 
 
Resumption of a Halted Study ............................................................................ 136 
 
Discontinuation of Study .......................................................................................... 137 
 
Withdrawal Criteria for an Individual Participant .................................................... 137 
 
Replacement of Withdrawn Participants or Participants Who Discontinue Study 
Agent 
138 
 
Safety Oversight ....................................................................................................... 138 
 
Safety Review and Communications Plan .......................................................... 138 
 
Sponsor Medical Monitor ................................................................................... 138 
 
Independent Safety Monitor (ISM) ..................................................................... 138 
 
Data and Safety Monitoring Board (DSMB) ...................................................... 139 
16 Site Monitoring Plan ........................................................................................................... 139 
17 Statistical Considerations .................................................................................................... 140 
 
Pilot Study ................................................................................................................ 140 
 
Primary Objective ............................................................................................... 140 
 
Secondary Objective ........................................................................................... 140 
 
Exploratory Objective ......................................................................................... 141 
 
Main Study ............................................................................................................... 141 
 
Primary Objective ............................................................................................... 141 
 
Secondary Objectives .......................................................................................... 142 
 
Exploratory Objectives ....................................................................................... 142 
 
Sample Size and Power Calculations ....................................................................... 143 
 
Pilot Study ........................................................................................................... 143 
 
Main Study .......................................................................................................... 144 
 
Randomization .......................................................................................................... 147 
18 Ethics/Protection of Human Subjects ................................................................................. 148 
 
Institutional Review Board (IRB) and Ethics Committee (EC) ............................... 148 
 
Informed Consent Process ........................................................................................ 148 
 
Mali Site Community Permission and Individual Informed Consent Process ... 149 
 
Informed Consent Process for Minors Who Reach the Age of Consent ............ 150 
 
Informed Consent Process for Emancipated Minors .......................................... 151 
 
Subject Confidentiality ............................................................................................. 151 
 
Potential Risks .......................................................................................................... 151 
 
Study Vaccine ..................................................................................................... 151 
 
Artemether/Lumefantrine (AL) .......................................................................... 154 
 
Venipuncture ....................................................................................................... 155 
 
Direct Skin Feeds ................................................................................................ 155 

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Other Risks.......................................................................................................... 155 
 
Potential Benefits ...................................................................................................... 156 
 
Compensation ........................................................................................................... 156 
19 Data Handling and Record Keeping ................................................................................... 158 
 
Data Capture and Management ................................................................................ 158 
 
Record Retention ...................................................................................................... 159 
 
Protocol Revisions .................................................................................................... 159 
Appendix A: Schedule of Procedures/Evaluations ..................................................................... 160 
Appendix B: Day to Day Schedule by Arms .............................................................................. 172 
Appendix C: Toxicity Tables ...................................................................................................... 208 
Appendix D: Institutional Normal Laboratory Values ............................................................... 216 
Appendix E: References ............................................................................................................. 218 
 
 
List of Tables 
Table 1. GMTs and sAUC for anti-CSP antibodies and GMTs for anti-HBs antibodies. Adapted 
from Owusu-Agyei, S et al.29 ........................................................................................................ 43 
Table 2. NIAID Protocol #15-I-0044 Enrollment and Vaccinations. ........................................... 45 
Table 3. Summary of Total AEs and Comparing Most Common AEs Among the Study Arms . 54 
Table 4. Rates of Solicited Local and Systemic Adverse Reactions of Interest Within Seven Days 
of Receiving Vaccine .................................................................................................................... 56 
Table 5. Rates of Solicited Local and Systemic Adverse Reactions of Interest Within Seven Days 
of Receiving Vaccine. ................................................................................................................... 58 
Table 6. Summary of Direct Skin Feed (DSF) results. ................................................................. 66 
Table 7. Summary of study progress for 19-I-N086 ..................................................................... 67 
Table 8. Malaria AE and parasitemia summary. ........................................................................... 69 
Table 9: Summary of LMIV Direct Skin Feed (DSF) Studies in Mali ......................................... 73 
Table 10. AS01 Adjuvant Systems. Adapted from Garcon et al. 2017, From discovery to 
licensure, the Adjuvant System story. ........................................................................................... 78 
Table 11. Adverse reactions reported after 3 doses of the vaccine58   Note: Very common ≥1/10; 
Common ≥1/100 to < 1/10; Uncommon ≥1/1000 to < 1/100 ....................................................... 79 
Table 12. Approximate census counts in Doneguebougou in June 2018. .................................... 87 
Table 13. Transmission-Blocking Vaccine (TBV) ..................................................................... 105 
Table 14. Transmission-Blocking Assay .................................................................................... 115 
Table 15. Solicited Adverse Events ............................................................................................ 127 
Table 16: Definitions for Severity of AE Grading ...................................................................... 128 
Table 17. Sample Size Power Calculations Based on Seasonal DSF Infection Rates and Seasonal 
Vaccine Activity. ........................................................................................................................ 145 
Table 18. Vaccine Efficacy Sample Size Power Calculations Based on Predicted Vaccine 
Efficacy ....................................................................................................................................... 147 
Table 19: Estimated Compensation Schedule  (Year 1)1 ............................................................ 157 

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Table 20. Estimated Compensation Schedule (Year 2)1 .............................................................. 158 
 
 

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List of Figures 
Figure 1: Protein Alignments of Recombinant Pfs230D1M to its Respective Native Protein or 
Protein Fragment. .......................................................................................................................... 29 
Figure 2: Frequency of Positive DSF Assays by Month and Age in Bancoumana, Mali (NIAID 
Protocol #17-I-N180). ................................................................................................................... 31 
Figure 3: Modeling estimated vaccine effects on prevalence and clinical incidence of P. 
falciparum malaria. ....................................................................................................................... 32 
Figure 4: Antibody dynamics and association with vaccine efficacy. .......................................... 33 
Figure 5: Spatial and temporal locations of infection clusters20: .................................................. 35 
Figure 6. Impact of number of Anopheles and gametocyte prevalence on number of human-to-
mosquito transmission events. ...................................................................................................... 40 
Figure 7. Percentage of solicited events post dose 1, 2, and 3 RTS,S (total vaccinated cohort). . 42 
Figure 8: Comparison of Transmission-Blocking Activity of Pfs25M-EPA/AS01 and 
Pfs230D1M-EPA/AS01 to Pfs25M-EPA/Alhydrogel and Pfs230D1M-EPA/Alhydrogel in CD1 
Mice. ............................................................................................................................................. 44 
Figure 9: Pfs230-specific Antibody Responses in Subjects Receiving Pfs230D1M, U.S. Cohort 
(#15-I-0044). ................................................................................................................................. 48 
Figure 10: Pfs25 and Pfs230 Functional Activity by Standard Membrane Feeding Assay 
(SMFA). ........................................................................................................................................ 49 
Figure 11: Pfs230 Functional Activity by Standard Membrane Feeding Assay in the Presence or 
Absence of Complement. .............................................................................................................. 49 
Figure 12: Pfs230-specific Antibody Responses in Subjects Receiving Pfs230D1M, 40 µg in 
Mali (#15-I-0044). ........................................................................................................................ 50 
Figure 13: Pfs230 Functional Activity by Standard Membrane Feeding Assay in Mali, #15-I-
0044............................................................................................................................................... 51 
Figure 14. Antibody Responses to Pfs230D1M-EPA/AS01 after Low Dose and High Dose 
Vaccinations of Adults in Sotuba, Mali during Pilot Phase trial. ................................................. 59 
Figure 15. Antibody Function by Standard Membrane Feeding Assay to Pfs230D1M-EPA/AS01 
after Low Dose and High Dose Vaccinations of Adults in Sotuba, Mali during Pilot Phase trial.
....................................................................................................................................................... 60 
Figure 16. Antibodies against Pfs230D1M measured by ELISA 12 weeks post 2nd and 3rd 
vaccination in Bancoumana/Doneguebougou, Mali during Main Phase trial. ............................. 61 
Figure 17. ELISA against Pfs230D1 antigen. ............................................................................... 62 
Figure 18. Transmission Reducing Activity Measured by SMFA 12 weeks post 2nd and 3rd 
Vaccinations in Bancoumana/Doneguebougou, Mali during Main Phase trial. ........................... 63 
Figure 19. Transmission blocking activity measured by SMFA 12 weeks post 2nd and 3rd 
vaccinations in Bancoumana/Doneguebougou, Mali during Main Phase trial. ............................ 64 
Figure 20. Serum transmission blocking activity (TBA) by SMFA 3 months after Pfs230/AS01 
booster dose (Day 560). ................................................................................................................ 65 

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Figure 21. Comparison of Pfs230D1M-EPA/AS01, 40 ug, in healthy Malian adults (17-I-N006) 
to healthy Malian children [5-8 year olds (green), 9-18 year olds (purple)] (19-I-N086). ........... 70 
Figure 22: Proportion of positive feeds (blue) with P. falciparum parasite (orange) and 
gametocyte density (yellow) over six years of direct skin feed (DSF) assays in Mali. ................ 74 
Figure 23. Percent P. falciparum Positive by Month and Age. .................................................... 76 
Figure 24. Percent P. falciparum Gametocyte Positive by Month and Age. ................................ 77 
Figure 25. Local and Systemic Symptoms Occurring 4 Days after Vaccination  ........................ 80 
Figure 26. Pilot Safety Group Study Schema. .............................................................................. 85 
Figure 27. Main Phase Study Schema. ......................................................................................... 86 
Figure 28. Fourth dose (Year 2) study schema. ............................................................................ 89 
Figure 29: Maps Showing the Location of Doneguebougou, Mali. ............................................. 94 
Figure 30: Aerial Map Showing Distribution of Family Compounds in Doneguebougou, Mali . 95 
Figure 31. Summary of human movement study enrollment. .................................................... 121 
Figure 32. Simulated power under assumptions of 0.4, 0.5, and 0.6 vaccine activity. ............... 146 
 
 

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List of Appendices 
Appendix A: Schedule of Procedures/Evaluations ..................................................................... 160 
Appendix B: Day to Day Schedule by Arms .............................................................................. 172 
Appendix C: Toxicity Tables ...................................................................................................... 205 
Appendix D: Institutional Normal Laboratory Values ............................................................... 216 
Appendix E: References ............................................................................................................. 218 
 
 

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List of Abbreviations 
AE 
adverse event 
ACIP 
Advisory Committee on Immunization Practices 
AGC 
absolute granulocyte count 
AL 
artemether/lumefantrine 
ALT 
alanine transaminase 
ANC 
absolute neutrophil count 
AR 
adverse reaction 
AS01 
Adjuvant System AS01 
β-hCG 
beta human choriogonadotropin 
BS 
blood smear 
CBC w/diff 
complete blood count with differential 
CFR 
Code of Federal Regulations 
cGMP 
current Good Manufacturing Practices 
Cr 
creatinine 
CRF 
case report form 
CSO 
Clinical Safety Office 
DEAP 
Epidemiology Department of Parasitic Diseases (FMPOS/USTTB) 
DSF 
direct skin feeds 
DSMB 
Data and Safety Monitoring Board 
EC 
ethics committee  
EKG 
electrocardiogram 
ELISA 
enzyme-linked immunosorbent assay 
EPA 
ExoProtein A 
ER 
emergency room 
FDA 
Food and Drug Administration 
FMPOS 
Faculté de Médecine Pharmacie d’OdontoStomatologie 
GCP 
Good Clinical Practice 
GEE 
generalized estimating equation 
GSK 
GlaxoSmithKline 
HIV 
human immunodeficiency virus 
HRPP 
Human Research Protection Program 
ICH 
International Conference on Harmonisation of Technical Requirements 
for Registration of Pharmaceuticals for Human Use 
Ig 
immunoglobulin 
IM 
intramuscular 
IND 
Investigational New Drug application 
IRB 
institutional review board 
ISM 
independent safety monitor 
IV 
intravenous 

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LMIV 
Laboratory of Malaria Immunology and Vaccinology (of NIAID) 
LMVR 
Laboratory of Malaria and Vector Research 
µg 
micrograms 
MPL 
monophosphoryl lipid 
MRTC 
Malaria Research and Training Center (Mali) 
n 
number (typically refers to subjects) 
NIAID 
National Institute of Allergy and Infectious Diseases (NIH) 
NIH 
National Institutes of Health 
NOCI 
new onset of chronic illness 
OCRPRO 
Office of Clinical Research Policy and Regulatory Operations 
OHRP 
Office for Human Research Protections 
OHSRP 
Office of Human Subjects Research Protections 
PBS 
phosphate-buffered saline 
PCR 
polymerase chain reaction 
Pfs25/Pfs230 
surface antigens of zygotes and ookinetes in the mosquito stage of 
Plasmodium falciparum 
PI 
principal investigator 
qPCR 
quantitative polymerase chain reaction 
RVF 
Rift Valley fever  
SAE 
serious adverse event 
SAR 
suspected adverse reaction 
SD 
standard deviation 
SERF 
Safety Expedited Report Form 
SMC 
seasonal malaria chemoprophylaxis 
SMFA 
standard membrane feeding assay 
SOP 
standard operating procedure 
SRCP 
Safety Review and Communications Plan 
SUSAR 
serious and unexpected suspected adverse reaction 
TBA 
transmission-blocking activity 
TBS 
TRIS-buffered saline 
TBV 
transmission-blocking vaccine 
TRA 
transmission-reducing activity 
T-TBS 
TRIS-buffered saline containing Tween-20 
UP 
unanticipated problem 
UPnonAE 
unanticipated problem that is not an adverse event 
USD 
United States dollar 
USTTB 
University of Sciences, Techniques, & Technologies of Bamako 
VIMT 
vaccine to interrupt malaria transmission  
VU 
Vaccine unit 
WBC 
white blood cell 
WHO 
World Health Organization 

Pfs230D1M Age De-escalation and Family Compound Trial 
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WMW 
Wilcoxon-Mann-Whitney test 
WSRT 
Wilcoxon signed rank test 
 
 
 

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Protocol Summary 
Short Title: 
Pfs230D1M Age De-escalation and Family Compound Trial 
 
Clinical Phase: 
2 
 
IND Sponsor: 
OCRPRO  
Conducted by: 
Laboratory of Malaria Immunology and Vaccinology 
(LMIV)/National Institute of Allergy and Infectious Diseases 
(NIAID)/National Institutes of Health (NIH) 
Clinical Sites: 
Malaria Research and Training Center (MRTC), University of 
Sciences, Techniques & Technologies of Bamako (USTTB), Mali, 
West Africa  
 
Doneguebougou and surrounding villages, Mali, West Africa 
Principal Investigators: 
Patrick Duffy, MD (LMIV/NIAID/NIH) 
 
Issaka Sagara MD, MSPH, PhD (MRTC/DEAP/FMPOS) 
Study Agent/ 
Intervention Description: 
Pfs230D1M-EPA/AS01, 40 μg, or matched comparator vaccine, 
administered via intramuscular (IM) injection on days 0, 28, 56, 
392/448.  
 
 
Pfs230D1M-EPA/AS01: recombinant Pfs230 domain 1 (a 
subdomain of a surface antigen of gametocytes, gametes, and 
zygotes, in the mosquito stage of Plasmodium falciparum) 
conjugated to a recombinant Pseudomonas aeruginosa ExoProtein 
A (EPA) and adjuvanted with Adjuvant System AS01B diluted to 
equivalent AS01E concentration directly before administration. 
Dosed on days 0, 28, 56, 392/448 
 
 
Comparator Vaccines 
 
 
HAVRIX: inactivated hepatitis A viral vaccine adsorbed on 
aluminum hydroxide. HAVRIX is indicated for active 
immunization against disease caused by hepatitis A virus (HAV). 
HAVRIX is approved for use in persons 12 months of age and 
older with 2 doses (0, 6 months). Dosed on days 0 and 392/448. 
 
 
 
TYPHIM Vi: polysaccharide vaccine containing the cell surface 
Vi polysaccharide extracted from Salmonella enterica serovar 
Typhi, S typhi Ty2 strains; non-adjuvanted. TYPHIM Vi is 
indicated for active immunization against typhoid fever in subjects 

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2 years of age and over and is given as a single dose. Dosed on day 
28. 
 
 
Menactra® (meningococcal serogroup A, C, Y, and W-135): is a 
sterile, intramuscularly administered vaccine that contains 
Neisseria meningitidis serogroup A, C, Y, and W-135 capsular 
polysaccharide antigens individually conjugated to diphtheria 
toxoid protein.  A single dose (0.5 mL) is recommended for those 
individuals 2 years of age and older who are otherwise healthy and 
are at increased risk for meningococcal disease (e.g., individuals in 
an epidemic or highly endemic country such as Mali). 
 
Sample Size: 
N~1960 (approximately 137 family compounds/vaccine units) 
N=60 (pilot) 
N~1500 (main, vaccine) 
N~400 (main, no vaccine) 
 
Accrual Ceiling: 
N=2380 
N=80 (pilot) 
N=1800 (main, vaccine) 
N=500 (main, no vaccine) 
 
Accrual Period: 
March 2019-September 2019 (primary series) 
 
June 2020-October 2020 (4th vaccination; continuation on study) 
Study Duration: 
Start Date: Approximately March 2019 
End Date: Approximately October 2021 
 
Study participants will be enrolled for a total of approximately 26 
to 28 months (for up to 12 months following the last vaccination) 
depending on vaccination schedule and arm assignment and timing 
of screening 
 
Study Population: 
Pilot: Malian children 5-18 years of age  
Main (vaccine): Malians 5 years of age and older 
Main (no vaccine): Malian children 1-4 years of age  
 
Study Design: 
Two phase study: pilot, main 
 
 
Pilot 
Age de-escalating, double-blind, randomized comparator 
controlled; pilot groups will join main at the time of vaccine #3  
 
 
 
 
Group 1:  9 to 18 years of age (n=30) 

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• Arm 1a (n=15), to receive 40 μg of Pfs230D1M-
EPA/AS01 on days 0, 28, 126, 448; receipt of 
artemether/lumefantrine (AL) on day -7 
• Arm 1b (n=15), to receive HAVRIX (day 0, 448), 
TYPHIM Vi (day 28), Menactra (day 126); receipt of 
AL on day -7 
Group 2:  5 to 8 years of age (n=30) 
• Arm 2a (n=15), to receive 40 μg of Pfs230D1M-
EPA/AS01 on days 0, 28, 126, 448; receipt of AL on 
day -7,112, 441 
• Arm 2b (n=15), to receive HAVRIX (day 0, 448), 
TYPHIM Vi (day 28), Menactra (day 126); receipt of 
AL on day -7,112, 441 
                                                Main 
Double-blind, family/compound-randomized comparator-
controlled trial  
                                               Group 3 (n~1500): Pfs230D1M-EPA/AS01 or comparator vaccine 
• Arm 3a: 5-8 years of age to receive 40 μg of 
Pfs230D1M-EPA/AS01 on days 0, 28, 56, 392; receipt 
of AL on day -7, 42, 385 
• Arm 3b: 5-8 years of age to receive HAVRIX (day 0, 
392), TYPHIM Vi (day 28), Menactra (day 56); receipt 
of AL on day -7, 42, 385 
• Arm 3c: 9-18 years of age to receive 40 μg of 
Pfs230D1M-EPA/AS01 on days 0, 28, 56, 392; receipt 
of AL on day -7  
• Arm 3d: 9-18 years of age to receive HAVRIX (day 0, 
392), TYPHIM Vi (day 28), Menactra (day 56); receipt 
of AL on day -7 
• Arm 3e: 19 years of age or older to receive 40 μg of 
Pfs230D1M-EPA/AS01 on days 0, 28, 56, 392; receipt 
of AL on day -7  
• Arm 3f: 19 years of age or older to receive HAVRIX 
(day 0, 392), TYPHIM Vi (day 28), Menactra (day 56); 
receipt of AL on day -7 

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Group 4 (n~400): AL treatment only; no vaccination (parasitemia) 
• Arm 4a: 1-4 years of age residing with/in same 
families/compounds as Arms 1a, 2a, 3a, 3c, 3e 
(Pfs230D1M-EPA/AS01 compounds) receipt of AL on 
day -14, 385 
• Arm 4b: 1-4 years of age residing with/in same 
families/compounds as Arms 1b, 2b, 3b, 3d, 3f 
(comparator compounds) receipt of AL on day -14, 385 
NOTE: each family compound/vaccine unit will be randomized as 
a whole, and all identified subjects eligible and enrolled from that 
family compound will be assigned that determination 
(Pfs230D1M-EPA/AS01 or comparator) 
 
Pilot Study Objectives: 
 
Primary objectives:  
• To assess safety and reactogenicity of administration of 
Pfs230D1M-EPA/AS01  
• To assess vaccine activity of Pfs230D1M-EPA/AS01 against P. 
falciparum transmission by direct skin feeding assay (DSF) (Arms 
1a/1b only) after receipt of 3rd and 4th vaccination and analyzed 
with the main group 
 
Secondary objectives: 
• To assess vaccine efficacy of Pfs230D1M-EPA/AS01 against P. 
falciparum infection measured by blood smear after receipt of 3rd 
and 4th vaccination and analyzed with the main group 
• To assess immunogenicity as measured by enzyme-linked 
immunosorbent assay (ELISA) titer response to Pfs230D1M  
• To assess functional antibody response by standard membrane 
feeding assay (SMFA) to Pfs230D1M  
 
Exploratory objectives: 
• To explore cellular and humoral responses to Pfs230D1M  
• To identify host and parasite factors associated with transmission  
• To track transmission of parasites from human to human using 
highly variant gene fragments  
• To explore the impact of human movement on transmission and 
parasitemia 

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Pilot Study Endpoints: 
Primary endpoints: 
• Incidence of local and systemic adverse events (AEs) and serious 
adverse events (SAEs)  
• P. falciparum functional activity as measured by DSF (Arms 1a/1b 
only) after receipt of 3rd and 4th vaccination and analyzed with the 
main group 
Secondary endpoints: 
• P. falciparum asexual parasitemia as measured by blood smears 
after receipt of 3rd and 4th vaccination and analyzed with the main 
group 
• Anti-Pfs230D1M immunoglobulin (Ig) G levels as measured by 
ELISA  
• Transmission-reducing activity (TRA)/Transmission-blocking 
activity (TBA) of induced antibody in SMFA  
 
Exploratory endpoints: 
• Cellular immune responses and antibody repertoire of functional 
antibody responses to vaccination  
• Measuring hemoglobin, hemoglobin variants, levels of 
parasitemia/gametocytemia, OMICS analyses (proteomics, 
genomics), clinical malaria infection and treatment  
• Sequences of highly variant parasite gene fragments  
• Evaluation of congregate movement measurements by GPS 
tracking on functional activity as measured by DSF and P. 
falciparum asexual parasitemia as measured by blood smears 
(subgroup from Arms 1a/1b only) 
 
Main Study Objectives: 
Primary objective: 
• To assess vaccine activity of Pfs230D1M-EPA/AS01 against P. 
falciparum transmission by direct skin feeding assay (DSF) (Arms 
3c/3d only) 
Secondary objectives: 
• To assess vaccine efficacy of Pfs230D1M-EPA/AS01 against P. 
falciparum infection measured by blood smear (All Arms except 
Arms 3e/3f) 
• To assess safety and reactogenicity of administration of 
Pfs230D1M-EPA/AS01 (All Arms except 4a/4b) 

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• To assess immunogenicity as measured by enzyme-linked 
immunosorbent assay (ELISA) titer response to Pfs230D1M (All 
Arms except Arms 4a/4b) 
• To assess functional antibody response by standard membrane 
feeding assay (SMFA) to Pfs230D1M (All Arms except Arms 
4a/4b) 
 
Exploratory objectives: 
• To explore cellular and humoral responses to Pfs230D1M (All 
Arms except Arms 4a/4b) 
• To identify host and parasite factors associated with transmission  
• To track transmission of parasites from human to human using 
highly variant gene fragments  
• To explore the impact of human movement on transmission and 
parasitemia 
Main Study Endpoints: 
Primary endpoints: 
• P. falciparum functional activity as measured by DSF (Arms 
3c/3d) 
 
Secondary endpoints: 
• P. falciparum asexual parasitemia as measured by blood smears 
(All Arms except Arms 3e/3f) 
• Incidence of local and systemic AEs and SAEs (All Arms except 
Arms 4a/4b) 
• Anti-Pfs230D1M immunoglobulin (Ig) G levels as measured by 
ELISA (All Arms except Arms 4a/4b) 
• Transmission-reducing activity (TRA)/Transmission-blocking 
activity (TBA) of induced antibody in SMFA (All Arms except 
Arms 4a/4b)  
 
Exploratory endpoints: 
• Cellular immune responses and antibody repertoire of functional 
antibody responses to vaccination (All Arms except Arms 4a/4b)  
• Measuring hemoglobin, hemoglobin variants, levels of 
parasitemia/gametocytemia, OMICS analyses (proteomics, 
genomics), clinical malaria infection and treatment  
• Sequences of highly variant parasite gene fragments  
• Evaluation of congregate movement measurements by GPS 
tracking on functional activity as measured by DSF and P. 

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falciparum asexual parasitemia as measured by blood smears 
(subgroup from Arms 3c/3d only) 
 
 
 
 

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Précis 
A vaccine to interrupt malaria transmission (VIMT), targeting disruption of both human and 
mosquito transmission, would be a valuable tool for local elimination or eradication of this 
disease. One strategy to design a VIMT is using components that block transmission of malaria 
to mosquitoes, such as Pfs230. Pfs230, a surface antigen of intracellular gametocytes, as well as 
extracellular gametes and zygotes in the mosquito stage of Plasmodium falciparum, is currently 
the leading candidate in clinical trials for a malaria transmission-blocking vaccine (TBV). 
Recombinant Pfs230D1M has been conjugated to a recombinant Pseudomonas aeruginosa 
ExoProtein A (EPA) and adjuvanted with AS01. When formulated in AS01, results from a recent 
first-in-human trial demonstrated that Pfs230-EPA induces functional transmission-reducing, and 
in a significant proportion of vaccinees, transmission-blocking serum activity that can be 
measured for months, the vaccine is well-tolerated and safe in adults, and our recent natural 
history data clearly indicate that children play a disproportionate role in malaria transmission. 
The next step in the development of Pfs230D1M-EPA as a TBV is therefore to conduct an age 
de-escalation trial to ensure that the vaccine is safe to administer to children and then to conduct 
a community clinical trial to assess efficacy in family groups.  
 
This Phase 2 study will first determine safety and tolerability of Pfs230D1M-EPA/AS01 in 
healthy Malian children of decreasing ages: 9-18 years old, followed by 5-8 years old. A total of 
60 subjects will be enrolled in Doneguebougou, Mali, West Africa. Children will be recruited 
from compounds/family that have agreed to participate in the main phase of the study and will 
enroll in a staggered manner to receive either Pfs230D1M-EPA/AS01 vaccine or comparator as 
assigned by their compound block randomization. Prior to receipt of vaccination #1, all subjects 
will receive a full treatment course of artemether/lumefantrine (AL). Safety and tolerability will 
be monitored and reported as local and systemic adverse events (AEs) and serious adverse events 
(SAEs) and reviewed by DSMB, sponsor, medical monitors, and study team prior to proceeding 
with enrollment of the main phase.  
 
If there are no safety concerns, in a staggered manner, the main phase will begin enrollment of 
approximately 137 compounds/vaccine units (~1500 vaccinees + ~400 under 5 years of age for 
parasite surveillance). Children enrolled during the pilot safety phase will join their main phase 
compounds/family for vaccination #3. Prior to receipt of first vaccination, all subjects will 
receive a full treatment course of AL. All vaccinated subjects will be monitored for safety and 
tolerability. Immunogenicity outcomes will be antibody responses as measured by enzyme-
linked immunosorbent assay (ELISA) against recombinant Pfs230D1M. Functional activity of 
the induced antibodies will be assessed by standard membrane feeding assays in select samples. 
Vaccine activity will be measured in children 9-18 years of age who will undergo direct skin 
feeds (DSF) starting 2 weeks post vaccination #3 for a total of 8 DSFs. 
 
 

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Prior to scheduled last vaccination in members of the compound/family, children 1-4 years of 
age and vaccinated children 5-8 years of age will receive a full treatment course of AL prior to 
the expected start of the transmission season and will then be followed every 2 weeks by blood 
smear (BS) along with all vaccinated children. Children 9-18 years of age will also be assessed 
for vaccine efficacy, but as a separate analysis from those 1-8 years of age. 
 
In Year 2, those who received vaccination during Year 1 (5 years of age and older at enrollment), 
if eligible and still on study, will receive a single fourth vaccination per their vaccine unit (VU) 
blinded arm assignment. No new individuals will be enrolled. Again, children 1-4 years of age 
and vaccinated children 5-8 years of age will receive a full treatment course of AL prior to the 
expected start of the transmission season and will then be followed every 2 weeks by BS along 
with all vaccinated children. Children 9-18 years of age will also be assessed for vaccine efficacy 
against parasitemia, but as a separate analysis from those 1-8 years of age. Immunogenicity 
outcomes will be antibody responses as measured by ELISA against recombinant Pfs230D1M. 
Functional activity of the induced antibodies will be assessed by standard membrane feeding 
assays in select samples. Vaccine activity will be measured in children 9-18 years of age who 
will undergo DSF starting 2 weeks post vaccination #4 for a total of 10 DSFs. 
 
 
 

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1 Background Information and Scientific Rationale 
 
Background Information 
According to the World Health Organization (WHO), global malaria control efforts have resulted 
in a reduction in the number of deaths since 2000. About 445,000 people are estimated to have 
died due to malaria in 2016, a decline of 37% since 2010 in the WHO regions of Africa. 1 
However, the progress in malaria control has stalled, with no reductions in the number of malaria 
cases worldwide in the past 2 years, per WHO World Malaria Reports in 2017 and 2018. 
Morbidity and mortality caused by malaria also has significant direct and indirect costs to the 
economic development of countries in which the disease is endemic. 2 These factors—as well as 
growing drug resistance of the parasite, widespread resistance of mosquitoes to insecticide, and 
increased human travel—necessitate new approaches to malaria elimination. A vaccine to 
interrupt malaria transmission (VIMT), targeting disruption of parasite transmission through both 
human and mosquito, would be a valuable additional resource in the fight to eliminate this 
disease. 3,4 
 
Transmission blocking vaccines (TBVs) induce anti-sporogonic antibodies that disrupt parasite 
transmission to the mosquito, thereby halting transmission to another human host. Pfs230, a 
parasite protein expressed by gametocytes in the human stage of P. falciparum, and a surface 
antigen of gametes and zygotes in the mosquito stage, is a target of polyclonal and monoclonal 
antibodies with transmission-blocking activity (TBA) in standard membrane feeding assay 
(SMFA.) 5,6,7 The full-length Pfs230 precursor of 360 kDa is expressed in gametocytes within 
erythrocytes, and is processed to become an approximately 300-kDa mature protein upon 
translocation to the surface of freshly emerged gametes from erythrocytes. 8 Malaria-exposed 
populations acquire antibody against Pfs230, which suggests that a Pfs230-based vaccine may be 
boosted by natural malaria infection.  
 
Development of the Study Agent, Pfs230D1M-EPA/AS01 
The recombinant protein Pfs230 domain 1 (Pfs230D1M) was developed at Laboratory of Malaria 
Immunology and Vaccinology (LMIV) and selected for clinical development. Pfs230 contains 
various amino acid substitutions throughout the protein; however, the function of these changes 
is unknown. Recombinant Pfs230D1M, which comprises about 10% of the whole Pfs230 
protein, contains minor allelic variants. 7 A comparative analysis with 11 Malian isolates shows 
two point mutations (G to S at position 64 and K to N at position 120) as well as the known N-
to-Q substitution at amino acid position 44 that LMIV engineered to remove the unique putative 
N-linked glycosylation site (Figure 1). These same mutations were also observed in an analysis 
of over 2,000 parasite isolates. 7 In West Africa, the minor allelic frequency was reported to be 
0.111 and 0.339 for G605S and K661N, respectively, shown Figure 1. Of note, rabbit antisera 
raised against Pfs230D1M blocked parasite transmission of a Thailand isolate with the G605S 

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mutation, suggesting efficacy of Pfs230D1M-EPA against the variant. The biological impact of 
the K661N mutation remains to be determined. 
 
 
Figure 1: Protein Alignments of Recombinant Pfs230D1M to its Respective Native Protein 
or Protein Fragment.  
Deduced amino acid sequence of Pfs230D1 3D7 and 11 other Malian isolates in addition to the amino acid sequence 
of recombinant Pfs230D1M (abbreviated as 230D1M). The amino acids highlighted in yellow denote point 
mutations relative to the 3D7 allele including those mutated N:Q to remove the putative N-linked glycosylation sites 
(i.e., NXS/T). 
 
Several N-terminal sub-domains within the 300-kDa protein were previously evaluated and 
found to induce functional antibodies to block transmission in animal studies. 9,10 Based on these 
findings, using a quality by design strategy, LMIV developed and manufactured a recombinant 
Pfs230D1M corresponding to amino acid sequence positions 542-736 of the full-length Pfs230 
with Pichia pastoris as the production system.  
 
LMIV investigators chemically conjugated Pfs230D1M to EPA, a recombinant mutant and 
detoxified protein from Pseudomonas aeruginosa. EPA is not a component of any licensed 
vaccines but has been extensively studied as a component of conjugated typhoid and shigellosis 
vaccines 11-13 and LMIV/MRTC’s previous phase 1 TBV studies involving Pfs25H, Pfs25M, and 
Pfs230D1M formulated with Alhydrogel or AS01 elicited strong TBAs in mice, rabbits, and 
Aotus monkeys. Pfs230D1M-EPA formulated in Alhydrogel has been evaluated in a Phase 1 
study in US adults (2015) and Malian adults (2015-2016) under National Institute of Allergy and 
Infectious Diseases (NIAID) Protocol #15-I-0044 and was demonstrated to be safe and 
immunogenic both in malaria-naïve and malaria-exposed adults (see Section 3.1). 
 
Pfs230D1M-EPA formulated in the more potent adjuvant AS01 has now completed recent 
evaluation over two years in the ongoing NIAID Protocol #17-I-N006 with initial results 

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supporting higher potency (enzyme-linked immunosorbent assay [ELISA] titers and antibody 
activity) for AS01 (see Section 3.2). AS01 is a liposome-based adjuvant system containing the 
immune-enhancers: MPL (3-O-desacyl-4’-monophosphoryl lipid A) and QS21 (a saponin 
molecule purified from the bark extract of Quillaja saponaria Molina tree). There are 
2 concentrations in the AS01 family, the AS01B Adjuvant System and AS01E Adjuvant System; 
this study will use AS01B diluted 1:1 with conjugated Pfs230D1M to deliver an AS01E dose 
concentration. Further discussion on clinical studies of the study vaccine is provided below 
(Section 3). 
 
Rationale for Study 
The ongoing Phase 1 study of Pfs230D1M-EPA/AS01 in adults (NIAID Protocol #17-I-N006) 
indicates that the vaccine is safe, tolerable, immunogenic, and provides high and durable 
transmission-blocking activity that can be increased with a fourth dose for another season. These 
initial results suggest that Pfs230D1M-EPA/AS01 can be employed in a community wide trial to 
evaluate its efficacy to reduce malaria infections in the community across two malaria seasons.  
 
Rationale for Study Design: Vaccination of School-Aged Children 
The major determinants of an individual’s contribution to onward malaria transmission are 
infectivity and exposure to mosquitoes. Schoolchildren are in general the most infectious age 
group, represent ~35% of the population, and are more often bitten by mosquitoes than toddlers. 
A recent study performed in Burkina Faso and Kenya confirms that after accounting for baseline 
infectiousness (measured by mosquito feeding assays), real-life mosquito exposure (by using 
molecular typing of blood meals) and demography, schoolchildren’s contribution to transmission 
is comparable to adults.14 In particular, in that study, the authors estimated that between 40 – 75% 
of mosquito infections originate from schoolchildren.  
 
Our studies of malaria transmission (NIAID Protocol #17-I-N180) using Direct Skin Feeding 
(DSF) assays (cups of mosquitoes are fed directly on study participant skin to quantify 
transmission) demonstrate that Malian schoolchildren disproportionately transmit parasites to 
mosquitoes (Figure 2). In DSF studies performed from March to August 2018, >95% of 
transmission events among the general Malian population were observed in children >4 years of 
age. For this reason, the impact of the malaria vaccine on transmission in this age group is 
particularly important to understand. Further, trials will need to vaccinate school-age children in 
order to interrupt malaria transmission and demonstrate efficacy (reduction in malaria infection 
incidence in the vaccinated group).  

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Figure 2: Frequency of Positive DSF Assays by Month and Age in Bancoumana, Mali 
(NIAID Protocol #17-I-N180).  
Malian children 5-18 years of age disproportionately transmit in DSF assays, being responsible for 27 of the 28 
transmission events  
Taking the vaccine activity measured via SMFA in Protocol #17-I-N006, in preliminary 
modeling studies (see Figure 3) Pfs230D1M-EPA/AS01, as a single malaria elimination tool, is 
estimated to reduce clinical malaria incidence by ~60% across a malaria season if administered 
to 80% of the target population. 
 

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Figure 3: Modeling estimated vaccine effects on prevalence and clinical incidence of P. 
falciparum malaria.   
Solid line is mass distribution (80% coverage); dashed line targets children <17 years of age. Transmission intensity 
driven by rainfall data from Bamako region. VE estimates from unpublished Pfs230-EPA/AS01 Phase 1 trial. 
Predictions will change with refined VE, study population data.  
 
Underpinning these plots is a developed mathematical model of malaria transmission.15 It is 
fitted to data on Plasmodium falciparum malaria prevalence and clinical incidence from across 
Africa, covering a range of transmission settings. In the model, a population develops immunity 
according to its level of exposure to the parasite. The model also allows for heterogeneity of 
exposure within the population (i.e. some individuals are bitten more often than others). The 
model also tracks the mosquito population (see supplement for Griffin et al paper15): adult 
female mosquitoes can be susceptible, latently infected, or infectious. 
 
The model has been used to estimate the impact of certain interventions, such as bed nets, indoor 
residual spraying and the RTS,S vaccine. Our aim was to take provisional data from the pilot 
study of Pfs230D1M-EPA/AS01 (presented at ASTMH 2017) to obtain a rough profile of the 
vaccine, describing the antibody dynamics & vaccine efficacy as a function of antibody titer (see 
Figure 4). For the latter, a Hill function relation was assumed, using the same 'shape parameters' 
as fitted to detailed RTS,S data. Essentially, we assume that the high transmission-reducing 
activity (TRA) observed for high antibody titers translates to high TBA (Figure 4B), which then 
wanes over time, as antibody levels decrease. 

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Figure 4: Antibody dynamics and association with vaccine efficacy.  
Black dots denote the data points from preliminary Phase I results. (A) Antibody dynamics predicted by the model 
after TBV vaccination schedule with two doses once per month and a booster dose 6 months after the first dose. (B) 
Estimated dose response relationship between antibody titers and vaccine efficiency after the second TBV dose. (C) 
Vaccine efficacy estimation over time based on the model predictions from panel A and dose response curve from 
panel B.  
 
This vaccine efficacy is useful to reduce the infectiousness (to mosquitoes) of those individuals 
in the model who are infected. The model is run with different scenarios for who receives the 
vaccine, to see how reducing the force of infection from humans to mosquitoes reduces 
subsequent human infections. A number of scenarios were considered, as described in the Figure 
4 caption above. We looked at the impact of TBV alone, and delivered along with the RTS,S 
vaccine.  
 
In all scenarios, we assumed 80% vaccine coverage in the population targeted in the intervention. 
We assumed that everyone who receives the vaccine received all doses. 
 
An important limitation of this model is that it does not account for spatial details. When an 
individual in the model is infected, the previous human host for those parasites could be any 
infected individual in the population, i.e. there is no sense in which one person is 'near' to 
another, or 'far away' from a third person. For example, in this model, it is inconsequential if 
people don't sleep in their compound, whereas in reality this could be quite important. 
 
This model underscores how important it is to test this vaccine by administering it to school-age 
(5 years of age and older) children and to adults in a small community setting (family household) 
and then assessing its safety, functional immunogenicity, and efficacy for reducing malaria 
infections within that community.  
 

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These results, together with evidence that transmission is local (as described in Section 1.2.2), 
suggest that for each malaria transmission unit, which corresponds to individual households, 
inclusion of schoolchildren in transmission-blocking intervention trials will directly impact 
infection incidence where they live, including their own risk of future infection in the same 
transmission season.  
 
Rationale for Study Design: Family Compound and Requirement of Broad 
Vaccine Coverage 
We believe TBV clinical trials will need to vaccinate individuals in groups (or “clusters”) in 
order to interrupt malaria transmission and demonstrate efficacy (i.e. efficacy to reduce malaria 
infection incidence in the vaccinated group). Vaccinating at the family compound level may be 
sufficient to achieve the effect, because clustering of malaria infection may occur at the family 
compound level. The hypothesized mechanism for compound infection clustering relates to the 
activity of female Anopheles mosquitoes, which after blood-feeding and then ovipositing in the 
nearest breeding site (water source), return to the same or neighboring houses, especially when 
there are nearby breeding sites. 
 
Clustering of infections is also the basis of the widely used strategy of reactive case detection, 
which involves testing and treatment of individuals living in the same or neighboring households 
of passively-detected clinical cases. The efficiency of this strategy in identifying infections 
corroborates the notion that infection in a household depends mostly on the infection status of its 
members.16 
 
The following evidence provides support that a malaria transmission unit corresponds to a 
household or at most neighboring households: 
• There is clustering of malaria infections within individual households17 
• Anopheles mosquitoes generally disperse less than 2 km18 
• Household-level variation in sporozoite infection rates have been observed, independent 
of mosquito numbers19 
• There are reports of non-P. falciparum malaria household clustering in areas with P. 
falciparum transmission20 
• Mathematical models that account for mosquito oviposition suggest that distribution of 
breeding sites is a determinant of locality of transmission21  
 
  
  
  
  
First, individuals living in households with passively detected cases are more likely to be 
infected, compared to control households with no cases. 17 Of note, the odds ratio of infection is 
increased in the households where the cases live compared to the neighboring households, and is 
also increased but to a lesser extent in neighboring households compared to randomly selected 
households. This pattern suggests that infection clustering at the household level is due to a 

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limited range of transmission for individual mosquitoes, and not due to other factors that would 
not explain the weaker association in neighboring households, such as multiple interrupted 
feeding episodes from the same mosquito or host genetics.  
 
Micro-epidemiological analysis in coastal Kenya confirms this pattern, and single-household 
clusters have been identified in that region.22 
 
Pertinent to our proposed trial, published analyses of longitudinal data from Mali show the 
presence of single-household clusters of transmission that lasted several months, further 
highlighting the narrow range of transmission for individual mosquitoes.20 When analyzed in 
relation to season, clustering was observed within individual or a few adjacent compounds (i.e., 
households) in the rainy season (panels A and B, Figure 5 below), while clustering was 
extended over a larger number of compounds after the rains or during the dry season (panels C 
and D). This is consistent with mosquitoes staying within individual compounds during the rains 
when oviposition/breeding sites are relatively ubiquitous, and then with mosquitoes dispersing 
more widely after the rains have ended when breeding sites become sparser. 
 
 
 
Figure 5: Spatial and temporal locations of infection clusters20:  
In this figure, temporal changes in the spatial scale of transmission in Bancoumana village, Mali, are shown. (a) 
October 1996, (b) October 1997, (c) December 1998, (d) May 2000. Dots represent households. Rate ratios are 
presented in brackets near each cluster. P.f.: cluster of P. falciparum infections (in red). gam: cluster of P. 
falciparum gametocyte carriages (in green). P.m.: cluster of P. malariae infections (in blue). The 4 time frames were 

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selected such that all the clusters were represented. For each of the 4 time frames, the x- and y-axis represent GPS 
coordinates. In the panels on the top (5a and 5b), clusters of transmission that consist of single or a few households 
are highlighted; these plots present data from the rainy season. After the rainy season subsides (panels 5c and 5d), 
clustering of infection extended through larger geographical areas, which is consistent with greater dispersal of 
mosquitoes as described in the arguments presented in this protocol.  
 
These patterns of infection clustering are consistent with entomological observations that, 
although long-distance mosquito dispersal has been observed, most mosquito movements are 
thought to be limited to a radius of less than 2 km,18 and may be more limited when sites for 
oviposition are nearby and readily accessible. Differences in household-level prevalence of 
infection in mosquitoes are also evidence of locality of transmission, as there is no evidence that 
infected mosquitoes’ movements would be systematically different from uninfected mosquitoes, 
and biased towards particular households. Data from a recent study performed in Burkina Faso 
showed that infection (sporozoite) rates in bloodfed mosquitoes that had head-thorax tested by 
polymerase chain reaction (PCR) vary between households independently of the number of 
mosquitoes,19 which supports the hypothesis that mosquitoes tend to return to the same house 
after feeding and ovipositing.  
 
Describing parasite genetic similarity at different spatial scales is another way of assessing the 
scale of transmission. Although these data are limited at the micro-epidemiological level, the 
distribution of non-falciparum malaria parasites in areas with predominant falciparum 
transmission is informative in this respect. In Mali, non-falciparum species are more likely to 
occur in a small fraction of households, where their household-level prevalence is high (see 
Figure 5 above), suggesting that these non-falciparum parasites, transmitted by the same vectors 
as Plasmodium falciparum, circulate locally, i.e. individuals in these households are infected by 
other members of the same household. Clustering data supports the idea that parasites circulate 
locally within households. 
 
Finally, mathematical models that have explicitly incorporated mosquito oviposition suggest that 
the distribution of breeding sites, whether they are productive or not, are an important 
determinant of the scale of transmission, i.e. the average distance between the locations where 
individual mosquitoes acquire infection and where they feed after full parasite development.21 In 
this respect, locality of transmission is likely to be more important in households near permanent 
breeding sites during the dry season, and throughout villages during the rainy season, since even 
temporary breeding sites can support oviposition and during this period it is more likely that 
mosquitoes find a suitable oviposition site near the house where they feed. 
 
The prominent role that children play in malaria transmission, as previously described in 
Section 1.2.1, combined with this evidence that transmission is local, suggests that for each 
malaria transmission unit, which corresponds to individual family compounds, inclusion of 
children 5 years and above in transmission-blocking intervention trials will impact the incidence 

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of malaria infections for all individuals residing in the family compound, including young 
unvaccinated children as well as vaccinated children and adults.  
 
Rationale for Study Design: Vaccine Dose and Regimen 
The vaccination dose and schedule were selected, and subsequently adjusted as described below 
in Section 1.2.3.1, based on initial results of the ongoing Phase 1 trial of Pfs230D1M-EPA/AS01 
and another investigational TBV, Pfs25M-EPA/AS01, in Malian adults (NIAID Protocol #17-I-
N006). The main phase of this study (#17-I-N006) evaluated Pfs230 antibody titers and function 
using a dose of 40 micrograms (µg) administered on study days 0, 28, 168 days, which is the 
same dose proposed to be used in this study in both children and adults.  
 
Preliminary analysis of antibodies against Pfs230 titers and function indicate that significantly 
high transmission reducing activity is observed for at least 12 weeks post the second dose 
compared to the activity observed in controls (Figure 16). However, it is only after receipt of the 
third dose when the transmission blocking activity becomes significantly higher compared to 
controls, indicating the benefit of a 3-dose regimen (Figure 17).  
 
Given this background, we plan to conduct an age de-escalating trial for Pfs230D1M-EPA/AS01 
in 2 different age groups: ages 9 to 18 years, and 5 to 8 years. Subjects will be recruited from 
compounds that have undergone initial consenting and screening and of which approximately 
80% of the compound/vaccine unit has agreed to participate and appears eligible to participate. 
In each group, subjects will receive three doses at 0, 28, 56 days of either 40 µg 
Pfs230D1M-EPA/AS01 or comparator vaccine. Safety data will be gathered, monitored, 
reviewed, and reported throughout the trial in order to proceed with further enrollment. Periodic 
blood collections will also be used to assess immunogenicity and functional activity of 
Pfs230D1M-EPA/AS01 over the course of the study. Subjects will be followed through at least 
six months after the last vaccination and will join their family compound during the main phase 
around vaccination #3.  
 
Upon determining that Pfs230D1M-EPA/AS01 is safe in children 5-18 years of age, all eligible 
residents 5 years of age and older in identified participating family compounds/vaccine units will 
be enrolled for vaccine administration to assess safety and efficacy. Subjects will receive study 
vaccine or comparator vaccine according to the family compound assignment at 0, 28, and 56 
days. Safety outcomes will be monitored and reported as local and systemic adverse events and 
serious adverse events (SAEs). Vaccine efficacy will be assessed by blood smear (BS) 
microscopy for detection of new P. falciparum infections in children 1-8 years of age living in 
the compound as well as 9-18 years of age. Vaccine activity will be assessed in children 9-18 
years of age by DSF assays whereby laboratory-raised mosquitoes are fed on study participants’ 
skin. 
 

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Our previous studies indicate that treatment with artemether-lumefantrine (AL) can reduce the 
rate of positive DSF assays for 4 or more weeks. For this reason, children 9-18 years of age who 
will undergo DSF to assess vaccine activity will not receive AL treatment before their 3rd or 4th 
vaccine dose. Since they are not receiving treatment before the transmission season, we will 
explore their reduced risk of infection as compared to controls on the basis of the proportion of 
positive BS. 
 
We hope that the results of this study will help us determine the safety and tolerability of 
Pfs230D1M-EPA/AS01 in Malian children and to assess its functional activity as well as its 
efficacy to prevent P. falciparum infection.  
1.2.3.1 Rationale for Change in Vaccine Dosing Schedule 
1.2.3.1.1 Malaria Transmission Dynamics in Mali 
The two epidemiological motivations for this change are: (i) expected temporal variation in 
locality of malaria transmission and (ii) changes in the number of human-to-mosquito 
transmission events over the course of a transmission season.  
1.2.3.1.1.1 Local, Seasonal Transmission 
There is evidence, that includes different types of studies (described in the Section of 1.2.2), that 
malaria transmission occurs at a very local scale, 18-20 that is, that the distance between the 
locations where mosquitoes acquire infection and where they feed after falciparum parasite 
sporogonic development, potentially inoculating sporozoites in uninfected individuals, is on 
average small. This key epidemiological observation provided the basis for the selection of 
compounds as units of randomization and analysis in this clinical trial, as the impact of 
transmission blocking vaccines is likely to be most evident at this scale. 
 
One of the determinants of the spatial scale in which malaria transmission occurs is the 
distribution of mosquito breeding sites. 21,23 Between blood meals, Anopheles mosquitoes lay 
eggs on water bodies, and the distance traveled for oviposition will depend on the presence of 
nearby breeding sites. During the rainy season, in areas with endemic malaria transmission, 
including in the village where this vaccine trial is being performed, the number of temporary 
breeding sites and their larval productivity increases, as observed in entomological studies across 
different setting24,25 and respective colleagues observed that the frequency of aquatic habitats and 
their stability were influenced by rainfall.26,27  This more widespread distribution of potential 
oviposition sites implies a high likelihood that there will be nearby breeding sites for mosquitoes 
ovipositing after feeding in study compounds/vaccine units. It is thus during this period that 
circulation of parasites within vaccine units (which may be one or more compounds) is expected 
to be highest (i.e. infected mosquitoes biting individuals living in the same compound/vaccine 
unit where they acquired their infection). As explained in detail in Section 1.2.2, there is 

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evidence from epidemiological studies in Mali that the degree of locality of malaria transmission 
varies seasonally (Figure 5).  
 
The primary endpoint for this trial and a measure of vaccine activity is the frequency of parasite 
transmission events measured by direct skin feed (DSF) assays in older children. As positive 
DSF assays are infrequent, maximizing antibody titers throughout the time period when positive 
DSF assays are most frequent will increase our power for detecting vaccine activity. A secondary 
endpoint of this trial and a measure of vaccine efficacy is the prevalence of malaria parasites in 
children living in the randomization units (P. falciparum asexual parasitemia measured by blood 
smears). Since malaria transmission is local during the rainy season, it is during this period that 
the vaccine, randomized to entire compounds, will have a more important effect on compound-
level human infection prevalence. For this reason, we propose to modify the schedule of vaccine 
dosing so that antibodies induced by the vaccine reach sufficiently high levels when direct 
benefit to vaccinated units is more likely. 
1.2.3.1.1.2 Changes in Human-to-Mosquito Transmission Events 
The optimal dosing schedule of transmission blocking vaccines with regards to their impact on 
infection numbers in mosquito populations requires that vaccine activity, measured by reduction 
in mosquito infection probability after blood feeding, reaches maximum levels during the periods 
when human-to-mosquito transmission is epidemiologically more important, that is, when most 
transmission events from humans to mosquitoes occur (Figure 6) In areas with seasonal 
transmission, two different processes determine malaria infection status in Anopheles 
mosquitoes: probability of transmission of parasites from humans in individual mosquito blood 
meals and age distribution of mosquito populations. While it has been argued that at the end of 
the transmission season the prevalence of malaria infection in mosquitoes is explained by the 
latter, mosquito age demographics, and the presence of, on average, older mosquitoes28 (as a 
direct consequence of the cumulative risk of infection, for mosquitoes, during the entire 
transmission season), human falciparum transmission to uninfected mosquitoes is essential 
throughout the wet season. Preventing mosquito infection at the start and peak of the rainy 
season is thus more likely to have an effect on infection levels compared to preventing human-
to-mosquito transmission at the end of the wet season. 
 

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Figure 6. Impact of number of Anopheles and gametocyte prevalence on number of 
human-to-mosquito transmission events.  
This figure illustrates temporal changes in two major determinants of the number of human-to-mosquito 
transmission events: number of Anopheles mosquitoes in a village and gametocyte prevalence, which is, for 
example, directly linked to infection prevalence. In the top panel, numbers of mosquitoes collected in the village of 
Bancoumana, Mali, with indoor live catches are shown (data from 2018). The second panel presents gametocyte 
prevalence in the villages of Bancoumana and Doneguebougou in 2018; note that parasitological data are not 
available for Doneguebougou before September. The third panel illustrates Pfs230-targeted immune responses with 
vaccine dosing on days 0, 28 and 56; the data presented is from experiments involving mice immunization, and here 
it is assumed that the vaccination would start in June. With the vaccination schedule proposed in this amendment, 
higher antibody levels will be present in individuals living in vaccinated randomization units during the time period 
when most transmission events occur. The bottom panel shows unpublished data from a clinical trial undertaken in 
the study area,  
 
Our data in both animal and human studies have shown that anti-Pfs230 ELISA titers correlate 
well with serum functional activity that blocks parasite transmission to mosquitoes, hence we 
seek to maximize the antibody levels and thereby 1) block parasite transmission during the time 

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window when within-household transmission may be maximal; 2) block parasite transmission 
during the time window when the frequency of positive DSF may be maximal. By changing the 
dosing schedule so that the second dose of vaccine is given early August and the third dose of the 
vaccine is given in early September 2019, we expect to induce high transmission reducing and 
blocking activities during peak transmission season, which will maximize the impact of the 
vaccine on parasite circulation within households in natura and on parasite transmission in the 
DSF assay. Indeed, as mentioned in the Section 3.2.2 of the study protocol, preliminary findings 
of a pilot vaccine trial indicate that Pfs230D1M-EPA/AS01 transmission reducing activity, as 
measured in standard membrane feeding assays, is statistically significantly higher 3 months 
after the 3rd dose compared to 3 months after the 2nd dose. Hence, giving the third vaccine dose 
one month rather than 5 months after the second dose will allow us to maximize antibody titers 
(and we therefore expect serum functional activity) during the periods of maximal intra-
household transmission and of most frequent DSF transmission events. 
 
These two factors, the expected relative increase in within-randomization unit parasite 
circulation during the rainy season and the proportionally high number of human-to-mosquito 
transmission events in the same period, implies that by administering a third dose of the 
Pfs230D1M-EPA/AS01 vaccine on study day 56 rather than on day 168, transmission blocking 
antibody levels will be sustainably high when the effect of the vaccine on prospective risk of 
human malaria infection would provide maximal benefit to the vaccinated units. 
1.2.3.1.2 Supporting Safety and Immunological Data from RTS,S/AS01E Experience 
In addition to these epidemiological arguments, there is evidence, from a malaria vaccine clinical 
study that used the same adjuvant as this trial (AS01E) that a vaccine schedule of 0, 1, 2 months 
is both safe and immunogenic.  
 
In a Phase 2 pediatric trial of RTS,S, a malaria vaccine using the same adjuvant (AS01E), that is 
being used in this protocol with Pfs230D1M-EPA, various vaccine dosing regimens were 
compared (0, 1 versus 0, 1, 2 versus 0, 1, 7 months) with the primary outcomes measured being 
the occurrence of SAEs until 10 months post dose 1.29   For the vaccine dosing regimens that 
included three vaccinations (0, 1, 2 months versus 0, 1, 7 months), Owusu-Agyei and colleagues 
observed similar numbers of AEs between the two vaccination regimens (Figure 7).  The 
proportion of study participants that developed pain at the injection site, swelling or fever were 
similar in the two vaccine dosing schedules as well. The occurrence of at least one SAE, at 10 
months post first vaccination and 19 months post vaccination, were also similar in the 0, 1, 2 
month vaccination regimen arm (14.4%, 7.9-23.4;  24.4%, 16.0-34.6) versus the 0, 1, 7 month 
vaccination regimen arm ( 15.6%, 8.8-24.7;  18.9%, 11.4-28.5).29 

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Figure 7. Percentage of solicited events post dose 1, 2, and 3 RTS,S (total vaccinated 
cohort).  
Yellow = RTS,S/AS02D; Green = RTS,S/AS01E;l; Grey = Rabies vaccine; Orange = Grade 3; Bars represent 95% 
confidence intervals. Adapted from Owusu-Agyei, S et al.29  
 
In that study, antibody levels against circumsporozoite protein reached higher peak levels when 
the third dose of the vaccine was administered a month after the second dose (0, 1, 2 months) 
compared to dosing of the same vaccine at 0, 1, 7 months (Table 1). The highest anti-CS GMTS 
seen in the study, comparing all regimens and adjuvants, was seen in the RTS,S/AS01E 0, 1, 2, 
month schedule (month 3 = 632 EU/mL, 95% CI 554, 720; compared with month 8 in the 0, 1, 7 
month schedule = 373 EU/mL, 95% CI 311, 447). 
 

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Table 1. GMTs and sAUC for anti-CSP antibodies and GMTs for anti-HBs antibodies. 
Adapted from Owusu-Agyei, S et al.29 
 
 
 
Rationale for Study Design: Inclusion of Children Under 5 Years of Age 
Children under 5 years of age are excluded from vaccination during this study because there are 
insufficient data regarding dosing or AEs from our trials in adults to judge the potential risk in 
children in this age group. Some evidence suggests an increased risk of febrile seizures with 
receipt of AS01 from RTS,S/AS01E studies in young children; since febrile seizures in the 
general population are most likely to occur in children under the age of 5 years, children under 
5 years old are excluded from vaccination in this study.  
 
In addition, Malian children under 5 years of age may make only a modest contribution to 
malaria transmission in the community. Recent studies suggest that mosquitoes feed infrequently 
on under-5 African children (compared to school age children or adults), hence their contribution 
to malaria transmission may be as low as 2-4% at some sites.14 Further, Malian children now 
receive monthly antimalarial treatments with long acting agents (termed Seasonal Malaria 
Chemoprevention or SMC), which limits their rates of gametocytemia, further reducing their 
contribution to malaria transmission. Thus, under-5 children are not a critical target demographic 
for vaccination. 
 
Notably, under-5 children are an important target to assess TBV efficacy even though they are 
not an important target for vaccination per se. Although under-5 children receive mosquito bites 

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infrequently, they have a high rate of infection in Mali as well as any African site, presumably 
because they lack immunity to prevent infection even when bitten by a single infectious 
mosquito. In our studies of Malian children who receive monthly SMC, we observed that 58% of 
children develop a patent parasitemia based on BS performed at the time of the monthly SMC 
treatments. 
2 Previous Preclinical Experience with Pfs230D1M-EPA/AS01 
In preclinical studies evaluating Pfs230D1M-EPA formulated with AS01 adjuvant in rabbits at 
two-week intervals over a 43-day study interval, the vaccine was determined to be safe, with 
expected mild, transient injection-site reactogenicity and mild, transient alterations in clinical 
laboratory parameters consistent with immune stimulation following vaccination.  
 
In animal studies conducted at LMIV, AS01 and AS01B adjuvanted products when compared to 
Alhydrogel consistently yielded the highest and longest sustained antibody responses with 
associated significant functional activity by SMFA (Figure 8). 
 
Figure 8: Comparison of Transmission-Blocking Activity of Pfs25M-EPA/AS01 and 
Pfs230D1M-EPA/AS01 to Pfs25M-EPA/Alhydrogel and Pfs230D1M-EPA/Alhydrogel in 
CD1 Mice.  

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A and B show antibody responses 14 days following Vaccination #2 to anti-Pfs25 (A) and anti-Pfs230 (B) 
immunoglobulin G (IgG) at increasing antigen doses adjuvanted with AS01E, Alhydrogel, and AS01B in CD1 mice 
that either received Pfs25 alone or Pfs25 and Pfs230 vaccinations. Circles represent antibody level per mouse and 
black bars indicate geometric mean of antibody levels. C and D report standard membrane feeding assay (SMFA) 
results without and with complement from sera samples 14 days following Vaccination #2 at increasing antigen 
doses adjuvanted with AS01E, Alhydrogel®, and AS01B in CD1 mice that either received Pfs25 alone, Pfs230 alone, 
or Pfs25 and Pfs230 vaccinations. In C and D, circles represent individual mosquitoes and black bars indicate 
geometric mean of oocyst/mosquito. AS01B and AS01E in this figure are designations that mean AS01B was diluted 
with conjugate/antigen in the formulation to have a dose equivalent to 50 µg monophosphoryl lipid (MPL) and 
50 µg QS21 in a 0.5 mL dose; and AS01E is the adjuvant AS01B diluted to a dose equivalent to 25 µg of MPL and 
25 µg of QS21 in a 0.5 mL dose. 
3 Previous Human Experience with Pfs230-EPA 
 
Pfs230D1M-EPA/Alhydrogel in Healthy Adults (#15-I-0044) 
A Phase 1 dose-escalating study evaluating the safety, tolerability, immunogenicity, and 
functional activity of Pfs230D1M-EPA adjuvanted with Alhydrogel was conducted in 2014-2017 
in both the U.S. and Bancoumana, Mali (NIAID Protocol #15-I-0044; clinicaltrials.gov: 
NCT02334462). Another TBV candidate, Pfs25M-EPA/Alhydrogel, was also assessed as a 
standalone vaccine and also co-administered with Pfs230D1M-EPA/Alhydrogel (Table 2).  
 
Table 2. NIAID Protocol #15-I-0044 Enrollment and Vaccinations.  
 
US 
Mali 
 
 
 
n 
n 
Vaccine 
Schedule 
(month) 
Pfs25 
5 
5 
16 µg Pfs25M-EPA/Alhydrogel 
0, 1, 6A, 
18A 
months 
5 
50 
47 µg Pfs25M-EPA/Alhydrogel A 
Pfs230 
5 
0 
5 µg Pfs230D1M-EPA/Alhydrogel 
5 
5 
15 µg Pfs230D1M-EPA/Alhydrogel 
5 
50 
40 µg Pfs230D1M-EPA/Alhydrogel A 
Pfs25 
+ 
Pfs230 
5 
5 
16 µg Pfs25M-EPA/Alhydrogel + 15 µg Pfs230D1M-
EPA/Alhydrogel 
5 
50 
47 µg Pfs25M-EPA/Alhydrogel + 40 µg Pfs230D1M-
EPA/Alhydrogel A 
Comparator 
0 
60 
TWINRIX, Menactra A 
A Arms that received the full 4 dose regimen (initial series + booster) during the vaccine activity phase (main) of the Mali 
study. 

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Safety of Pfs230D1M-EPA/Alhydrogel in Healthy Adults 
In both the U.S. and Mali safety dose-escalation of Pfs230D1M-EPA/Alhydrogel, vaccinations at 
increasing doses were well-tolerated, with minimal local and systemic reactogenicity. The 
majority of the reported AEs were mild (Grade 1) or moderate (Grade 2). Overall, the reported 
local reactogenicity by vaccination and dose appeared to increase in the frequency of reporting 
and duration of symptoms with increasing antigen dose of Pfs230, but not with subsequent 
vaccinations. Solicited systemic symptoms were reported in all dosing arms and did not increase 
in frequency, severity, nor duration with increasing antigen dose. Various laboratory 
abnormalities, the majority being Grade 1, were noted across the dosing arms, and when 
compared to a control vaccine in Mali, Pfs230 appeared to have an increase in abnormal 
laboratory values, though only one was deemed related to Pfs230 vaccination. In a single Pfs230 
vaccinated subject in Mali, a Grade 3 gastroenteritis was reported with associated Grade 4 
laboratory abnormalities (leukocytosis, blood creatinine increased), all deemed unlikely related 
to vaccination and all of which resolved shortly after resolution of the gastroenteritis symptoms. 
Other than the one noted above, there were no Grade 3 or 4 AEs. No SAEs were reported in the 
Pfs230 vaccinated arms. 
 
Safety analysis of the high dose (40 µg) of Pfs230D1M-EPA/Alhydrogel showed that many 
reported AEs have been mild (Grade 1; 708/1431; 49%), with the most commonly reported AEs 
being injection site pain, headache, malaria, neutropenia, nasopharyngitis, and rhinitis. The most 
commonly reported related AEs were injection site reactogenicity (pain, induration, pruritus, and 
edema), leukopenia, neutropenia, and headache, which have been all Grade 1 or 2. Most reported 
related AEs were injection site pain, which did not increase in frequency with subsequent 
vaccination. Overall, in comparison to the comparator arms, Pfs230D1M vaccinees, alone or in 
combination, reported more related AEs, the majority of which were Grade 1 or 2 local 
reactogenicity. During the study period, 3 serious adverse events were reported in the high dose 
arms; Pfs230D1M: a snake bite; Pfs230D1M: peritonsillar abscess; Pfs25M+Pfs230D1M: 
cerebrovascular accident which resulted in death - all were determined to be unrelated to 
vaccination. All of these subjects completed the 4 vaccinations. No participants were removed 
from study participation due to a related AE of any severity. The cerebrovascular accident which 
resulted in death case was a healthy 51-year-old woman enrolled in a trial conducted from 2014 
to 2016 who developed symptoms of a stroke approximately a week after receiving her fourth 
and last vaccination on study. She died the following day as a result of the stroke. This serious 
event was reviewed by Sponsor, Institutional Review Board (IRB), Faculté de Médecine 
Pharmacie d’OdontoStomatologie (FMPOS) Ethics Committee (EC), DSMB, and U.S. Food and 
Drug Administration (FDA) and was determined unrelated to the vaccine she received. 
 
As mentioned above, Protocol #15-I-0044 also evaluated the use of Pfs230D1M-EPA in 
combination (co-administration at separate sites) with Pfs25M-EPA at 2 different dose regimens: 
Pfs25M 16 µg + Pfs230D1M 15 µg and Pfs25M 47 µg + Pfs230D1M 40 µg. In general, there 

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were more AEs reported overall given the increased reporting of local reactogenicity per each 
vaccination received (2 versus 1). Overall, the reported local reactogenicity by vaccination and 
dose appeared to increase in the frequency of reporting with increasing doses and was more 
likely to be attributed to Pfs230 than Pfs25, but not significantly so. However, with increasing 
antigen dose, the duration of symptoms and severity of symptoms remained unchanged. Solicited 
systemic symptoms were reported in all dosing arms and did not increase in frequency, severity, 
nor duration with increasing antigen dose. 
 
Overall, there has been no significant difference in reported local or systemic solicited 
reactogenicity or laboratory abnormalities between Pfs230D1M-EPA/Alhydrogel alone, 
Pfs25M-EPA/Alhydrogel alone, or co-administration of Pfs25M-EPA/Alhydrogel and 
Pfs230D1M-EPA/Alhydrogel. In conclusion, in malaria-naïve and malaria-exposed adults, 
Pfs25M-EPA/Alhydrogel administered alone, Pfs230D1M-EPA/Alhydrogel administered alone, 
or co-administration of Pfs25M-EPA/Alhydrogel and Pfs230D1M-EPA/Alhydrogel is safe and 
tolerable. 
 
Immunogenicity and Functional Activity of Pfs230D1M-EPA/Alhydrogel in 
Healthy Adults 
Pfs230D1M induced antibody responses in most US vaccinees (Figure 9C-E) and resulted in 
high transmission blocking activity in 2/5 individuals (100%, 98% transmission-reducing activity 
[TRA]) and significant transmission reducing activity (73%, 62% TRA) in 2 others (Figure 10A) 
after just 2 vaccine doses. In the Pfs25M + Pfs230D1M combination group, antibody responses 
were similar to the individual antigen arms (Figure 9F-I) and 2 individuals had appreciable 
functional activity after 2 vaccinations (one individual had 90% and one had 68% (Figure 10A). 
The activity correlated well with anti-Pfs230D1M titers, demonstrating that the functional 
activity is due to the vaccine (Figure 10C). Pfs230D1M functional activity dependency on 
complement was confirmed with the Pf230D1M immune sera samples, as heat-inactivated sera 
markedly reduced inhibitory activity from these individuals (Figure 11). 

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Figure 9: Pfs230-specific Antibody Responses in Subjects Receiving Pfs230D1M, U.S. 
Cohort (#15-I-0044).  
Results presented in enzyme-linked immunosorbent assay (ELISA) units for each arm. Vaccinations occurred on 
Days 0, 28. Day 0 was drawn pre-vaccination; Day 42 is 14 days post Vaccination #2. Each individual datapoint 
represents an individual subject anti-Pfs230 ELISA response. 
 

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Figure 10: Pfs25 and Pfs230 Functional Activity by Standard Membrane Feeding Assay 
(SMFA).  
Samples obtained 14 days following receipt of Vaccination #2 in the highest antigen dose arms (Pfs25M 47 µg 
alone, Pfs230D1M 40 µg alone, and Pfs25M 47 µg + Pfs230D1M 40 µg co-administered). Each individual column 
represents an individual subject. Each individual datapoint represents a single mosquito dissected. 
 
 
Figure 11: Pfs230 Functional Activity by Standard Membrane Feeding Assay in the 
Presence or Absence of Complement.  
Each individual column represents assay results for an individual subject using Day 0 (before Vaccination #1) and 
Day 42 (14 days post Vaccination #2) serum samples. Each individual datapoint represents a single mosquito 
dissected. Top set of figures is the assay completed with complement present, the bottom set of figures is the assay 

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completed without complement present in the assay. Only the highest antigen dose arms (Pfs230D1M 40 µg alone, 
n=5, and Pfs25M 47 µg + Pfs230D1M 40 µg co-administered, n=5) are presented. 
 
Evaluation of immunogenicity by ELISA in healthy Mali adults showed a few individuals did 
have pre-existing baseline responses to Pfs230. The majority of vaccinated subjects developed 
responses to Pfs25 or Pfs230 following 2 doses of vaccine (Figure 12). When comparing 
Pfs25M-EPA to Pfs230D1M-EPA in Alhydrogel during the main phase of the Mali study, we 
found that Pfs230 alone compared to Pfs230 and Pfs25 in combination produced similar results 
in regard to immunogenicity (peak ELISA responses; Figure 12), functional activity (SMFA; 
Figure 13), and percentage of responders (detectable antibody responses).  
 
 
 
Figure 12: Pfs230-specific Antibody Responses in Subjects Receiving Pfs230D1M, 40 µg in 
Mali (#15-I-0044).  
Results presented in enzyme-linked immunosorbent assay (ELISA) units for each arm. Vaccinations occurred on 
Days 0, 28, 168, 540. Day 0 was drawn pre-vaccination; Each individual datapoint represents an individual subject 
anti-Pfs230 ELISA response. 
 

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Figure 13: Pfs230 Functional Activity by Standard Membrane Feeding Assay in Mali, #15-
I-0044.  
Each individual column represents assay results (calculated TRA, TBA) for an Arm of the Main Phase in Mali. Each 
individual datapoint represents a single subject’s TRA or TBA for that time point. Vaccinations were received on 0, 
28, 168, 540 and samples for SMFA taken 2 weeks post dose 3 and 4 as well as 10 weeks post dose 4. 
 
Overall, there was no statistically significant difference between Pfs230 alone versus Pfs230 and 
Pfs25 given in combination, though Pfs230 alone did have a trend to higher overall peak ELISA 
responses and consistently higher SMFA responses. 
 
 
Pfs230D1M-EPA/AS01 in Healthy Malian Adults (#17-I-N006) 
The double-blind comparator-controlled Phase 1 trial of Pfs230D1M-EPA/AS01 in Malian 
adults (NIAID Protocol #17-I-N006) evaluated Pfs230D1M-EPA/AS01 at escalating doses of 13 
µg and 40 µg administered on a schedule of 0, 1, and 6 months in a pilot study. Once safety was 
reviewed, the main phase started and looked at Pfs230D1M 40 µg at full (3-doses of 40 µg at 0, 
1, and 6 months) and fractional dosing (2 vaccinations at 0 and 1 month of 40 µg 
Pfs230D1M/AS01 and third vaccination at 6 months of 1/5 of the full dose) and then ~12 months 
post last vaccination all received Pfs230D1M 40 µg at full or 4 vaccinations of Engerix-B + 
Menactra® (controls). 

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Safety of Pfs230D1M-EPA/AS01 in Healthy Adults (Primary Series) 
In the pilot phase of the study, there was a staggered dose escalation of Pfs230D1M and Pfs25M, 
given individually and in combination. In the Pfs230D1M arms alone, vaccinations with the low 
dose of Pfs230D1M (13 µg) and high dose of Pfs230D1M (40 µg) were overall well-tolerated. 
Most of the related AEs reported have been mild (Grade 1) (13 µg: 23/29, 79%) and (40 µg: 
42/51, 82%), with the majority being injection site pain. One subject in the low dose arm and 
3 subjects in the high dose arm experienced a total of 4 Grade 3 events (neutropenia, gastritis, 
respiratory tract infection, and malaria), all of which were determined not related to vaccination.  
 
When Pfs25M and Pfs230D1M were combined, the vaccinations were also safe and well tolerated 
with the most commonly reported AEs being local injection site abnormalities. The frequency of 
local reactogenicity reported is increased compared to other Arms, given that in these Arms, 
2 vaccinations are given at each vaccination time point, 1 in each arm. No Grade 3 AEs have been 
reported. 
The rate of AE reporting in subjects receiving Pfs230 were similar to what was seen in the 
comparator Arm that received Engerix-B. Overall, the most common vaccine-related AE seen in 
the TBV arms have been expected mild local reactogenicity post vaccination that appeared on the 
day of vaccination or one day post vaccination and resolved within 2-3 days. As expected, solicited 
systemic reactogenicity (such as headache, arthralgia, fatigue) were reported and usually were 
Grade 1 (mild) and transient in presentation.  
Laboratory abnormalities were also observed, with the majority being transient, asymptomatic 
Grade 1 (mild) and Grade 2 (moderate) neutropenias. In the TBV Arms, a few Grade 3 AEs (5 out 
of 45 subjects, 11%) were reported overall (versus 3 out of 20 subjects in the comparator arm, 
15%), all of which were determined not related to study product/procedures.  
As the high dose (40 µg) of the Pfs230D1M/AS01 was determined safe and tolerable in the pilot 
study, it was selected to be used for the main phase of the study.  
 
In the full dose Arm (n=56), overall vaccinations were well tolerated. There was a total of 383 
AEs, 252 (66%) of which were mild (Grade 1) AEs. The most common Grade 1 AE (95/252; 
38%) was injection site pain. Other common Grade 1 AEs were headache and malaria. There 
was a total of 127 (33%) Grade 2 AEs. Of these, only 24/127 (19% of Grade 2 AEs; 6% of total 
AEs) were designated possibly, probably or definitely related to the study procedures. Of the 
related Grade 2 AEs, 16/24 (67%) were injection site pain. Other related Grade 2 AEs were 
2 episodes each of fever, headache, arthralgias and neutropenia. Of the 103/127 unrelated 
Grade 2 AEs, the most common were malaria, respiratory infections and headache. There were 4 
Grade 3 AEs of fever, hypertension, malaria and bronchitis that were not related to study 
procedures.  
 

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In the fractional dose Arm (n=61) alone, overall vaccinations were well tolerated. There was a 
total of 342 AEs, 190 (55%) of which were mild (Grade 1) AEs. Similar to the full dose Arm, the 
most common Grade 1 AE (78/190; 41%) was injection site pain. Notably, fewer episodes 
(16/78, 21%) of injection site pain were reported after the third vaccination in which the 
participant received a fractional dose of the vaccine. Other common Grade 1 AEs were headache 
and malaria. There was a total of 150 (44%) Grade 2 AEs, of these only 20/150 (13% of Grade 2 
AEs; 6% of total AEs) were designated possibly, probably or definitely related to the study 
procedures. Of the related Grade 2 AEs, 12/20 (60%) were injection site pain. Other related 
Grade 2 AEs were headache and one episode of increased total white blood cell count. Of the 
130/150 (87%) unrelated Grade 2 AEs, the most common were malaria, headache, respiratory 
infections and dental caries. There were 2 Grade 3 AEs of malaria that were not related to study 
procedures.  
 
In the comparison, the control Arm, which received Engerix-B (n=119), overall vaccinations 
were well tolerated. There was a total of 524 AEs, 268 (51%) of which were mild (Grade 1) AEs. 
In comparison to the full and fractional dose Arms, Grade 1 injection site pain occurred at a 
much lower rate of 39/268; 15%. Other most common Grade 1 AEs in an almost equal 
distribution were headache, malaria and respiratory infections. There was a total of 247 (47%) 
Grade 2 AEs, of these only 15/247 (6% of Grade 2 AEs; 3% of total AEs) were designated 
possibly, probably or definitely related to the study procedures. Of the related Grade 2 AEs, 3/15 
(20%) were injection site pain. Other related Grade 2 AEs were headache (6/15; 40%), 
abdominal pain, fatigue and neutropenia. Of the 232/247 (94%) unrelated Grade 2 AEs, similar 
to full and fractional arms as expected, the most common were malaria, headache, respiratory 
infections and dental caries. There were 9 Grade 3 AEs, 7/9 were of malaria, and 1 episode each 
of systolic hypertension and snake bite that were not related to study procedures. These AEs are 
summarized in Table 3 below. A few laboratory abnormalities were also observed during the 
follow up period, with the majority being transient, asymptomatic Grade 1 (mild) and Grade 2 
(moderate) neutropenias and leukopenias. There were no Grade 3 laboratory abnormalities noted 
(Table 4).  
 

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Table 3. Summary of Total AEs and Comparing Most Common AEs Among the Study 
Arms 
  
Arm 2c 
Pfs230/AS01 full 
dose 
Arm 2d 
Pfs230/AS01 
fractional dose 
Arm 4c  
Engerix-B 
Total 
  
N=56 
N=60 
N=120 
N= 236 
  
  
Total # AEs 
383 (55) 98% 
342 (59) 98% 
524 (113) 94% 
1249 (227) 96% 
Classification 
  
  
  
  
Local Reactogenicity 
111 (51) 91%* 
90 (48) 80%* 
42 (31) 26% 
243 (130) 55% 
Systemic Reactogenicity 
10 (9) 16%* 
4 (3) 5% 
2 (2) 2% 
16 (14) 6% 
Laboratory Abnormalities 
14 (13) 25%* 
3 (3) 5% 
16 (13) 11% 
33 (29) 12% 
Unsolicited AEs 
188 (51) 91% 
200 (54) 90% 
408 (110) 92% 
796 (215) 91% 
Severity and Relationship 
  
  
  
  
Grade 1 
  
Injection Site Pain 
95 (47) 84%* 
78 (43) 72%* 
39 (29) 24% 
212 (119) 50% 
Headache 
20 (14) 25%* 
13 (13) 22%* 
6 (5) 4% 
39 (32) 14% 
Pyrexia 
8 (7) 13%* 
4 (3) 5% 
2 (2) 2% 
14 (12) 5% 
Grade 2 
  
Injection Site Pain 
16 (14) 25%* 
12 (11) 18%* 
3 (2) 2% 
31 (27) 11% 
Headache 
2 (2) 4% 
7 (7) 12% 
6 (5) 4% 
15 (14) 6% 
Pyrexia 
2 (2) 4% 
0 
0 
2 (2) 1% 
Grade 3 
  
Injection Site Pain 
0 (0) 
0 (0) 
0 (0) 
0 (0) 
Headache 
0 (0) 
0 (0) 
0 (0) 
0 (0) 
Pyrexia 
0 (0) 
0 (0) 
0 (0) 
0 (0) 
Grade 4 
  
Injection Site Pain 
0 (0) 
0 (0) 
0 (0) 
0 (0) 
Headache 
0 (0) 
0 (0) 
0 (0) 
0 (0) 
Pyrexia 
0 (0) 
0 (0) 
0 (0) 
0 (0) 
1)  X(X)X% = absolute number of AE (number of subjects experiencing AEs) Percentage of subjects with AEs 
2) * indicates a significant difference between the vaccine arm and the control by Fisher's Exact test, p<0.05 
 
In closer analysis of solicited local and systemic reactogenicity (occurring within 7 days of 
vaccination), there were some significant differences between the vaccine and control arms. 
Participants in the full dose arm experienced significantly more Grade 1 injection site pain (p= 
0.02), than the fractional dose arm. Grade 1 injection site pain was reported in the full dose arm in 
95/168 (57%) doses, fractional dose arm in 78/180 (43%) doses and control arm in 39/360 (11%) 
doses. Reports of Grade 1 headaches were significantly greater in the full dose arm than the control 
(p< 0.0001), and similarly were greater in the fractional arm versus the control arm (p= 0.0019), 
there was no significant difference between full and fractional arms (p= 0.15). Pyrexia was 

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reported more frequently in the full dose arm than the control arm (p=0.01), but the fractional dose 
was not significantly different from the control (p=0.1), and there were no significant differences 
in pyrexia between the full and fractional dose arms. 
The most common Grade 2 AEs were headache and injection site pain. There were no significant 
differences between arms for Grade 2 reports of headache. Grade 2 injection site pain was 
significantly greater in the full and fractional dose arms compared to the control, p<0.01 and p= 
0.01, respectively.  
Although minor differences can be seen between the full and fractional dose arms, the dosing for 
both arms was similar for the first and second vaccination as previously described. The dosing 
difference only occurred during the third vaccination. Thus, we can dissect the AEs further by dose 
if we look exclusively at the post-dose 3 solicited AEs. Grade 1 AEs for headache were reported 
in 5/56 (9%) full dose participants, 1/60 (2%) fractional dose participants and 2/120 (2%) control 
participants; these were not significantly different by arm. Grade 1 injection site pain was reported 
in 27/56 (48%) full dose participants, 16/60 (27%) fractional arm participants and 5/120 (4%) 
control participants. Grade 1 injection site pain was significantly more frequent in the full dose 
arm (p<0.01) and fractional dose arm (p<0.01) compared to the control. Grade 2 injection site pain 
was significantly different between the full and control arms (p=0.01), but was not significantly 
different between the fractional and control arms (p=0.26). Thus, in performing this further 
analysis, it is possible to deduce that there is a direct relationship between dosage and injection 
site pain (Table 4).  
 

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Table 4. Rates of Solicited Local and Systemic Adverse Reactions of Interest Within Seven Days of Receiving Vaccine 
  
 Pfs230/AS01 full dose (N=56) 
 Pfs230/AS01 fractional dose (N=61) 
Engerix-B (N=119) 
  
Dose 1 
Dose 2 
Dose 3 
Dose 1 
Dose 2 
Dose 3 
Dose 1 
Dose 2 
Dose 3 
Grade 1 
  
  
  
  
  
  
  
  
  
ABDOMINAL PAIN 
- 
- 
1(1) 2% 
- 
- 
- 
- 
- 
- 
ALANINE AMINOTRANSFERASE INCREASED 
- 
- 
- 
- 
- 
- 
1 (1) 1% 
- 
- 
ARTHRALGIA 
1 (1) 2% 
3 (3) 5% 
1(1) 2% 
- 
2 (2) 3% 
- 
- 
- 
- 
BLOOD CREATININE INCREASED 
- 
- 
- 
- 
- 
- 
- 
1 (1) 1% 
- 
CHILLS 
- 
- 
1(1) 2% 
- 
- 
- 
- 
- 
- 
FATIGUE 
- 
1 (1) 2% 
- 
- 
1 (1) 2% 
- 
- 
- 
- 
HEADACHE 
5 (5) 9% 
10 (10) 18% 
5 (5) 9% 
2 (2) 3% 
10 (10) 17% 
1 (1) 2% 
3 (3) 3% 
1 (1) 1% 
2 (2) 2% 
HEMOGLOBIN DECREASED 
- 
- 
- 
- 
1 (1) 2% 
- 
- 
- 
- 
INJECTION SITE MOVEMENT IMPAIRMENT 
- 
3 (3) 5% 
- 
- 
1 (1) 2% 
- 
- 
- 
- 
INJECTION SITE OEDEMA 
- 
2 (2) 4% 
- 
- 
- 
- 
- 
- 
- 
INJECTION SITE PAIN 
37 (37) 66% 
31 (31) 55% 
27 (27) 48% 
39 (39) 65% 
23 (23) 38% 
16 (16) 27% 
21 (21) 18% 
13 (13) 11% 
5 (5) 4% 
INJECTION SITE PRURITUS 
- 
- 
- 
- 
- 
- 
- 
1 (1) 1% 
- 
LEUKOPENIA 
2 (2) 4% 
- 
3 (3) 5% 
- 
- 
- 
1 (1) 1% 
3 (3) 3% 
2 (2) 2% 
MYALGIA 
- 
4 (4) 7% 
- 
- 
- 
- 
- 
1 (1) 1% 
- 
NEUTROPENIA 
1 (1) 2% 
3 (3) 5% 
2 (2) 4% 
- 
1 (1) 2% 
- 
- 
3 (3) 3% 
2 (2) 2% 
PAIN 
- 
- 
1 (1) 2% 
- 
- 
- 
- 
- 
- 
PYREXIA 
1 (1) 2% 
6 (6) 11% 
1 (1) 2% 
1 (1) 2% 
3 (3) 5% 
- 
1 (1) 1% 
1 (1) 1% 
- 
THROMBOCYTOPENIA 
- 
- 
1 (1) 2% 
- 
- 
- 
- 
1 (1) 1% 
- 
URTICARIA 
- 
- 
- 
- 
1 (1) 2% 
- 
- 
- 
- 
Grade 2 
  
  
  
  
  
  
  
  
  
ABDOMINAL PAIN 
- 
- 
- 
- 
- 
- 
1 (1) 1% 
- 
- 
ARTHRALGIA 
2 (2) 4% 
- 
- 
- 
- 
- 
- 
- 
- 
FATIGUE 
- 
- 
- 
- 
- 
- 
- 
1(1) 1% 
- 
HEADACHE 
- 
2 (2) 4% 
- 
1 (1) 2% 
3 (3) 5% 
2 (2) 3% 
4 (4) 3% 
2 (2) 2% 
- 
HYPERLEUKOCYTOSIS 
- 
- 
- 
1 (1) 2% 
- 
  
- 
- 
- 
INJECTION SITE PAIN 
2 (2) 4% 
9 (9) 16% 
5 (5) 9% 
3 (3) 5% 
7 (7) 12% 
2 (2) 3% 
1 (1) 1% 
1 (1) 1% 
1 (1) 1% 
NEUTROPENIA 
- 
1 (1) 2% 
- 
- 
- 
- 
- 
- 
1 (1) 1% 
PYREXIA 
- 
2 (2) 4% 
- 
- 
- 
- 
- 
- 
- 
Grade 1 Total 
47 
63 
43 
42 
43 
17 
27 
25 
11 
Grade 2 Total 
4 
14 
5 
5 
10 
4 
6 
4 
2 
Grade 3 and 4 Total 
0 
0 
0 
0 
0 
0 
0 
0 
0 
TOTAL 
51 
77 
48 
47 
53 
21 
33 
29 
13 
1)  X(X)X% = absolute number of AE (number of subjects experiencing AEs) % of subjects with AEs; 2) “ – “ is not observed 
 

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Safety of Pfs230D1M-EPA/AS01 in Healthy Adults (Year 2, 4th Dose) 
The following year (2018) a fourth dose was provided to all eligible participants with those 
randomized to Pfs230D1M arms (full and fractional dosing) receiving a single Pfs230D1M 
40 µg full dose (n=82) and those in the control arm receiving a single vaccination with 
Menactra® (controls; n=80) ~12 months post dose 3.   
 
The fourth dose of Pfs230D1M-EPA/AS01 was well tolerated; similar to the prior Pfs230D1M 
doses with a total of 363 AEs; most of which were mild (Grade 1 AEs; 160/363, 44%).  The 
most commonly reported Grade 1 AE was injection site pain, which was observed in about half 
of Pfs230D1M vaccinees (full dosing: 22/39, 56%; fractional dosing: 20/42, 48%).  
 
A total of 197 Grade 2 AEs (54%) were reported in Pfs230D1M vaccinees.  Of these only 11/197 
(6%) were designated possibly, probably or definitely related to vaccination. Of the related 
Grade 2 AEs, as expected injection site pain (7/11, 63%) was most commonly reported, followed 
by headache (2/11, 18%), fatigue (1/11, 9%) and injection site movement impairment (1/11, 9%).  
There were six Grade 3 AEs reported (fever, malaria, sinobronchitis and systolic hypertension) 
that were not related to vaccination.  
 
In the comparator arm (Menactra®), a total of 305 AEs were reported, of which 117 (38%) were 
Grade 1. The most commonly reported Grade 1 AEs were headache and malaria both with 
(20/117, 17%), followed by injection site pain and rhinitis with (10/117, 8%).  A total of 185 
(60%) Grade 2 AEs were reported.  Of the related Grade 2 AEs (n=6) injection site pain (3/6, 
50%) was most commonly reported, followed by injection site movement impairment (2/6, 
33%), and headache (1/6, 17%).  There were 3 grade 3 AEs (dysentery and malaria) that were 
not related to vaccination. 
 
 

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Table 5. Rates of Solicited Local and Systemic Adverse Reactions of Interest Within Seven 
Days of Receiving Vaccine. 
 
X(X)X% = absolute number of AE (number of subjects experiencing AEs) Percentage of subjects with AEs; 2) * 
indicates a significant difference between the vaccine arm and the control by Fisher's Exact test, p<0.05 
 
Analysis of the solicited local and systemic reactogenicity (occurring within 7 days of 4th 
vaccination) showed there were some significant differences between the Pfs230D1M vaccine 
and control arms (Table 5).  Participants in the Pfs230D1M booster arm experienced 
significantly more Grade 1 injection site pain (p <0.0001) and headache (p <0.0001) than the 
comparator arm.  Reports of Grade 2 injection site pain was also reported more frequently in the 
Pfs230D1M arm but was not significantly different from the comparator (p=0.3285).  Similarly 
Grade 2 headaches were not reported significantly differently from the control arm (p=1). There 
was no statistical difference in occurrence of fevers seen between the two arms.  
 
Immunogenicity and Functional Activity (SMFA) of Pfs230D1M-EPA/AS01 in 
Healthy Adults (Primary Series + 4th Dose; Year 1 + 2) 
Vaccination induced detectable antibody titers 2 weeks after the first dose, which increased 
further after dose 2, but the peak after a third dose was not significantly higher (Figure 14). 
There were no differences in titers between the low and high vaccine doses. Antibody function 
was assessed 2 weeks and 12 weeks after dose 2 (Figure 15). Anti-Pfs230 was sufficient to 
induce 100% TRA 2 weeks post-dose 2, which was still >90% 12 weeks post-dose 2. In contrast, 

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anti-Pfs25 did not exceed 80% TRA weeks post-dose 2. The combination of Pfs25 and Pfs230 
was not superior to Pfs230 alone for inducing functional serum activity.  
 
 
Figure 14. Antibody Responses to Pfs230D1M-EPA/AS01 after Low Dose and High Dose 
Vaccinations of Adults in Sotuba, Mali during Pilot Phase trial.  
Response assessed by enzyme-linked immunosorbent assay (ELISA). (A) shows antibody responses of Malian 
adults after administration of low-dose vaccinations of Pfs230D1M-EPA/AS01 (either 13 µg Pfs230D1M alone or 
16 µg Pfs230D1M-EPA/AS01 plus Pfs25M) at various time points out to 6 months. (B) shows antibody responses 
after administration of high-dose vaccinations of Pfs230D1M-EPA/AS01 (either 40 µg Pfs230D1M alone or 47 µg 
Pfs230D1M-EPA/AS01 plus Pfs25M) at various timepoints out to 6 months. Red arrows indicate immunization 
administrations at 0, 1, and 6 months. Dotted lines represent antibody titers to Alhydrogel-adjuvanted Pfs230D1M-
EPA obtained in previous studies. 
 

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Figure 15. Antibody Function by Standard Membrane Feeding Assay to Pfs230D1M-
EPA/AS01 after Low Dose and High Dose Vaccinations of Adults in Sotuba, Mali during 
Pilot Phase trial.  
(A) shows numbers of malaria oocysts recovered per mosquito after being fed on blood collected from Malian adults 
2 weeks after the second administration of low-dose (13 µg) Pfs230D1M-EPA/AS01 vaccine with a control of 
malaria-naïve sera collected from volunteers in the U.S. and a comparison arm after immunization with ENERGIX-
B, a hepatitis B vaccine. (B) similarly shows results after high-dose (40 µg) vaccination with Pfs230D1M-
EPA/AS01. (C) and (D) show results at 12 weeks after the second administration of low-dose and high-dose 
vaccinations, respectively. 
 
As was seen with the pilot phase, higher antibody titers were seen with AS01 adjuvant compared 
with what has been observed previously with other adjuvants. A significantly higher antibody 
titer measured by ELISA against Pfs230 was observed in both the full and fractional dose arms 
compared to the control arm after only 2 doses given at 0 and 1 months (p<0.01 in both; Figure 
16) Antibody titers were also assessed after the 3rd vaccination, and as expected, the full and 
fractional dose arms had significantly higher titers compared to control arms (p<0.01 in both). 
Interestingly, although there was a trend in the full dose arm, the antibody titer in both full and 
fractional arms were not significantly higher after the 3rd vaccination compared to 2nd 
vaccination (p=0.20 for full dose group; p=>0.99 for fractional dose group; Figure 16). Notably, 

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Pfs230D1 titers were significantly higher in the full versus fractional dosing regimen at 12 weeks 
post-dose 3.  
 
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Figure 16. Antibodies against Pfs230D1M measured by ELISA 12 weeks post 2nd and 3rd 
vaccination in Bancoumana/Doneguebougou, Mali during Main Phase trial.  
Note: Error bars are median and 95% CI.  
 
Immune responses, as defined by antibody titers and functional activity in SMFA (standard 
membrane feeding assay), have been also assessed post vaccination #4.  Higher Pfs230 antibody 
responses were seen post Vaccination #4 than post Vaccination #3 in both the original full 
Pfs230 dose and fractional Pfs230 dose arms (Figure 17). 
 

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Figure 17. ELISA against Pfs230D1 antigen.  
Titer increase was greater with full versus fractional regimen after the 3rd vaccine dose. The booster was full dose 
for both vaccination arms and antibody titers increased equally for full and fractional arms. Full dose = Pfs230D1M-
EPA/AS01 at 40 µg at 0, 1, 6, 18 months. Fractional dose = Pfs230D1M-EPA/AS01 at 40 µg at 0, 1,18 months, 
receipt of fractional dose of 8 µg Pfs230D1-EPA/AS01 at dose #3 at 6 months. 
 
The functional activity was also measured by SMFA at 12 weeks post both 2nd and 3rd 
vaccination. Both fractional and full dose arms were found to have significantly higher TRA 
(Figure 18), the decrease in the number of oocyst per infected mosquito, after both 2nd and 3rd 
vaccination, compared to control arms (p<0.01). As has been seen in the antibody titers, there 
was significantly higher TRA in the full dose arm (p=0.04) after 3rd vaccination compared to 2nd 
vaccination; this difference was not observed in the fractional dose arms (Figure 18). Together 
with the ELISA data (Figure 16), these results indicate that the full dose arm regimen may be 
inducing better antibody responses than the fractional dose regimen.  
0
200
400
600
800
0
1
2
3
4
Study Day
ELISA (log 10 scale)
Full Dose
Fractional Dose
Control

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Figure 18. Transmission Reducing Activity Measured by SMFA 12 weeks post 2nd and 3rd 
Vaccinations in Bancoumana/Doneguebougou, Mali during Main Phase trial.  
Note: Error bars are median and 95% CI. 
Interestingly, when evaluating functional activity as TBA, it is not until after the 3rd vaccination 
that both arms were significantly different from control arm (p<0.01 for both; Figure 19). After 
the 2nd vaccination, both the full and fractional dose arms were not significantly different from 
controls.  
 
 
F u ll D o s e  1 2  w k s  p o s t V a x  2
F u ll 1 2  w k s  p o s t V a x  3
F ra c tio n a l D o s e  1 2  w k s  p o s t V a x  2
F ra c tio n a l D o s e  1 2  w k s  p o s t V a x  3
C o n tro l1 2  w k s  p o s t V a x  2
C o n tro l 1 2  w k s  p o s t V a x  3
-1 0 0
-5 0
0
5 0
1 0 0
T R A
A d j p =  0 .0 3 8 7

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Figure 19. Transmission blocking activity measured by SMFA 12 weeks post 2nd and 3rd 
vaccinations in Bancoumana/Doneguebougou, Mali during Main Phase trial.  
Note: Error bars are median and 95% CI 
Functional activity, measured by SMFA by transmission reducing activity, was maintained after 
fourth dose at a high level of activity. Transmission blocking activity (reduction in infected 
mosquitoes) by SMFA was significantly higher in both Pfs230D1M vaccine arms compared to 
the comparator at three months post dose 4 but did not significantly differ between full and 
fractional dosing regimens (Figure 20). 
 
 
 

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Figure 20. Serum transmission blocking activity (TBA) by SMFA 3 months after 
Pfs230/AS01 booster dose (Day 560).  
TBA (% reduction in infected mosquitoes) was significantly higher in Pfs230D1 vaccine arms versus comparator 
group. There was no significant difference between full or fractional dosing. 
 
These promising results demonstrate that Pfs230D1M-EPA/AS01 has the potential to provide 
transmission reducing activity as well as transmission blocking activity across two malaria 
seasons.  
 
Functional Activity by Direct Skin Feeds 
Direct skin feeds (DSF) were also used to assess vaccine activity. During the rainy season in 
2017 (Year 1, Main Phase) and 2018 (Year 2, fourth dose), a total of 4861 DSFs were performed 
in 2017 and another 5056 DSFs were completed in 2018. There was a total of 40 positive DSFs 
(0.82%) from 19 unique individuals in 2017; and a total of 88 positive DSFs (1.74%) from 
25 unique individuals in 2018 (Table 6). A trend of lower infections in the full dose regimen was 
observed in 2017 but not significant. Positive DSFs were significantly less frequent in the full 
dose regimen in 2018, a first and an extremely important achievement in the field of malaria 
transmission blocking vaccines – in vivo functional activity. 
 
 
 
-50
0
50
100
150
TBA
Full dose
Fractional Dose
 Control

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Table 6. Summary of Direct Skin Feed (DSF) results. 
Twice weekly feeds for 12 weeks in 2017 and for 16 weeks in 2018. *, ^ proportion of infection between 
groups were significantly different by chi-square test. 
 
 
Pfs230D1M-EPA/AS01 in Healthy Malian Children and Adults (19-I-N086) 
The Phase 2 study of the safety, immunogenicity, vaccine activity, and vaccine efficacy of 
Pfs230D1M-EPA/AS01 against Plasmodium falciparum malaria in VUs in Doneguebougou, 
Mali and surrounding villages started the age de-escalation Pilot Phase in April 2019 (9-18 yo) 
and May 2019 (5-8 yo) followed by Main Phase enrollment and vaccinations in June 2019.  
 
For the initial Pilot Phase safety arms, 30 subjects in each age group received Vaccinations #1, 
#2 on a 0, 28 day schedule and then were scheduled to receive Vaccination #3 along with the 
majority of their vaccine unit (VU) members, resulting in a final Vaccination schedule of 0, 28, 
126 days. Main Phase subjects (vaccinees; Arms 3a-3f) started enrollment in June 2019 and 
completed enrollment in September 2019. Subjects in the Main Phase are scheduled to receive 
vaccinations on a 0, 28, 56 day schedule. 
 
All study vaccinations for the primary series have been completed as of 30 November 2020. 
Vaccination #3 was completed in 97.9% of vaccinees (1078/1101) who received Vaccination #1 
and 1066/1101 (97%) of participants had all three doses of the vaccine.  Children aged 1-4 year 
old (Arm 4a/4b; n=192) enrolled with their VU prior to dose #3. Direct skin feeds started in 9-18 
year old subjects (Arms 1a/1b/3c/3d) in Sep 2019 and ended in March 2020. Summary of study 
enrollment, vaccination, DSF progress is summarized below in Table 7.  
 
 
 

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Table 7. Summary of Study Progress for 19-I-N086 
Pilot Phase 
Main Phase 
Arm 2a/2b  
5-8 yo 
Arm 1a/1b  
9-18 yo 
Arm 3a/3b  
5-8 yo 
Arm 3c/3d  
9-18 yo 
Arm 3e/3f 
≥19 yo 
Arm 4a/4b 
1-4 yo 
Number subjects 
enrolled 
30 
30 
185 
369 
495 
192 
Timing of enrollment May 2019 
Apr 2019 
Jun to Sep 2019 
Aug to Nov 
2019 
Number subjects 
received ≥1 vaccine 
30 
30 
183 
368 
490 
-- 
Timing of 
vaccination 
May to Oct 
2019 
Apr to Sep 
2019 
Jun to Nov 2019 
-- 
Number subjects AL 
dosed prior to Vax #3 
29 
-- 
181 
-- 
-- 
-- 
Timing of AL dosing 
prior to Vax #3 
Aug to Oct 
2019 
-- 
Aug to Nov 
2019 
-- 
-- 
-- 
Number subjects 
underwent DSF ≥1 
-- 
26 
-- 
358 
-- 
-- 
Timing of start -end 
of DSFs 
-- 
Sep to Dec 
2019 
-- 
Sep 2019 to 
Mar 2020 
-- 
-- 
 
Safety in Healthy Malian Children and Adults  
At the time of the fourth dose amendment (V5.0), safety data post dose 1, 2, and 3 as well as 
malaria episodes (patent parasitemia, symptomatic malaria) and direct skin feeding events were 
reviewed by the study team, Sponsor, NIAID DSMB, an external scientific advisory board, and 
internal scientific review committee without any reported safety concerns about proceeding with 
a fourth dose and repeat study procedures for an other year of evaluation. 
3.3.1.1 1-4 Years Old (Unvaccinated) 
At the time of the fourth dose amendment (V5.0), 192 children 1-4 years of age had enrolled in 
the study. Children in this age group did not nor will not receive vaccinations but did receive AL 
treatment at enrollment and underwent regular blood draws during the study. Most commonly 
reported AE has been malaria and rhinitis. Grade 3 AEs have been reported in 14 kids (14/192, 
7.3%) with the most commonly reported Grade 3 AE being malaria, as expected. One SAE 
(malaria) has been reported with associated anemia; unrelated to study participation. 
3.3.1.2 5-8 Years Old (Pilot + Main; Vaccinated) 
At the time of the fourth dose amendment (V5.0), 213 subjects aged 5-8 years old have received 
at least one vaccination, with 210 receiving 2 doses, and 210 receiving 3 doses. 

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Vaccinations have been well tolerated with majority of the reported AEs being mild (Grade 1) 
and most being expected local site reactogenicity. For solicited AEs, as seen with similar 
vaccines with the adjuvant AS01 in young children (Figure 7) and our previous experience in 
adults, the number of solicited AEs within 7 days post vaccination, in particular interest fevers, 
increased in frequency and severity from Vaccination #1 (5/213 subjects, 2.3%; all Grade 1) to 
Vaccination #2 (18/210 subjects, 8.6%; Grade 1 to 3) and then decreased post Vaccination #3 
(7/210 subjects, 3.3%; Grade 1 to 3). These fevers tend to occur within 24 hours post vaccination 
and resolved within 1-3 days. Majority of fevers experienced are mild/moderate, but Grade 3 
fevers can be seen post vaccination as previously reported, but overall in a small proportion of 
the population (4/213, 1.9%). Most commonly reported unsolicited AE has been malaria and 
rhinitis, unrelated to vaccination. 
 
No SAEs nor Grade 4 or higher AEs have been reported in this age group. Grade 3 AEs have 
been reported in 16 subjects (16/213, 7.5%) during the course of the study with only 4 related 
Grade 3 AEs, all of which were Grade 3 pyrexia (4/213, 1.9%) as noted above. Most commonly 
reported not related Grade 3 AE by subject (5/213, 2.3%), as expected, has been malaria. 
3.3.1.3 9-18 Years Old (Pilot + Main, Vaccinated) 
At the time of the fourth dose amendment (V5.0), 398 subjects aged 9-18 years old have received 
at least one vaccination, with 391 receiving 2 doses, and 386 receiving 3 doses.  
 
Vaccinations were also well tolerated in this age group with majority of the reported AEs being 
mild and majority of Grade 1 AEs being expected local site reactogenicity. As noted in the 5-8 
year olds, but in a smaller proportion of vaccinees and with decreased severity, pyrexia was more 
commonly reported post Vaccination #2, with 18 AEs reported (Grade 1: 10/18; Grade 2: 7/18; 
Grade 3: 1/18) than post dose 1 or subsequently for Vaccination #3.  Most commonly reported 
unsolicited AE has also been malaria and rhinitis. 
 
Grade 3 AEs have been reported in 23 subjects (23/398, 5.8%) during the course of the study 
with only 3 related Grade 3 AEs, including Grade 3 pyrexia, n=1 and Grade 3 neutropenia, n=2 
(3/398, 0.8%). Most commonly reported not related Grade 3 AE by subject (5/213, 2.3%), as 
expected, has been malaria. One Grade 4 AE has been reported as a SAE (Retained Placenta or 
Membranes; not related to vaccination). 
3.3.1.4 ≥19 Years Old (Main, Vaccinated) 
At the time of the fourth dose amendment (V5.0), 490 subjects aged ≥19 years old have received 
at least one vaccination, with 479 receiving 2 doses, and 482 receiving 3 doses.  
 

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Vaccinations were well tolerated with majority of the reported AEs being mild and majority of 
Grade 1 AEs reported being expected local site reactogenicity. As noted in the pediatric age 
groups, adults also had an increase in pyrexia reporting post dose #2, though not as frequent or 
severe. As seen with the younger kids, as expected, most commonly reported unsolicited AEs 
have been malaria and rhinitis.  
 
Grade 3 AEs have been reported in 11 subjects (11/490, 2.2%) during the course of the study 
with 1 related Grade 3 AE (neutropenia) reported. One SAE, which was also a Grade 4 AE, 
(increased creatinine, not related to vaccination) has been reported. 
3.3.1.5 Clinical Malaria and Parasitemia   
Malaria, measured by scheduled blood smears in 1-18 year olds, as well as unscheduled sick 
visits in all participants, was prospectively followed in vaccinated and unvaccinated population 
and summarized below for the first malaria transmission post dose 3 (2019-2020) (Table 8). 
 
Table 8. Malaria AE and Parasitemia Summary 
 
Arms 4a/4b 
1-4 yo 
Arm 2a/2b/13a/3b  
5-8 yo 
Arm 1a/1b/3c/3d  
9-18 yo 
Arm 3e/3f 
≥19 yo 
 
AL day -14 
AL day -7 + 
AL day 42 or 112 
AL day -7 
AL day -7 
Post Vaccination #1 
N/A 
n=213 
n=398 
n=490 
Malaria AE 
N/A 
6 (2.8%) 
13 (3.3%) 
3 (0.6%) 
Positive P. falciparum 
N/A 
17 (8.0%) 
28 (7.0%) 
19 (3.9%) 
Post Vaccination #2 
N/A 
n=210 
n=391 
n=479 
Malaria AE 
N/A 
25 (11.9%) 
45 (11.5%) 
21(4.4%) 
Positive P. falciparum 
N/A 
30 (14.3%) 
83 (21.2%) 
43 (9.0%) 
Post Vaccination #3 
 
n=210 
n=386 
n=482 
Malaria AE 
 
135 (64.3%) 
282 (73.1%) 
129 (26.8%) 
Positive P. falciparum 
 
109 (51.9%) 
275 (71.2%) 
163 (33.8%) 
1 Numbers are unique individuals unless otherwise stated. 
 
 
Immunogenicity in Healthy Malian Children  
Anti-Pfs230 antibody testing was implemented to be conducted in Mali in early 2020. At the 
time of the fourth dose amendment (V5.0) ELISA assays from the Pilot Phase (Pediatric Arms, 
n=30 9-18 years old, n=30 5-8 years old; 1:1 randomization to Pfs230D1M-EPA/AS0, n=15/age 
arm and comparator, n=15/age arm) have been completed prior to premature cessation of 
laboratory activities secondary to COVID-19 pandemic. Figure 21 presents the antibody 
responses seen in the evaluated population (both 9-18 yo Pilot Phase subjects and 5-8 yo Pilot 
Phase subjects). Children, on initial analysis, appear to have similar antibody responses to 
Pfs230D1M-EPA/AS01 vaccine as healthy adults.  

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Figure 21. Comparison of Pfs230D1M-EPA/AS01, 40 ug, in Healthy Malian Adults (17-I-
N006) to Healthy Malian Children [5-8 year olds (green), 9-18 year olds (purple)] (19-I-
N086).  
For 19-I-N086, only blinded data are presented given study status; figure represents only those values above the 
level of ELISA detection.  
 
Functional Activity by DSF in Healthy Malian Children 
Direct Skin Feeds (DSF) were completed in March 2020. The study remains blinded, but during 
the DSF follow-up there have been 70 positive DSFs (2.4%) in the 2875 DSFs completed. These 
positive DSFs occurred in 45 unique individuals and resulted in 550 infected mosquitoes. A plan 
for additional analysis of these positive DSFs, including oocyst speciation, sieving analysis of 
Pfs230, and parasite genotyping is currently pending.  
4 Malaria Transmission Dynamics in Mali 
As stated previously, in order for a TBV to have a significant impact on malaria transmission, it 
would have to be administered at a community level and include a high proportion of community 
participants that are significantly contributing to transmission events. Thus, it is important to 
understand malaria transmission dynamics in order to maximize the vaccine efficacy. 
 
Transmission of malaria is influenced by environmental, social and behavioral factors of the 
host, vector, and parasite.30-36 Mosquito factors that influence transmission include innate 
susceptibility to Plasmodium, host choice, mosquito longevity, and mosquito feeding and resting 
behavior. Once a mosquito has an established malaria infection in its salivary glands, the 
probability of transmitting malaria to a new human host is thought to be regulated by mosquito 
parasite density factors.37 Human genetic factors also play a role. For example, different 
hemoglobinopathies, such as sickle cell trait, hemoglobin C and E, and α-thalassemia, are known 
to confer protection against malaria infection.38,39 
 

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DSF have been traditionally used to study malaria transmission dynamics across a number of 
field sites worldwide.40-43 Several studies have been conducted in Mali enrolling hundreds of 
participants in or near Bancoumana with no significant adverse effects. Touré et al conducted 
DSF on 72 children as young as 2 years old in 1994-1995, with no safety risks identified.44 Diallo 
et al. conducted DSF from 1996-1998 on 372 children 4-18 years of age, also with no safety 
issues.45 DSF experiments had also been conducted in Bancoumana and a neighboring village in 
2002-2003 on a total of 44 gametocyte carriers 6-18 years of age without any safety problems.46  
 
By the end of 2017, MRTC/LMIV teams had performed nearly 10,000 DSFs on subjects 5 years 
and older under a number of different protocols completed in the past 6 years (NIAID Protocols 
11-I-N143, 13-I-N109, 14-I-N159, and 15-I-0044) as summarized in Table 9 and Figure 22 
below. All DSF participants were actively followed 24 hours post feed and were passively 
followed for 2 weeks post feed on the observational studies (11-I-N143, 14-I-N159) and seen 
frequently (at least 6 times over the course of 2 months) on the vaccine protocols (13-I-N109, 
15-I-0044). Except for 1 case of definitely related grade 2 erythema at the DSF site that was 
resolved within 48 hours, there have been no other adverse events (AEs) recorded as related 
(definitely, probably, or possibly) to the feeding procedures in either study. 
 
From 2011-2015, LMIV conducted a transmission-blocking assay study in Bancoumana 
(protocol 11-I-N143; NCT01360112) where 500 adults and children ages 3 months to 50 years 
were enrolled and underwent monthly visits to assess parasitaemia. When found to be parasite- 
and/or gametocyte-positive, individuals underwent DSF assays to explore transmission dynamics 
on an individual level as well as compare mosquito infectivity by DSF to that of mosquitoes fed 
in membrane feeding assays in Mali and the US. As part of this study, we optimized the 
parameters that contribute to successful mosquito feeding outcomes in DSF. Such parameters 
include the following: 1) mosquito age at time of feeding, 2) duration of mosquito starvation 
prior to feeding, 3) anatomical location of DSF feeding (arm, calf, and ankle), and 4) time of day 
for DSF (dawn or dusk). We found that younger mosquitoes were significantly associated with 
higher feeding, survival, and infection rates. Longer starvation times were positively, but not 
significantly, associated with higher infection rates, but starvation times of 20 hours were 
negatively associated with feeding and survival. Although dusk was found to be associated with 
higher infection rates, this may be confounded by the time from positive BS. Based on these 
findings, we make specific recommendations for optimal feeding parameters to maximize the 
chance of detecting parasite transmission in a standardized manner. 
 
In 2013, LMIV employed DSFs in a vaccine study whereby gametocyte-positive vaccine 
participants were invited to undergo DSFs post third vaccination of a Pfs25 vaccine product (13-
I-N109). In 2014, DSFs were expanded to cover all vaccine participants enrolled in the Pfs25 
trial for a 6 week period post vaccination 4 to explore whether DSF assays could be used to 
measure functional activity endpoints.47,48  

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Also in 2014, LMIV conducted a PfSPZ vaccine trial in Doneguebougou, Mali, and conducted 
DSF assays on all vaccine participants for 6 weeks post vaccination 4 to determine if the vaccine 
had an effect on interrupting malaria transmission (14-I-N159).  
 
In 2015, LMIV initiated a second TBV trial using the combination of Pfs25 and Pfs230 antigens 
(15-I-0044). Vaccine enrollees were again invited to undergo DSFs twice weekly post 
vaccinations 3 and 4 for a 6-week period. This represented the largest number of DSFs 
conducted by the Mali-LMIV team to date with 2,005 conducted in 2015 and 1,844 conducted in 
2016. 
 
A Phase 1 trial of Pfs25M-EPA/AS01 combined with Pfs230D1M-EPA/AS01 in adults in 
Bancoumana and Doneguebougou enrolled in April 2017, and an important secondary objective 
of the trial will be to compare mosquito infectivity in vaccinated subjects versus the comparator 
group. To date 4,865 DSFs have been performed in this study. 
 
Research at LMIV examining what factors lead to a positive DSF are ongoing, but the strongest 
correlate to a positive DSF is host gametocyte density: Individuals with a higher gametocyte 
prevalence in their peripheral BS are more likely to give a positive DSF. This, however, is not 
always the case and some individuals with high gametocyte burdens do fail to infect mosquitoes. 
A possible explanation may be recent ingestion of antimalarial drugs which are known to ablate 
transmission.49 Likewise, positive DSFs do occur from individuals who are BS–negative for 
gametocytes, indicating that sub-patent gametocyte levels are still able to infect mosquitoes in 
some cases.  
 
Gametocyte carriage varies with age, with younger age groups (<10 years old) having higher 
gametocyte carriage rates and densities.50 We have also been able to better understand the 
epidemiology of malaria transmission in this area. Based on data collected in the first 2 years 
(2011-2012) of our transmission-blocking assay study (11-I-N143), gametocyte carriage rates are 
higher in children 5-17 years old and in adults below the age of 35 years, compared to rates in 
children under 5 years. Mapping data has also identified housing clusters with higher carriage 
rates. 

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Table 9: Summary of LMIV Direct Skin Feed (DSF) Studies in Mali 
 
 
 
 
Year 
Site 
Protocol 
No. 
Total 
No. 
DSF 
No. 
Positive 
No. Unique 
Infected 
Individuals 
Participant 
Age Range 
Feeding 
Criteria 
Time 
of 
Year 
2011 
Bancoumana 
11-I-N143 
73 
13 
12 
5-50 years 
Gametocyte-
positive only 
Jun - 
Dec 
2012 
Bancoumana 
11-I-N143 
41 
17 
17 
5-50 years 
Gametocyte-
positive only 
Jan - 
Dec 
2013 
Bancoumana 
11-I-N143 
16 
1 
1 
5-50 years 
Gametocyte-
positive only 
Jan - 
Jun 
2014 
Bancoumana 
11-I-N143 
17 
7 
6 
5-50 years 
Gametocyte-
positive only 
Jan - 
Oct 
2015 
Bancoumana 
11-I-N143 
2 
0 
0 
5-50 years 
Gametocyte-
positive only 
Jan - 
Feb 
2013 
Bancoumana 
13-I-N109 
109 
10 
8 
18-45 years 
Parasite- or 
gametocyte-
positive 
Jul - 
Dec 
2014 
Bancoumana 
13-I-N109 
516 
16 
12 
18-45 years 
All individuals 
Jan - 
Dec 
2015 
Bancoumana 
13-I-N109 
9 
0 
0 
18-45 years 
All individuals 
Feb - 
Mar 
2014 
Doneguebougou 
14-I-N159 
458 
19 
16 
18-35 years 
All individuals 
Aug - 
Dec 
2014 
Doneguebougou 
14-I-N159 
42 
1 
1 
18-35 years 
All individuals 
Jan 
2015 
Bancoumana 
15-I-0044 
2005 
58 
18 
18-50 years 
All individuals 
Sept - 
Dec 
2016 
Bancoumana 
15-I-0044 
1844 
30 
18 
18-50 years 
All individuals 
Sept – 
Nov 
2017 
Bancoumana/Doneguebougou 
17-I-N006 
4865 
40 
19 
18-50 years 
All individuals 
Sept - 
Dec 

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Figure 22: Proportion of positive feeds (blue) with P. falciparum parasite (orange) and gametocyte density (yellow) over six 
years of direct skin feed (DSF) assays in Mali.  
Note: Feeding criteria changed over time with initial studies requiring individuals to be gametocyte-positive to participate in feeds; later studies including vaccine 
trials fed on all individuals irrespective of blood smear status.  
 

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We have initiated a study that surveys measures of malaria infection and transmission across the 
community in order to prepare for community trials of transmission-blocking vaccines. In this 
survey of unprecedented scope, all residents within selected compounds of Bancoumana village 
were invited to participate in the study, and a similar survey has been initiated in 
Doneguebougou village. Both Bancoumana and Doneguebougou villages have been the sites for 
initial evaluations of our transmission-blocking vaccines in adults. All study participants provide 
a monthly blood sample for microscopic evaluation of malaria infection gametocytemia by 
Giemsa-stained BS. All individuals 5 years and older are invited to undergo DSFs with 
laboratory-raised mosquitoes on a monthly basis, and all huts where study participants reside are 
visited monthly for capture of blood-fed Anopheles mosquitoes. 
 
The study results have emphasized that children between the ages of 5-18 years are the primary 
target for a TBV, as well as the primary beneficiaries of tools such as TBV that will reduce the 
incidence of malaria infection across the community. 
 
Studies commenced in Bancoumana with BS collections in February 2018, which is early in the 
dry season, and direct skin feeding assays were started in March 2018. Across the dry season and 
into the rainy season, which begins around July, children 5-18 years of age consistently have the 
highest prevalence of malaria infection (measured as the proportion of children who have 
detectable P. falciparum parasites on a BS at a scheduled visit or an unscheduled visit (due to 
illness) at least once during the month (Figure 23). Thus, these children appear to be the most 
likely of all the age groups to benefit by a reduction in the force of infection in the community. 
 

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Figure 23. Percent P. falciparum Positive by Month and Age.  
 
Children between 5-18 years of age also have higher measures of malaria transmission. Thus far 
in the study, children 5-18 years of age consistently have the highest prevalence of P. falciparum 
gametocytemia (Figure 24), suggesting they are a key reservoir of ongoing transmission to 
mosquitoes. Notably, children under 5 have a relatively low rate of P. falciparum gametocytemia 
throughout follow-up, and gametocytemia has been absent in this demographic from August 
onward (Figure 24). This latter effect of reduced gametocytemia may be the result of seasonal 
malaria chemoprevention (SMC), which entails monthly doses of sulfadoxine-pyrimethamine + 
amodiaquine given to all under 5 children in Mali during the malaria season; in Bancoumana, 
these treatments started at the end of July during the current study. The effect of SMC can also 
be seen in the relatively low rates of asexual P. falciparum parasitemia seen in under 5 children 
during the malaria season (Figure 23). The use of SMC implies that Malian children under 
5 years of age may not be a key target for TBV administration during trials to measure vaccine 
activity and efficacy, and this notion is supported by the low rates of gametocytemia observed in 
this population during the malaria transmission season thus far. 
 

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Figure 24. Percent P. falciparum Gametocyte Positive by Month and Age.  
 
More strikingly, the DSF studies, which have thus far been limited to participants 5 years of age 
and older, have highlighted the disproportionate role of children 5-18 years of age in parasite 
transmission. From March through August 2018, there were 28 positive DSF assays out of 3,806 
performed (Figure 2). Remarkably, 27 of these positive DSF assays were recorded in children 5-
18 years of age, while only one was recorded in an adult. Notably, the rate of parasite 
transmission by DSF in children was higher in the dry season (2% positive in March) than was 
previously seen in adults during the rainy season (0.9% positive in 2017). The rates of DSF 
positivity in children 5-18 years of age in July-August suggest that we can expect an overall rate 
throughout the transmission season of 2.5% or higher during follow-up. 
5 Experience in Children with AS01 Adjuvant 
GlaxoSmithKline (GSK) has developed several vaccine adjuvants under the auspices of its 
Adjuvant System program, with 5 products being used in clinical trials to date and 3 currently 
used in licensed vaccines: AS01, AS03, and AS04 (Table 10). Our current trial is using the 
AS01 system, which contains 2 immunostimulatory components, 3-O-desacyl-4'-
monophosphoryl lipid A (MPL) and the saponin QS-21 formulated with liposomes. AS01 was 
designed to provoke a strong humoral immune response with an acceptable safety profile. AS01 
is prepared by GSK in 2 forms: AS01B (provided in 100 µg/mL in 1 mL to provide in 0.5 mL 50 
µg MPL + 50 µg QS-21 in liposomal formulation), which is used in the licensed herpes zoster 
vaccine Shingrix®, and AS01E, which contains half the dose of MPL and QS-21 as AS01B and is 
used in the candidate malaria vaccine MosquirixTM.  
 

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Table 10. AS01 Adjuvant Systems. Adapted from Garcon et al. 2017, From discovery to licensure, 
the Adjuvant System story. 
   
 
 
 
RTS,S/AS01E Pediatric Safety Data 
RTS,S/AS01E is the most studied investigational vaccine in infants and children that utilizes the 
AS01 adjuvant system. Following initial development, safety, immunogenicity, and protective 
efficacy assessment in adults in the US, Europe, and Africa, RTS,S has undergone subsequent 
8 Phase 1/2 and 3 Phase 3 clinical studies in subjects aged at least 5 months and 4 Phase 1/2 and 
2 Phase 3 clinical studies in infants less than 5 months of age have been completed. 
 
In these studies, the AS01 adjuvant has been shown to be more immunogenic than the AS02 
adjuvant used in the initial studies and RTS,S has continued to be well tolerated and efficacious 
in younger age groups.51-54 These studies led to a large scale Phase 3 study, as previously noted, 
with the final product Mosquirix™ (RTS,S 25μg/AS01E) evaluated in over 15,000 children 
across 11 sites in 7 countries.55 Overall, the vaccine had an acceptable safety profile, even when 
given to young children and infants and in co-administration with the Expanded Programme on 
Immunization (EPI) routine vaccines. Following the large Phase 3 studies, imbalances were seen 
in the vaccinees versus controls that are now being further explored specifically in regards to 
cases of meningitis, febrile seizures, and all cause mortality in female children.55-57 
 
 

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The most common side effects seen in participants who are vaccinated with Mosquirix™ are low 
grade fever (27%), irritability (14%), injection site pain (16%) and injection site swelling (7%), 
and these occur at rates similar to when children receive standard vaccines as summarized in 
Table 11 below. In children (5- 17 months of age), there was notably higher incidence of fever 
during the primary series of vaccination and during a booster 4th dose within 7 days of receiving 
the vaccine compared to comparator groups. Some of these fever incidences were associated 
with generalized convulsive seizures (2.5 per 1000 doses in those receiving the 4th booster dose 
at 20 months versus 1.2 per 1000 doses in those who received comparator vaccine at the 20-
month timepoint vs 0.4 per 1000 doses in those who received only comparator vaccine doses at 
all 4 time points). In infants (6 to 12 weeks of age), the rate of febrile seizures was also noted to 
be 2.2 per 1000 doses following the 4th dose. All the seizure episodes resolved without long-term 
sequalae.  
 
The frequency of overall SAEs was not significantly different between vaccinated participants 
and control participants. However, there was a significant difference in the number of meningitis 
SAEs, with 21 having occurred in vaccinated children versus 1 in control children.56 No clear 
relationship between Mosquirix™ and meningitis has been identified and further analysis and 
investigation is ongoing. In question is still whether it is the antigen or the adjuvant that may be 
related to these (febrile seizures and meningitis) adverse events.  
 
Table 11. Adverse reactions reported after 3 doses of the vaccine58   Note: Very common ≥1/10; 
Common ≥1/100 to < 1/10; Uncommon ≥1/1000 to < 1/100 
System Organ Class 
Frequency 
Adverse Reactions 
Metabolism and nutrition 
disorders 
Common 
Decreased appetite 
Psychiatric disorders 
Very common 
Irritability 
Nervous system disorders 
Common 
Somnolence 
Uncommon 
Febrile convulsions (within 7 
days post vaccinations) 
Gastrointestinal disorders 
Common 
Diarrhea 
Uncommon  
Vomiting 
General disorders at 
administration site conditions 
Very common 
Fever, injection site reactions 
(including swelling, erythema 
and pain) 
Uncommon 
Injection site induration 
 
The European Medicines Agency has reviewed the safety profile of Mosquirix™ and has stated 
that, “The safety profile of this vaccine is acceptable and quite similar to others apart from a 
higher risk for febrile convulsions in the older age group within 7 days after a dose (mostly the 
third dose) of Mosquirix™”59. Childhood febrile seizures are common in children between the 

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age of 6 months and 5 years with peak episodes between 12 and 18 months. Although 
investigations are ongoing in order to determine the relationship between Mosquirix™ and 
febrile seizures, due to ongoing concerns about this relationship, the proposed study which uses 
the same adjuvant AS01, will not enroll children under the age of 5 years.  
 
A Phase I double-blind, randomised controlled, staggered, dose-escalation study using 
RTS,S/AS02A was completed in older children, 6-11 years, in Gambia. In this study, the older 
children were divided into 3 groups (n=20 in each group) to receive 10 µg RTS,S dose (10 µg 
RTS,S in 0.1 mL AS02A), 25 µg dose (25 µg RTS,S in 0.25 mL AS02A) or 50 µg dose (50 µg 
RTS,S in 0.5 mL AS02A) at 0, 1 and 3 months (Figure 25). A comparator group (n=30) received 
rabies vaccine. The vaccine was found to be safe and well tolerated, there were no reported SAEs 
related to the study. The most common AEs reported was injection site pain (>80% of the doses), 
headache (18-24% vs 12% in rabies vaccine) and fever (10% vs 6% in rabies vaccine) in all dose 
groups. There were few Grade 3 AEs that resolved within 24 hours. The rate of unsolicited AEs 
was similar between the vaccine groups and control group. There was only one SAE in the 
control group, bronchopneumonia, that resolved without sequelae (Figure 25).60    
Figure 25. Local and Systemic Symptoms Occurring 4 Days after Vaccination  

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Although there is sparse data regarding the safety of AS01 in children above the age of 5 years, 
the safety data from the study above with AS02A is encouraging. As had been seen in adults, the 
most common AEs were mild local reactogenicity and fever.  
 
Clinical Development Plan 
LMIV has a mandate to develop vaccines that will interrupt malaria transmission, and therefore 
will be useful for malaria elimination campaigns. Vaccines can target either pre-erythrocytic 
malaria parasites to prevent human infection or sexual stage malaria parasites to prevent 
transmission to mosquitoes (TBV) in order to interrupt malaria transmission. 
 
LMIV has the world’s most advanced program for clinical development of TBVs. TBVs are 
intended to interrupt malaria transmission by producing antibodies in humans that block parasite 
development in mosquitoes. The immunological data from a Phase 1 Pfs25H-EPA/Alhydrogel 
study in the US and in Mali showed a consistent increase in Pfs25 antibody response by ELISA 
following each subsequent vaccination, but these responses appear short lived. Data from these 
studies also showed that increasing antibody titers were correlated with increased functional 
transmission reducing activity by SMFA. Further studies in the US and Mali also showed that 
adding another transmission blocking target, Pfs230D1M, either alone or in combination with 
Pfs25M is safe and immunogenic. The immunological data from a Phase 1 Pfs25M-
EPA/Alhydrogel and Pfs230D1M-EPA/Alhydrogel study in the US and Mali has likewise shown 
a consistent increase in Pfs25 and Pfs230D1M antibody response by ELISA following each 
subsequent vaccination that correlate with functional activity with SMFA. Specifically, vaccine 
regimens containing Pfs230D1M can achieve high functional activity by SMFA compared to 
regimens containing Pfs25. However, the duration of the antibodies was shorter than intended. 
Although there was no evidence of interference of activity when Pfs25 and Pfs230D1M were 
combined, it did not appear that Pfs25 enhanced the activity of Pfs230D1M.  
 
A more potent adjuvant, AS01, was then evaluated in a Phase 1 clinical trial in Mali, induced 
higher titers as well as longer lasting functional antibodies for both Pfs25M and Pfs230D1M 
assessed individually or in combination. Since Pfs230D1M appeared superior for inducing 
functional antibody, it was selected to continue with the future community studies to assess the 
efficacy of this vaccine in reducing transmission of malaria at a community level. However, to 
assess vaccine efficacy that reduces malaria incidence in the community, we need to ensure that 
a large portion of the population is enrolled and vaccinated, especially individuals who transmit 
malaria at high rates. Ongoing LMIV studies have identified children as contributing 
disproportionately to transmission events, hence it will be essential to also include children in 
future community trials of a transmission-blocking vaccine.  
 
At this time, we will evaluate for the first time the vaccine antigen Pfs230D1M formulated with 
AS01 to see whether it is safe in healthy Malian children, and if so, we will proceed to enroll all 

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willing residents within selected family compounds to receive vaccine or comparator to further 
assess safety, and to assess vaccine efficacy, activity and immunogenicity. Vaccine efficacy will 
be assessed by detecting P. falciparum infections by BS, vaccine activity will be assessed by 
DSF assays and SMFA, and lastly immunogenicity will be assessed by testing sera in ELISA. 
Given the promising results, in particular seen with DSF activity after a fourth dose of 
Pfs230D1M-EPA/AS01 in healthy adults, we will assess this vaccine regimen over two malaria 
seasons (2019-2020, 2020-2021).  
6 Study Objectives 
 
Pilot Objectives 
 
Primary Objectives 
• To assess safety and reactogenicity of administration of Pfs230D1M-EPA/AS01  
• To assess vaccine activity of Pfs230D1M-EPA/AS01 against P. falciparum transmission 
by direct skin feeding assay (DSF) (Arms 1a/1b only) after receipt of 3rd and 4th 
vaccination and analyzed with the main group 
 
Secondary Objectives 
• To assess vaccine efficacy of Pfs230D1M-EPA/AS01 against P. falciparum infection 
measured by blood smear after receipt of 3rd and 4th vaccination and analyzed with the 
main group 
• To assess immunogenicity as measured by enzyme-linked immunosorbent assay (ELISA) 
titer response to Pfs230D1M  
• To assess functional antibody response by standard membrane feeding assay (SMFA) to 
Pfs230D1M  
 
Exploratory Objectives 
• To explore cellular and humoral responses to Pfs230D1M  
• To identify host and parasite factors associated with transmission  
• To track transmission of parasites from human to human using highly variant gene 
fragments 
• To explore the impact of human movement on transmission and parasitemia  
 
Main Objectives 
 
Primary Objectives 
• To assess vaccine activity of Pfs230D1M-EPA/AS01 against P. falciparum transmission 
by direct skin feeding assay (DSF) (Arms 3c/3d only) 

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Secondary Objectives 
• To assess vaccine efficacy of Pfs230D1M-EPA/AS01 against P. falciparum infection 
measured by blood smear (All Arms except Arms 3e/3f) 
• To assess safety and reactogenicity of administration of Pfs230D1M-EPA/AS01 (All 
Arms except 4a/4b) 
• To assess immunogenicity as measured by enzyme-linked immunosorbent assay (ELISA) 
titer response to Pfs230D1M (All Arms except Arms 4a/4b) 
• To assess functional antibody response by standard membrane feeding assay (SMFA) to 
Pfs230D1M (All Arms except Arms 4a/4b) 
 
Exploratory Objectives 
• To explore cellular and humoral responses to Pfs230D1M (All Arms except Arms 4a/4b) 
• To identify host and parasite factors associated with transmission  
• To track transmission of parasites from human to human using highly variant gene 
fragments  
• To explore the impact of human movement on transmission and parasitemia 
 
7 Study Design 
 
Description of the Study Design 
This is a Phase 2, double-blind, block (compound/vaccine unit)-randomized, comparator-
controlled study designed to evaluate the safety, reactogenicity, immunogenicity, transmission-
blocking activity, and subsequent vaccine efficacy of Pfs230D1M conjugate formulated on 
AS01. Subjects will be drawn from approximately 137 compounds/vaccine units (extended 
families/neighboring compounds) in Doneguebougou and surrounding villages that have agreed 
as a family/compound to participate in the study. Individuals within the compound will not be 
randomized to individual arm assignments, but rather an entire family/compound will be 
randomized as a whole to receive either Pfs230D1M-EPA/AS01 or comparator vaccine in 
eligible and willing residents in that family/compound (vaccine unit) as well as the enrolled 
young children (1-4 years of age) in the parasitemia cohort (Group 4).  
 
As previously noted, all eligible and enrolled pilot and main phase subjects ≥5 years of age or 
older will receive AL treatment prior to first vaccination as well as either Pfs230D1M-
EPA/AS01 or comparator vaccine at 0, 28, 56, and 392 days (main phase) and 0, 28, 126, and 
448 days (pilot phase). All vaccinated subjects will be followed for safety and tolerability, as 
well as immunogenicity via ELISA and functional antibody responses via SMFA.  
 
Subjects 5-8 years of age will also receive AL treatment ~14 days prior to vaccination #3 and 
~7 days prior to vaccination #4 in their compound, regardless of other antimalarial treatments 

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administered prior, subject to clinician’s discretion. Children 1-4 years of age will be enrolled 
just before the malaria transmission season and will receive AL concomitantly with children 5-8 
years of age who will be treated ~14 days before the 3rd vaccine dose and ~7 days prior to 
vaccination #4. All enrolled subjects (except adults, ≥19 years old) will be monitored for patent 
parasitemia every 2 weeks, and all enrolled subjects during unscheduled visits if clinically 
indicated, for at least 6 months up to 12 months post vaccination, with time to patent parasitemia 
being evaluated as a vaccine efficacy timepoint in children 1-8 years of age as well as 9-18 years 
of age within the compounds (separate secondary evaluation given expected nighttime mobility 
and no pre-transmission season AL treatment). Preteen and adolescent subjects will also be 
assessed by DSF for vaccine activity starting 2 weeks post receipt of vaccination #3 and post 
receipt of vaccination #4; DSFs will be completed every 2 weeks for a total of 8 DSF (post 
vaccination #3) and for a total of 10 DSF (post vaccination #4). 
 
Study Groups 
 
Pilot Safety Group 
We will enroll, drug treat with AL prior to vaccination #1, and vaccinate a total of 60 children. 
Subjects will be enrolled in 2 different age groups, as follows: 
Group 1: Subjects 9 to 18 years of age (n=30) 
• Arm 1a (n=15), to receive 40 μg of Pfs230D1M-EPA/AS01 on days 0, 28, 126, 448; 
receipt of AL on day -7 
• Arm 1b (n=15), to receive HAVRIX (day 0, 448), TYPHIM Vi (day 28), Menactra (day 
126); receipt of AL on day -7 
Group 2: Subjects 5 to 8 years of age (n=30) 
• Arm 2a (n=15), to receive 40 μg of Pfs230D1M-EPA/AS01 on days 0, 28, 126, 448; 
receipt of AL on day -7, 112, and 441 
• Arm 2b (n=15), to receive HAVRIX (day 0, 448), TYPHIM Vi (day 28), Menactra (day 
126); receipt of AL on day -7, 112, and 441 
 
The age groups will be enrolled in descending order, oldest to youngest, with at least 2 weeks 
between the previous vaccination of one group (Group 1) and the first enrollment of the next 
group (Group 2). The age de-escalation design allows assessment of the risk level in pre-teens 
and teenagers based on data from adults and, subsequently, the risk level in younger children 
based on data from teenagers. Teenagers and older children who are able to provide assent will 
participate before the study opens to younger children who cannot assent.  
 
After receipt of vaccination #1 in the older age group (Group 1: Arms 1a/1b), the DSMB will 
conduct an interim safety review of all safety data collected to date before the vaccination #1 is 

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administered in younger children (Group 2: Arms 2a/2b) and second vaccination in the older 
group (Group 1: Arms 1a/1b) age groups respectively. The DSMB will also review the safety 
data collected to date prior to administration of vaccination #2 in the younger age group (Group 
2: Arms 2a/2b; Figure 26). 
 
 
Figure 26. Pilot Safety Group Study Schema 
Once the main phase of the study starts, children enrolled in the pilot study will join the main 
phase and complete their third vaccination with their respective family compounds. Those 
children enrolled in Group 2 (Arms 2a/2b), in alignment with all others 5-8 years of age, will 
also receive a treatment course of AL prior to vaccination #3 and those in Group 1 (Arms 1a/1b) 
will join the main phase Arms 3c/3d (9-18 years of age) for vaccination #3. 
 

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Main Study Group 
We will enroll, drug treat with AL prior to vaccination #1, and vaccinate a total of approximately 
137 compounds (~1500 subjects) that have approximately 80% consented, eligible subjects. 
Subjects will be enrolled and vaccinated by family compound and specified to Arms as noted 
below by age at time of enrollment (Figure 27). Younger children (Group 4; n~400) will also be 
enrolled at the same time as their corresponding family compound. While our goal is to enroll 
the maximum number of children in this age bracket, there are currently children 1-4 years of 
age in Doneguebougou per the most recent census (Table 12); enrollment may occur in adjacent 
villages if required to meet minimum sample sizes. If there are younger children in the 
compound that will reach the age of study participation prior to the family compounds’ 
scheduled 3rd vaccination, they can be enrolled on a continuous basis until then once they 
become of age. 
 
 
Figure 27. Main Phase Study Schema. 

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Table 12. Approximate Census Counts in Doneguebougou in June 2018. 
Age (years) 
<1 
1-4 
5-8 
9-18 
19+ 
Population 
29 
295 
229 
584 
867 
Actual Enrollment 
0 
192 
215 
399 
495 
 
Group 3 (n~1500): Pfs230D1M-EPA/AS01 or comparator vaccine 
• Arm 3a: 5-8 years of age to receive 40 μg of Pfs230D1M-EPA/AS01 on days 0, 28, 
56, 392; receipt of AL on day -7, 42, and 385 
• Arm 3b: 5-8 years of age to receive HAVRIX (day 0, 392), TYPHIM Vi (day 28), 
Menactra (day 56); receipt of AL on day -7, 42, and 385 
• Arm 3c: 9-18 years of age to receive 40 μg of Pfs230D1M-EPA/AS01 on days 0, 28, 
56, 392; receipt of AL on day -7  
• Arm 3d: 9-18 years of age to receive HAVRIX (day 0, 392), TYPHIM Vi (day 28), 
Menactra (day 56); receipt of AL on day -7 
• Arm 3e:  19 years of age or older to receive 40 μg of Pfs230D1M-EPA/AS01 on days 
0, 28, 56, 392; receipt of AL on day -7  
• Arm 3f: 19 years of age or older to receive HAVRIX (day 0, 392), TYPHIM Vi (day 
28), Menactra (day 56); receipt of AL on day -7 
 
Group 4 (n=400): blood smear + AL treatment (parasitemia) 
• Arm 4a: 1-4 years of age residing with/in same families/compounds as Arms 1a, 2a, 
3a, 3c, 3e (Pfs230D1M-EPA/AS01 compounds) receipt of AL on day -14, 385 
• Arm 4b: 1-4 years of age residing with/in same families/compounds as Arms 1b, 2b, 
3b, 3d, 3f (comparator compounds) receipt of AL on day -14, 385 
 
To ensure further safety, family compounds will be enrolled in a staggered manner with 
acceleration over time, with a planned DSMB review after approximately 1/3 enrollment (~30 
family compounds/vaccine units). Five compounds will be enrolled within the first 1-2 days of 
the main phase enrollment, then will await 2 days from first vaccination in that wave of 
enrollment until enrolling an additional 10 compounds over 1-2 days. Following waiting at least 
2 days from the first vaccination during that second wave of enrollment, a final additional 15 
compounds will be enrolled. Given our current estimates of family compound make up, this 
initial study enrollment (Pilot Phase) will result in an additional vaccination of a smaller subset 
of 5-8 years of age and 9-18 years of age for extra safety data prior to accelerated enrollment of 
the remaining family compounds ~1 week later. 
 
Children in Arms 2a/2b, 3a/3b, and 4a/4b (1-8 years of age) will also receive a treatment course 
of AL ~14 days prior to the time of vaccination #3 in the relevant Arms (AL dosing prior to 

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Vaccination #3 = study day 112/42 for Arms 2a/2b, 3a/3b; for Arms 4a/4b this is study day -14). 
Preteen and adolescent subjects in Arms 1a/1b, 3c/3d will also be assessed by DSF for vaccine 
activity starting 2 weeks post receipt of vaccination #3; DSFs will be completed every 2 weeks 
for a total of 8 DSFs. 
 
All enrolled subjects (Groups 1-4, except adults on a less regular active follow-up) from the 
family compound will be monitored for patent parasitemia every 2 weeks, and during 
unscheduled visits if clinically indicated, for at least 6 months up to 12 months post vaccination, 
with time to patent parasitemia being evaluated as a vaccine efficacy timepoint in children 1-8 
years of age as well as 9-18 years of age within the compounds (separate secondary evaluation 
given expected nighttime mobility and no pre-transmission season AL treatment).  
 
Year 2: Pilot and Main Study Groups 
In Year 2, during the last scheduled follow-up visit from Year 1, each VU will be approached for 
reconsenting and focus rescreening to continue on for a 4th vaccination (Arms 1a/1b, 2a/2b, 
3a/3b/3c/3d/3e/3f) and followed for the malaria season (2020) and for a year post last 
vaccination (Figure 28). 
 
 

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Figure 28. Fourth Dose (Year 2) Study Schema    
Updated study schedule for fourth dose. Pilot and Main Phase will align with their VUs for timing of AL dosing and 
vaccination. 
8 Study Endpoints 
 
Pilot Endpoints 
 
Primary Endpoints 
• Incidence of local and systemic adverse events (AEs) and serious adverse events (SAEs)  
• P. falciparum functional activity as measured by DSF (Arms 1a/1b only) after receipt of 
3rd and 4th vaccination and analyzed with the main group 
 
Secondary Endpoints 
• P. falciparum asexual parasitemia as measured by blood smears after receipt of 3rd and 4th 
vaccination and analyzed with the main group 
• Anti-Pfs230D1M immunoglobulin (Ig) G levels as measured by ELISA  
• Transmission-reducing activity (TRA)/Transmission-blocking activity (TBA) of induced 
antibody in SMFA  
Jun 2020
Aug 2020
Jul 2020
Sep 2020
Oct 2020
Nov 2020
Dec 2020
Feb 2021
Jan 2021
Pfs230D1M-EPA/AS01
Comparator Vaccine
Artemether/Lumefantrine
Mar 2021
Arm 2a/3a: 40µg Pfs230D1M-EPA/AS01 
Arm 2b/3b: Comparator (HAVRIX)
Arm 1a/3c: 40µg Pfs230D1M-EPA/AS01 
Arm 1b/3d: Comparator (HAVRIX)
DSMB
9-18 years of age
5-8 years of age
DSF
DSF
Arm 3e: 40µg Pfs230D1M-EPA/AS01 
Arm 3f: Comparator (HAVRIX)
19 years of age or older
Arm 4a: 40µg Pfs230D1M-EPA/AS01 Vaccine Units
Arm 4b: Comparator (HAVRIX Vaccine Units)
1-4 years of age
Direct Skin Feeds every 2 
weeks
Apr 2021
Jun 2021
Aug 2021
Jul 2021
May 2021

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Exploratory Endpoints 
• Cellular immune responses and antibody repertoire of functional antibody responses to 
vaccination  
• Measuring hemoglobin, hemoglobin variants, levels of parasitemia/gametocytemia, 
OMICS analyses (proteomics, genomics), clinical malaria infection and treatment  
• Sequences of highly variant parasite gene fragments  
• Evaluation of congregate movement measurements by GPS tracking on functional 
activity as measured by DSF and P. falciparum asexual parasitemia as measured by blood 
smears (subgroup from Arms 1a/1b only) 
 
Main Endpoints 
 
Primary Endpoints 
• P. falciparum functional activity as measured by DSF (Arms 3c/3d) 
 
Secondary Endpoints 
• P. falciparum asexual parasitemia as measured by blood smears (All Arms except Arms 
3e/3f) 
• Incidence of local and systemic AEs and SAEs (All Arms except Arms 4a/4b) 
• Anti-Pfs230D1M immunoglobulin (Ig) G levels as measured by ELISA (All Arms except 
Arms 4a/4b) 
• Transmission-reducing activity (TRA)/Transmission-blocking activity (TBA) of induced 
antibody in SMFA (All Arms except Arms 4a/4b)  
 
Exploratory Endpoints 
 
• Cellular immune responses and antibody repertoire of functional antibody responses to 
vaccination (All Arms except Arms 4a/4b)  
• Measuring hemoglobin, hemoglobin variants, levels of parasitemia/gametocytemia, 
OMICS analyses (proteomics, genomics), clinical malaria infection and treatment  
• Sequences of highly variant parasite gene fragments  
• Evaluation of congregate movement measurements by GPS tracking on functional 
activity as measured by DSF and P. falciparum asexual parasitemia as measured by blood 
smears (subgroup from Arms 3c/3d only) 
 
 
Sample Size and Estimated Duration of Study 
A total of 60 subjects will be vaccinated in the age de-escalation pilot phase of the trial. At the 
time of enrollment, all children will receive a treatment course of AL prior to first vaccination 
and will be randomized at the time of vaccination #1. Since children may be replaced if enrolled 
but not randomized, more subjects may be initially enrolled, but no more than 60 subjects will 

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receive at least 1 vaccine. Each subject will be monitored actively for at least 12 months after the 
last vaccination, for a total of 13 to 18 months of participation depending on timing of screening 
for the study. Up to 80 subjects will be screened to accommodate possible screening failures. 
 
For the main phase of the study, approximately 137 total compounds/vaccine units will be 
approached for enrollment for an estimated total of ~1500 vaccinated subjects 5 years of age and 
older and ~400 subjects 1-4 years of age. Given each compound is of a variable population 
(ranging from 5-90+ years of age), this is an estimate of enrollment with expected enrollment in 
the total main phase to be ~1500 subjects +/- 15%. Subjects will be monitored actively for at 
least 12 months after the last vaccination, for a total of 13 to 18 months of study participation 
depending on timing of screening for the study. Up to 2380 subjects will be screened to 
accommodate possible screening failures. 
 
For the fourth dose and continuation on study, the enrolled compounds/vaccine units will be 
approached for reconsenting and re-screening. No new enrollments will be permitted. Despite 
adjusted ages over the last year, subjects will remain in their assigned arms from enrollment for 
evaluation of study endpoints (e.g. DSFs, blood smears), receipt of AL, and receipt of 
vaccinations. All subjects who received at least 1 vaccination during 2019-2020 and are on study 
may be eligible for continuation. All enrolled children (1-4 year olds) who received antimalarial 
treatment with their VU will be eligible for continuation. Subjects not wanting to continue on 
study for another year or are found to be ineligible will not be unblinded until the end of Year 2, 
and will be provided the opportunity to complete a final visit with their VU at that time. 
 
Study Definitions 
Screened: Subjects will receive a screening identification number (SC-XXX-XXX) when the 
informed consent is signed, and will either be determined as “enrolled” or “screen failures” as 
noted below; subjects within the same vaccine unit will be noted by the same first 3 numbers 
(SC-XXX-xxx) of their screening identification. 
• 
Screening may be completed over the course of multiple visits. 
• 
Screening will occur within 56 days prior to enrollment into the study. 
• 
If the screening visit is >56 days prior to enrollment (determined by the 
date of each component), then an updated medical review, physical 
exam/vital signs, repeat Malaria Comprehension Exam, and repeat 
laboratory evaluation (inclusive of safety labs, human 
immunodeficiency virus [HIV], EKG) will be completed to determine 
eligibility for enrollment. 
 
Enrolled: Subjects will be considered enrolled beginning with the receipt of the first blood draw 
and first AL dose.  

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• 
Final study number assigned – MV-19-XXX-XXX-X [MV-19-(vaccine 
unit #)-(study # per age)-(letter for QC)] 
o Given the last three numbers of the study number are dependent on age 
and requirement by the electronic database to have a study number during 
screening, if a child/young adult changes age categorization (45-year-
old, 89-year-old, 1819-year old) between screening and enrollment a 
new study ID will be assigned categorizing the study subject as above. 
The screening identification number will not change.  
 
Randomized: Subjects are considered randomized when their family compound/vaccine unit 
undergoes randomization collectively at the time of first vaccination of their first family member. 
A compound/family may be randomized once the following criteria are met: 
• 
Approximately 80% of the family compound/vaccine unit preliminarily 
eligible to be vaccinated has signed consent, agreed to participate in the 
study, and has been found preliminarily eligible for enrollment 
• 
Entire family/compound will be randomized as a collective whole at the 
time of first vaccination in any family compound/vaccine unit 
participant 
• 
To note: prior to the start of the study, the study geographers, based off 
GPS coordinates and village census assigned family compounds into 
vaccine units which were randomized as a unit. 
 
Re-enrolled: Subjects are considered re-enrolled for Year 2 of follow-up when they complete 
the following: 
• 
Re-consent 
• 
Successfully rescreen and meet inclusion/exclusion criteria for Year 2 
• 
Receive either AL (1-8 years old only) and/or vaccine (scheduled dose 
#4) 
Screen Failures: Subjects are considered screen failures when they meet 1 of the following 
criteria after signing consent: 
• 
Screening results reveal that the subject is ineligible per Section 11.1 
• 
Subject withdraws consent before being randomized. 
• 
Family compound/Vaccine unit preliminary enrollment is significantly 
lower than approximately 80%, including low estimated pediatric 
participation. 
 
Discontinued: Subjects are considered discontinued when they meet 1 or more of the following 
criteria: 

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• 
Subject withdraws consent after being vaccinated and/or randomized. 
• 
Subject is withdrawn by the PI/sponsor after being vaccinated and/or 
randomized. 
 
Completed: Subjects are considered completed when they complete the final study visit for their 
arm. 
9 Study Population 
 
Study Site 
The study will be carried out in collaboration between the LMIV/National Institutes of Health 
(NIH) and the MRTC headquartered in Bamako, Mali. The study will be conducted by the 
MRTC at one location in Mali, West Africa (see Figure 29). The MRTC staffing at the Mali site 
is employed by NIAID/ University of Sciences, Techniques, & Technologies of Bamako 
(USTTB) programs and assigned to the Doneguebougou site for the duration of the study to 
provide clinical care and execute the protocol. 
 
Doneguebougou is a community located 30 km north of Bamako and has a population of about 
2,000 people, with another 2,000 inhabitants in the surrounding villages. For the purpose of 
vaccine trials and epidemiology studies, adequate facilities have been put in place at 
Doneguebougou within walking distance to the residents’ homes. At Doneguebougou, malaria 
transmission is highly seasonal, with the transmission season taking place from June until 
December. Doneguebougou is situated in a high transmission area, with entomological 
inoculation rates (determined by human landing catch) as high as 137 to 167 infectious bites per 
person over one transmission season. There is a high study participation rate per compound in 
Doneguebougou, thus this site greatly fits the goal of a community-wide vaccination strategy 
such as what is being proposed for these TBV vaccines. 
 
The additional nearby village to Doneguebougou, named Sirababougou, will also be approached 
for enrollment. Sirababougou is located at about 5km northeast of Doneguebougou village, the 
main site (see Figure 29). The population of Sirababougou is about 700 inhabitants based on the 
census performed at the beginning of this year by the study team. The population’s primary 
occupation is mainly farming and the sociocultural behavior of the Sirababougou village is 

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similar to Doneguebougou. Malaria transmission intensity is expected to be similar to 
Doneguebougou village. 
 
 
 
 
Figure 29: Maps Showing the Location of Doneguebougou, Mali.  
(A) In reference to other West African countries and within Mali; (B) Context of location of Doneguebougou and 
Sirababougou to one another.  
 
A family compound in Mali is a composite of different houses, and members are usually related 
by paternal relationship. The family compound can contain members of several related 
generations and be as large as 100+ or as small as 5-10. The family compound has a chief of 
family (usually the oldest man), while houses within the family compound are led by the chief of 
the household (usually the husband of the household). Important decisions are typically made by 
the chief of family. Spatial distribution and categorization of inhabitants by family compounds in 
Doneguebougou are provided below in Figure 30. As noted prior, a single family compound 
may compose a single vaccine unit or multiple family compounds may compose a single vaccine 
unit. 
 

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Figure 30: Aerial Map Showing Distribution of Family Compounds in Doneguebougou, 
Mali 
 
Rationale for Subject Selection  
TBVs must be given to a large proportion of any given community to be successful in preventing 
the spread of malaria. Teenagers and children, especially school-aged children, are major 
transmitters of the malaria parasite. Thus, it is important to study whether a candidate TBV is 
safe, well tolerated, and effective in children as well as adults. By enrolling young children who 
live and often stay within the confines of these compounds, the study will illustrate a measurable 
direct effect of the Pfs230D1M-EPA/AS01, as seen by a reduction in the rate of malaria 
infections.  
 
To ensure adequate cohorting, healthy children living in compounds/vaccine units in which 
approximately 80% of those preliminarily eligible for vaccination have agreed to participate in 

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the study, signed informed consent, and are preliminarily eligible for enrollment (e.g. screening 
of available vaccine unit for major medical problems, pregnancy, and time availability) will be 
recruited for this study. 
 
Recruitment Plan 
Community permission will be obtained from village elders and other community members in 
Doneguebougou after explanation and discussion of the study at a community meeting. A 
general announcement inviting household and family members to the participating clinic to learn 
about the study will be made at the time of community permission, using local radio or any 
traditional channel of communication. 
 
Inclusion Criteria for Groups 1, 2, 3 
All of the following criteria must be fulfilled for a volunteer to participate in this trial: 
1. 
Meets age requirements for Arm currently being enrolled. 
2. 
Available for the duration of the trial. 
3. 
Family compound known resident or long-term resident (more than 1 year) of 
Doneguebougou, Mali or surrounding villages. 
4. 
Able to provide proof of identity to the satisfaction of the study clinician 
completing the enrollment process. 
5. 
In good general health and without clinically significant medical history in the 
opinion of the investigator. 
6. 
Females of childbearing potential must be willing to use reliable contraception 
from 21 days prior to Study Day 0 and until 1 month after the last vaccination.  
• A reliable method of birth control includes one of the following:  
1. Confirmed pharmacologic contraceptives (parenteral) 
delivery;  
2. Intrauterine or implantable device  
EXCEPTIONS to required pregnancy prevention includes the following:  
1. Postmenopausal state: defined as no menses for 12 months 
without an alternative medical cause   
2. Surgical sterilization 
3. Unmarried AND not sexually active AND menstruating 
OR not menstruating females 12-17 years of age 
4. NOTE: if a female of childbearing potential’s status 
changes during the course of vaccination through 1 month 
post vaccination (e.g. they become ≥18 years of age, 

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married, or sexually active), the female will be required to 
start reliable contraception 
7. 
Willing to have blood samples stored for future research. 
 
Inclusion Criteria for Group 4 
All of the following criteria must be fulfilled for a volunteer to participate in this trial: 
1. Meets age requirements for Arm currently being enrolled. 
2. Available for the duration of the trial. 
3. Family compound known resident or long-term resident (more than 1 year) of 
Doneguebougou, Mali or surrounding villages. 
4. Able to provide proof of identity to the satisfaction of the study clinician 
completing the enrollment process. 
5. In good general health and without clinically significant medical history in the 
opinion of the investigator. 
6. Willing to have blood samples stored for future research. 
 
Inclusion Criteria for Groups 1, 2, 3, (Year 2) 
All of the following criteria must be fulfilled for a volunteer to continue to participate in this 
trial: 
1. Previously enrolled in the study and has received at least 1 vaccination. 
2. Available for the duration of the trial. 
3. In good general health and without clinically significant medical history in 
the opinion of the investigator. 
4. Females of childbearing potential must be willing to use reliable 
contraception from 21 days prior to scheduled vaccine dose #4 and until 
1 month after the 4th vaccination.  
• A reliable method of birth control includes one of the following:  
1. Confirmed pharmacologic contraceptives (parenteral) 
delivery;  
2. Intrauterine or implantable device  
EXCEPTIONS to required pregnancy prevention includes the following:  
1. Postmenopausal state: defined as no menses for 12 months 
without an alternative medical cause   
2. Surgical sterilization 
3. Unmarried AND not sexually active AND menstruating 
OR not menstruating females 12-17 years of age 

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4. NOTE: if a female of childbearing potential’s status 
changes during the course of vaccination through 1 month 
post vaccination (e.g. they become ≥18 years of age, 
married, or sexually active), the female will be required to 
start reliable contraception 
5. Willing to have blood samples stored for future research. 
 
Inclusion Criteria for Group 4 (Year 2) 
All of the following criteria must be fulfilled for a volunteer to continue to participate in this 
trial: 
1. Previously enrolled in the study  
2. Available for the duration of the trial. 
3. In good general health and without clinically significant medical history in the 
opinion of the investigator. 
4. Willing to have blood samples stored for future research. 
 
Exclusion Criteria for Groups 1, 2, 3 
An individual will be excluded from participating in this trial if any one of the following criteria 
is fulfilled: 
1. 
Pregnant, as determined by a positive urine or serum beta human 
choriogonadotropin (β-hCG) test (if female). 
NOTE: Pregnancy is also a criterion for discontinuation of any further vaccine 
dosing 
2. 
Menstruating females 11 years of age and younger. (In order to avoid cultural 
implications of further assessing pregnancy potential i.e. sexual activity in this 
age group.)  
3. 
Behavioral, cognitive, or psychiatric disease that in the opinion of the 
investigator affects the ability of the subject to understand and comply with 
the study protocol at a level appropriate for the subject’s age. 
4. 
Hemoglobin, white blood cell (WBC), absolute neutrophil count, or platelet 
levels outside the local laboratory–defined limits of normal. (Subjects may be 
included at the investigator’s discretion for “not clinically significant” values 
outside of normal range and ≤ Grade 2.) 
5. 
Alanine transaminase (ALT) or creatinine (Cr) level above the local 
laboratory–defined upper limit of normal. (Subjects may be included at the 
investigator’s discretion for “not clinically significant” values outside of 
normal range and ≤ Grade 2.) 
6. 
Infected with HIV 

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7. 
Evidence of clinically significant neurologic, cardiac, pulmonary, hepatic, 
endocrine, rheumatologic, autoimmune, hematological, oncologic, or renal 
disease by history, physical examination, and/or laboratory studies including 
urinalysis. 
8. 
Clinically significant prolonged QTc (>450 milliseconds) on screening EKG 
9. 
History of receiving any investigational product within the past 30 days. 
10. 
Current or planned participation in an investigational vaccine study until the 
last required protocol visit. 
11. 
Medical, occupational, or family problems as a result of alcohol or illicit drug 
use during the past 12 months. 
12. 
History of a severe allergic reaction or anaphylaxis. 
13. 
Known: 
o Severe asthma, defined as asthma that is unstable or required emergent 
care, urgent care, hospitalization, or intubation during the past 2 years, or 
that has required the use of oral or parenteral corticosteroids at any time 
during the past 2 years. 
o Autoimmune or antibody-mediated disease including but not limited to: 
systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, 
Sjögren’s syndrome, or autoimmune thrombocytopenia. 
o Immunodeficiency syndrome. 
o Seizure disorder (exception: history of simple febrile seizures) 
o Asplenia or functional asplenia. 
o Use of chronic (≥14 days) oral or intravenous (IV) corticosteroids 
(excluding topical or nasal) at immunosuppressive doses (i.e., prednisone 
>10 mg/day) or immunosuppressive drugs within 30 days of Study Day 0. 
o Allergy to latex or neomycin 
o Adverse reaction to hepatitis A and/or typhoid and/or meningococcal 
vaccine in the past 
o Adverse reaction to artemether/lumefantrine in the past 
14. 
Receipt of: 
o Live vaccine within the past 4 weeks or a killed vaccine within the past 
2 weeks to enrollment. 
o Immunoglobulins and/or blood products within the past 6 months. 
o Investigational malaria vaccine in the last 2 years. 
15. 
Any other condition that in the opinion of the investigator would jeopardize 
the safety or rights of a subject participating in the trial, interfere with the 

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evaluation of the study objectives, or would render the subject unable to 
comply with the protocol. 
 
Co-enrollment guidelines: Co-enrollment in other trials is restricted, other than enrollment on 
observational studies. Consideration for co-enrollment in trials evaluating the use of a licensed 
medication will require the approval of the PI. Study staff should be notified of co-enrollment on 
any other protocol as it may require the approval of the investigator. 
 
Exclusion Criteria for Group 4 
An individual will be excluded from participating in this trial if any one of the following criteria 
is fulfilled: 
1. Behavioral, cognitive, or psychiatric disease that in the opinion of the investigator 
affects the ability of the subject to understand and comply with the study protocol 
at a level appropriate for the subject’s age. 
2. Evidence of clinically significant neurologic, cardiac, pulmonary, hepatic, 
endocrine, rheumatologic, autoimmune, hematological, oncologic, or renal 
disease by history, physical examination, and/or laboratory studies including 
urinalysis. 
3. Clinically significant prolonged QTc (>450 milliseconds) on screening EKG 
4. History of adverse reactions to artemether/lumefantrine in the past 
5. History of receiving any investigational product within the past 30 days. 
6. Current or planned participation in an investigational vaccine study until the last 
required protocol visit. 
7. Receipt of: 
o Investigational malaria vaccine in the last 2 years. 
8. Any other condition that in the opinion of the investigator would jeopardize the 
safety or rights of a subject participating in the trial, interfere with the evaluation 
of the study objectives, or would render the subject unable to comply with the 
protocol. 
 
Exclusion Criteria for Groups 1, 2, 3 (Year 2) 
An individual will be excluded from participating in this trial if any one of the following criteria 
is fulfilled: 
1. Pregnant, as determined by a positive urine or serum beta human 
choriogonadotropin (β-hCG) test (if female). 
NOTE: Pregnancy is also a criterion for discontinuation of any further vaccine 
dosing 

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2. Menstruating females 11 years of age and younger. (In order to avoid 
cultural implications of further assessing pregnancy potential i.e. sexual 
activity in this age group.)  
3. Behavioral, cognitive, or psychiatric disease that in the opinion of the 
investigator affects the ability of the subject to understand and comply 
with the study protocol at a level appropriate for the subject’s age. 
4. Hemoglobin, white blood cell (WBC), absolute neutrophil count, or 
platelet levels outside the local laboratory–defined limits of normal. 
(Subjects may be included at the investigator’s discretion for “not 
clinically significant” values outside of normal range and ≤ Grade 2.) 
5. Alanine transaminase (ALT) or creatinine (Cr) level above the local 
laboratory–defined upper limit of normal. (Subjects may be included at the 
investigator’s discretion for “not clinically significant” values outside of 
normal range and ≤ Grade 2.) 
6. Infected with HIV 
7. Evidence of clinically significant neurologic, cardiac, pulmonary, hepatic, 
endocrine, rheumatologic, autoimmune, hematological, oncologic, or renal 
disease by history, physical examination, and/or laboratory studies 
including urinalysis. 
8. History of receiving any investigational product within the past 30 days. 
9. Medical, occupational, or family problems as a result of alcohol or illicit 
drug use during the past 12 months. 
10. History of a severe allergic reaction or anaphylaxis. 
11. Known: 
o Severe asthma, defined as asthma that is unstable or required emergent 
care, urgent care, hospitalization, or intubation during the past 2 years, or 
that has required the use of oral or parenteral corticosteroids at any time 
during the past 2 years. 
o Autoimmune or antibody-mediated disease including but not limited to: 
systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, 
Sjögren’s syndrome, or autoimmune thrombocytopenia. 
o Immunodeficiency syndrome. 
o Seizure disorder (exception: history of simple febrile seizures) 
o Asplenia or functional asplenia. 
o Use of chronic (≥14 days) oral or intravenous (IV) corticosteroids 
(excluding topical or nasal) at immunosuppressive doses (i.e., prednisone 
>10 mg/day) or immunosuppressive drugs within 30 days of Study Day 0. 

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o Allergy to latex or neomycin 
o Adverse reaction to hepatitis A and/or typhoid and/or meningococcal 
vaccine in the past 
o Adverse reaction to artemether/lumefantrine in the past 
12. Receipt of: 
o Live vaccine within the past 4 weeks or a killed vaccine within the past 
2 weeks to enrollment. 
o Immunoglobulins and/or blood products within the past 6 months. 
13. Any other condition that in the opinion of the investigator would 
jeopardize the safety or rights of a subject participating in the trial, 
interfere with the evaluation of the study objectives, or would render the 
subject unable to comply with the protocol. 
 
 
Exclusion Criteria for Group 4 (Year 2) 
An individual will be excluded from participating in this trial if any one of the following criteria 
is fulfilled: 
1. Behavioral, cognitive, or psychiatric disease that in the opinion of the investigator 
affects the ability of the subject to understand and comply with the study protocol 
at a level appropriate for the subject’s age. 
2. Evidence of clinically significant neurologic, cardiac, pulmonary, hepatic, 
endocrine, rheumatologic, autoimmune, hematological, oncologic, or renal 
disease by history, physical examination, and/or laboratory studies including 
urinalysis. 
3. History of adverse reactions to artemether/lumefantrine in the past 
4. History of receiving any investigational product within the past 30 days. 
5. Current or planned participation in an investigational vaccine study until the last 
required protocol visit. 
6. Receipt of: 
o Investigational malaria vaccine in the last 2 years. 
7. Any other condition that in the opinion of the investigator would jeopardize the 
safety or rights of a subject participating in the trial, interfere with the evaluation 
of the study objectives, or would render the subject unable to comply with the 
protocol. 
 

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Justification for Exclusion of Special Populations 
 
Justification of Exclusion of Pregnant Women 
This study will not vaccinate pregnant women during the course of the study; they will be 
excluded from this cohort. The effects of Pfs230D1M-EPA/AS01 on the developing human fetus 
are unknown with the potential for teratogenic or abortifacient effects. In a reproductive and 
developmental toxicity study in female rats who were administered SHINGRIX or the AS01B 
adjuvant alone, no adverse effects on preweaning development up to post-natal day 25 were 
observed, and there were no vaccine-related fetal malformations or variations.  
 
In addition, hepatitis A vaccine (HAVRIX) and typhoid vaccine (TYPHIM Vi) and 
meningococcal vaccine (Menactra) are classified as FDA pregnancy risk category C. Though, 
according to the Advisory Committee on Immunization Practices (ACIP), administration of 
inactivated virus vaccines to pregnant women have not resulted in adverse effects in the fetus. 
The ACIP recommends vaccination during pregnancy when the likelihood of disease exposure is 
high, potential infection would cause harm to mother or fetus, and when the vaccine is unlikely 
to cause harm. The manufacturer recommends administration of the vaccine to pregnant women 
only if clearly needed.  
 
Justification for Exclusion of Children Under the Age of Five (Vaccination)  
Children under the age of 5 years are excluded from this study because there are insufficient data 
regarding dosing or AEs available in adults to judge the potential risk in children in this age 
group. Some evidence suggests an increased risk of febrile seizures with receipt of AS01 from 
RTS,S/AS01 studies in young children; since febrile seizures in the general population are most 
likely to occur in children under the age of 5 years, children under 5 years of age are excluded 
from vaccination. In addition, Malian children under 5 years of age receive monthly antimalarial 
treatments with long acting agents (termed Seasonal Malaria Chemoprevention), which limits 
their rates of parasitemia and especially gametocytemia, and as a consequence these children are 
not a primary target for transmission blocking vaccine assessment. 
10 Study Agents 
 
Pfs230D1M-EPA/AS01 
 
Manufacturing 
PpPfs230D1M and EcEPA lots, both manufactured at the Walter Reed Bioproduction facility 
(Silver Spring, Maryland) in current Good Manufacturing Practices (cGMP) compliance, were 
used to manufacture the conjugate. PpPfs230D1M is a Pichia-expressed recombinant 
subsegment (S542-G736) of Pfs230 with a molecular mass of 21,854 Daltons. EcEPA is an 
Escherichia coli-expressed recombinant protein with a molecular mass of 66,975 Daltons. The 
Pfs230D1M-EPA conjugate was produced by reaction between thiolated PpPfs230D1M and 

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maleimide-activated EcEPA, followed by purification using size-exclusion chromatography. The 
Pfs230D1M-EPA conjugate was manufactured at Walter Reed Bioproduction facility in cGMP 
compliance in May 2015 and July 2016. 
 
The Pfs230D1M-EPA was formulated as conjugated Pfs230D1M in 4 mM phosphate-buffered 
saline (PBS) to a 2X dilution of the high dose (160 µg/mL in 0.5 mL volume) in cGMP 
compliance at Walter Reed Bioproduction facility in April 2016 or at the Biopharmaceutical 
Development Program (BDP) in December 2018 and is provided as a single use vial. AS01B 
adjuvant was manufactured for use in the SHINGRIX vaccine by GSK as 100 µg/mL MPL and 
100 µg/mL QS21 in a liposomal formulation in a volume of 0.625 mL. For each dose of vaccine, 
the two single-use vials, one of Conjugated Pfs230D1M in 4mM PBS, and one of AS01B, will be 
mixed 1:1 at bedside. The vaccine will be mixed and drawn up into the syringes directly prior to 
injection. A final dose of 0.5 mL will administer 40 µg conjugated Pfs230D1M, 31 µg or 36 µg 
conjugated EPA in AS01 (25 µg MPL+ 25 µg QS21 liposomal formulation). 
 
Disposition and Dispensation 
The Pfs230D1M-EPA vials will be supplied to the study site pharmacist by the Sponsor or 
Sponsor Representative. The Sponsor receives the product from Pilot Bioproduction Facility, 
Walter Reed Army Institute of Research, Silver Spring, Maryland, where the materials were 
formulated and packaged, or from ThermoFisher BioServices in Gaithersburg, MD where 
additional product manufactured at BDP is stored. The AS01B vials will be purchased as 
manufactured for the SHINGRIX vaccine by GSK. 
 
Formulation, Packaging, and Labeling 
Each single-use vial of Pfs230D1M-EPA contains 160 µg/mL of conjugated Pfs230D1M, 
124 µg/mL or 143 ug/mL of conjugated EPA in 4-mM PBS in a volume of 0.5 mL. The vial 
label reads: 160 µg/mL Conjugated Pfs230D1M in 4-mM PBS. Vaccines will be labeled 
“Caution: New Drug – Limited by Federal (or United States) law to investigational use.”  
 
There are 2 formulations in the AS01 family: AS01B and AS01E. While AS01B contains 50 µg of 
MPL and 50 µg of QS21 per one human dose, AS01E contains half the quantity of each immune-
enhancer (i.e., 25 µg of MPL and 25 µg of QS-21). For this study, the AS01B (50 µg of MPL and 
50 µg of QS21 per one human dose) adjuvant will be used at a diluted concentration similar to 
the concentration of AS01E (i.e., 25 µg of MPL and 25 µg of QS-21), but not the same 
formulation as AS01E.  
 
Storage, Shipping, and Stability 
Study vaccine Pfs230D1M-EPA must be stored at -65°C to -90°C except during vaccine 
transports (on days of vaccination/use), when the range must remain within -60°C to +9°C. This 
allows for the vaccine to thaw during transport to the field. The vaccine is then stored at 2-8°C 

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until use, and cannot be refrozen for later use. Once thawed, the vaccine vial must be labeled and 
quarantined as unusable. Shipping specifications in the standard operating procedures (SOPs) 
allow for a range of -90°C to -40°C for dry ice shipments. The long-term storage in a freezer 
cannot exceed -65°C, however. Vaccines are always shipped on dry ice, and generally remain 
below -70°C. Comparator vaccines HAVRIX® or TYPHIM Vi®, and adjuvant AS01B must be 
stored at temperatures of 2-8°C, except during vaccine transports, when the range must remain 
within 0.5°C to 9.0°C. There are ranges built into the individual SOPs for shipping, transport and 
direct use.  
 
Vials will be transported and stored at temperature-controlled conditions, according to standard 
operating procedures (SOPs). Temperature data loggers will accompany the vaccines at all times 
to ensure storage temperature limits have not been violated. Refrigerator and freezer 
temperatures will be continuously monitored. Access to study vaccine will be limited to 
authorized study personnel. Any temperature excursion outside the defined range must be 
reported to the Sponsor. The impacted products must not be used and must be stored in 
quarantine at label temperature conditions until usage approval has been obtained from the 
Sponsor. 
 
Preparation, Dosage, and Administration of Study Agents 
The TBV conjugates are stored at -80°C until just before transport to the field for use, then 
stored at 2 to 8°C as described above. AS01 adjuvant is stable in the refrigerator at +2°C to 
+8°C. The final vaccine for administration is obtained by admixing Pfs230D1M-EPA with AS01 
and must be given via intramuscular (IM) injection within 4 hours of reconstitution. The 
formulation of the study vaccine is presented in Table 13. 
 
Table 13. Transmission-Blocking Vaccine (TBV)  
Vaccine 
Product Name 
Formulation 
Volume to be 
administered 
Number of 
doses 
TBV/AS01 
(Arms 1a, 2a, 3a, 
3c, 3e) 
Pfs230D1M-EPA 
Pfs230D1M = 40 µg 
0.5 mL 
4 
EPA= 31 µg 
AS01B 
AS01 = 25 µg MPL + 25µg 
QS21 liposomal formulation 
 
Administration 
Study agent or comparator vaccines will be administered as IM injections into the deltoid muscle 
or anterolateral thigh. Arms may be alternated with successive vaccinations. When choosing an 
arm for the vaccine injection, clinicians should consider whether there is an arm injury, local 
skin problems such as scarring or rash, or significant tattoo that precludes administering the 
injection or will interfere with evaluating the arm after injection. In keeping with the MRTC 
practices and procedures and good medical practice, acute medical care will be provided to 

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subjects for any immediate allergic reactions or other injury resulting from participation in this 
research study.  
 
Comparator vaccine 
The comparator vaccines (HAVRIX® and TYPHIM Vi® and Menactra®) will be distributed to 
the study in single-use or multi-use vials per the manufacturer’s recommendation and will be 
administered by IM injection on the schedules outlined in the Study Design (Section 7). 
MenAfriVac comparator (Menactra® was previously used but another meningitis vaccine, the 
MenAfriVac® will be used) vaccine will also be offered to participants randomized to the Pfs230 
arms at the end of the study. 
 
HAVRIX® (Hepatitis A Vaccine) 
HAVRIX (Hepatitis A Vaccine; HAV) is produced by GSK and is a sterile suspension of 
inactivated virus for IM administration. The virus (strain HM175) is propagated in MRC-5 
human diploid cells. After removal of the cell culture medium, the cells are lysed to form a 
suspension. This suspension is purified through ultrafiltration and gel permeation 
chromatography procedures. Treatment of this lysate with formalin ensures viral inactivation. It 
is then adsorbed on aluminum hydroxide. HAVRIX is FDA approved for active immunization 
against disease caused by HAV for persons 12 months of age and older. In children and 
adolescents (1-18 years of age) a single 0.5-mL dose will be administered on day 0, day 392/448. 
In adults (≥19 years of age) a single 1-mL dose will be administered on day 0, day 392/448.   
 
Additional information is provided in the HAVRIX® package insert provided. 
 
If HAVRIX in not available for the study, another equivalent FDA approved or WHO certified 
HAV will be used and the package insert submitted to NIH IRB and FMPOS EC prior to 
administration.  
 
TYPHIM Vi (Salmonella typhi vaccine) 
TYPHIM Vi (Typhoid Vi Polysaccharide Vaccine), produced by Sanofi Pasteur SA, for IM use, 
is a sterile solution containing the cell surface Vi polysaccharide extracted from Salmonella 
enterica serovar Typhi, S typhi Ty2 strain (inactivated, subunit vaccine). TYPHIM Vi vaccine is 
indicated for active immunization for the prevention of typhoid fever caused by S typhi and is 
FDA approved for use in persons 2 years of age or older. The recommended immunizing dose 
for adults and children is a single injection of 0.5 mL, this is the dose that will be given on day 
28. The dose for adults will be given by IM route in the deltoid, and the dose for children will be 
given IM either in the deltoid or the anterolateral thigh.  
 
Additional information is provided in the TYPHIM Vi package insert provided. 
 

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If TYPHIM Vi is not available for the study, another equivalent FDA approved or WHO 
certified Salmonella typhi inactivated, subunit vaccine will be used and the package insert 
submitted to NIH IRB and FMPOS EC prior to administration.  
 
Menactra® 
Menactra® (Sanofi Pasteur) is a sterile, intramuscularly administered vaccine that contains 
Neisseria meningitidis serogroup A, C, Y, and W-135 capsular polysaccharide antigens 
individually conjugated to diphtheria toxoid protein.  No preservative or adjuvant is added during 
the manufacturing process.  Menactra® is FDA approved for active immunization to prevent 
invasive meningococcal disease caused by Neisseria meningitidis serogroups A, C, Y, and W-
135 (but does not protect against serotype B) for use in individuals 9 months through 55 years of 
age.  
 
Previously, Advisory Committee on Immunization Practices recommended only the quadrivalent 
meningococcal polysaccharide vaccine (MPSV4) for use in adults age 56 years and older. The 
most recent recommendations recommend the use of Menactra® in adults age 56 years and older 
who were vaccinated previously with conjugated vaccine (e.g. Menactra® or MenAfriVac) and 
now need revaccination or are recommended to receive multiple doses. The use of these 
conjugated vaccines in people age 56 and older is off-label but recommended by ACIP.  CDC 
expands that recommendation to any child or adult at increased risk for meningococcal disease 
(e.g. individuals in an epidemic or highly endemic country such as Mali).  A single dose 
(0.5 mL) is recommended for all individuals regardless of age. The dose for adults will be given 
by IM route in the deltoid, and the dose for children will be given IM either in the deltoid or the 
anterolateral thigh. 
 
Additional information is available in the Menactra® package insert provided. 
 
Contraindications to Vaccination 
The following criteria should be checked prior to each study vaccination and are 
contraindications to further vaccination:  
• Hypersensitivity reaction following administration of the study vaccine or comparator. 
• Positive urine or serum β-hCG test prior to vaccination. 
 
Subjects will be encouraged to remain in the study for safety evaluation of the dose(s) already 
received and complete research visits for immunogenicity and functional activity if deemed safe 
by the PI. 

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Indications for Deferral of Vaccination 
If any of the following AEs occur at the time of the scheduled study vaccination, the subject may 
be either vaccinated at a later date within the allowable visit window specified in the protocol or 
withdrawn at the discretion of the investigator: 
• Oral temperature >38.0ºC at the time of vaccination. 
• Receipt of a prohibited medication/procedure as described in Section 10.6 
• Any other condition that in the opinion of the investigator poses a threat to the individual 
if immunized or that may complicate interpretation of the safety of vaccine following 
immunization. 
 
Such individuals will be followed in the clinic until the symptoms resolve or the window for 
immunization expires. No further vaccination will be performed if the subject does not recover 
(i.e., temperature ≤38.0ºC and/or lack of symptoms) within the vaccination window. Subjects 
will be encouraged to remain in the study for safety evaluation of the dose(s) already received 
and complete research visits for immunogenicity and functional activity if deemed safe by the PI. 
 
If the subject meets any of the above criteria for deferral on the day of first immunization, the 
investigator may elect to exclude the subject from further participation in the study and that 
subject may be replaced. If the subject meets any of the above criteria for deferral on the day of 
subsequent immunizations, the investigator may elect to continue to follow them per protocol 
without additional vaccinations for the remainder of the study. Subjects who miss vaccinations 
after the first vaccination cannot be replaced.  
 
Concomitant Medications and Procedures 
All concomitant prescription and nonprescription (including over-the-counter) medications taken 
during study participation will be recorded. For this protocol, a prescription medication is 
defined as a medication that can be prescribed only by a properly authorized/licensed clinician.  
 
Prohibited Medications and Procedures 
Treatment with any of the following medications/procedures could potentially interfere with 
vaccine-induced immunity and will not be permitted. Use of any of these during the study may 
exclude a subject from receiving further doses of the study vaccine. However, the subject will be 
encouraged to remain in the study for safety evaluations. 
• Licensed killed vaccines in the 2-week period prior to and following each vaccination or 
licensed live vaccines in the 4-week period prior to and following each vaccination. 
• Receipt of immunoglobulins or any blood products up to 6 months prior to the first study 
vaccination and continuing for 30 days after administration of the last vaccination. 

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• Chronic oral or IV administration (≥30 days) of immunosuppressive doses of steroids 
(i.e., prednisone >10 mg per day), immunosuppressants, or other immune-modifying 
drugs from each day of vaccination to 2 weeks following each vaccination. 
• Any investigational drug or investigational vaccine other than the study vaccine during 
the study period. 
• Required surgical removal of the spleen or the development of a hematologic or other 
disease that would interfere with normal immunity. 
 
Over-the-counter medications such as acetaminophen or ibuprofen may be used to help relieve 
symptoms from vaccination and are not considered prohibited. 
 
Use of antimalarial medications or antibiotics that have antimalarial activity during the study 
period is not exclusionary but will be documented by clinical staff.  
 
Vaccine Accountability 
After administration of a vaccine dose, the single-dose vials of antigen and adjuvant or 
comparator vaccine will be accounted for according to the site SOPs and in agreement with the 
study IND sponsor for appropriate monitoring. 
 
Accurate inventory and accountability record of vaccine supplies for this study will be 
maintained by the study site pharmacist (or designee). Partially used vials may not be 
administered to other subjects. 
11 Study Schedule 
The study schedule and approximate amounts of blood drawn are summarized in Appendix A 
and detailed in Appendix B.  
 
Screening 
The purpose of the screening visit is to determine volunteer eligibility for study participation. 
Screening procedures include the informed consent process, Malaria Comprehension Exam, 
laboratory assessments, and clinical assessments. Screening activities can occur over multiple 
visits if necessary, including the day of enrollment.  
 
In the event that HIV infection or other chronic illnesses are discovered during the course of 
screening, long-term treatment and care will not be reimbursed by the study, but referral for 
continuing care can be provided to subjects. 
 
Per national requirements for reporting communicable diseases, confirmed positive test results 
for HIV will be reported to the local health department according to applicable laws and 
appropriate medical referrals initiated. 

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Screening for Arms 1, 2, 3 
The following screening evaluations must be completed for all subjects within the 56 days prior 
to first vaccination (Arms 1, 2, 3):  
• Explain the study and informed consent/assent documents to the subject and their 
parent/guardian. (Year 1 + 2) 
• Ensure that the subject (aged 7-17 years of age and unmarried) has acknowledged assent 
by signing or fingerprinting the assent document and that the parent/guardian 
acknowledges permission for their children or for themselves to participate by signing or 
fingerprinting the informed consent form. Ensure that the subject and parents/guardians 
receive a signed copy of the informed consent and assent (when applicable). (Year 1 + 2) 
• Ensure the subject and/or parents/guardians have correctly answered ≥80% of the 
questions on the Malaria Comprehension Exam. (Year 1 only) 
• Elicit a complete medical history, including menstrual and contraceptive history and/or 
history of surgical sterility for females, sexual activity and marital status for females 12-
17 years of age, and medication use. (Year 1 + 2) 
• Females of childbearing potential must be willing to use reliable contraception from at 
least 21 days prior to first and fourth vaccination through 1 month after the last 
vaccination. (Year 1 + 2) 
• 
EXCEPTIONS to required pregnancy prevention includes the following:  
• Postmenopausal state: defined as no menses for 12 months without 
an alternative medical cause   
• Surgical sterilization 
• Unmarried AND not sexually active AND menstruating OR not 
menstruating females 12-17 years of age 
NOTE: if a female of childbearing potential’s status changes during the course of 
vaccination through 1 month post vaccination (e.g. they become ≥18 years of age, 
married, or sexually active), the female will be required to start reliable 
contraception 
• Administer a complete physical examination, including vital signs (blood pressure, 
temperature, and heart rate), height/length, and weight. (Year 1 +2) 
• HIV pre- and post-test counseling as indicated including follow-up contact with subject 
to report the results and referral for appropriate medical care if indicated. (Year 1 + 2) 
• Obtain blood for complete blood count with differential (CBC w/diff) and platelet count, 
ALT, Cr, and HIV antibody. (Year 1 + 2) 
• Obtain urine for urinalysis/urine dipstick for protein and blood and, for females of 
childbearing potential, obtain urine (or serum) for pregnancy testing. (UA, Year 1 only; 
Pregnancy testing Year 1 + 2) 

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• 
Pregnancy testing (urine or serum) will be completed on all females 9 
years of age and older as indicated in Appendix A. 
• Obtain a 12-lead electrocardiogram (EKG). (Year 1 only) 
 
If screening laboratories are completed within ≤2 days prior to Study Day -7 prior to vaccination 
(AL dosing), these clinical laboratory values (CBC w/diff, ALT, Cr) may be used for Study Day 
-7 assessments and do not need to be repeated.  
 
If initial screening is completed >56 days prior to Study Day 0 (day of vaccination #1), the 
following screening procedures (per procedure; only those >56 days prior to Study Day 0) will 
need to be repeated: repeat a new Malaria Comprehension Exam, updated medical history, repeat 
screening labs (except HIV unless determined change in risk), repeat physical exam, and repeat 
EKG. Subjects will then be reassessed for eligibility based on the re-screening information. 
 
Screening for Arm 4 
The following screening evaluations must be completed for all subjects within the 56 days prior 
to enrollment (Arm 4):  
• Explain the study and informed consent documents to parent/guardian. (Year 1 + 2) 
• Ensure that the parent/guardian has acknowledged consent by signing or fingerprinting 
the consent document and that the parent/guardian acknowledges permission for the 
subject to participate by signing or fingerprinting the informed consent form. Ensure that 
the parent/guardian receives a signed copy of the informed consent. (Year 1 + 2) 
• Ensure the subject’s parent/guardian has correctly answered ≥80% of the questions on the 
Malaria Comprehension Exam. (Year 1 only) 
• Elicit a complete medical history and medication use. (Year 1 + 2) 
• Administer a complete physical examination, including vital signs (blood pressure, 
temperature, and heart rate), height/length, and weight. (Year 1 + 2) 
• Obtain a 12-lead electrocardiogram (EKG). (Year 1 only) 
 
If initial screening is completed >56 days prior to Study Day 0, the following screening 
procedures will need to be repeated before enrollment: repeat a new Malaria Comprehension 
Exam, updated medical history, repeat physical exam, and repeat EKG. Subjects will then be 
reassessed for eligibility based on the re-screening information. 

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Assignment to Groups 
 
Pilot Safety Study 
The subjects in Pilot Safety Study will be assigned randomization per their family compound 
randomization. In order for a compound to qualify for randomization, approximately 80% of the 
residents within a family compound/vaccine unit who are preliminarily eligible for vaccination 
must have signed consent and be preliminarily eligible for enrollment (e.g. screening available 
vaccinee unit for major medical problems, pregnancy, and time availability). When the first child 
(<19 years of age) from the family compound is scheduled for vaccination, the family compound 
as a whole will then be randomized to Pfs230D1M-EPA/AS01 or comparator.  
 
The Pilot Study cohorts will be enrolled in the following order: 
1. 
Arms 1a (n=15) and 1b (n=15); then 
2. 
Arms 2a (n=15) and 2b (n=15) 
 
Main Study 
If a family compound has already been randomized secondary to enrollment of a child in the 
Pilot Safety Study (Group 1/2), these family compounds will be prioritized for the start of the 
Main Study to enroll, undergo AL drug treatment, and receive their first vaccination as a unit. As 
new family compounds/vaccine units are considered preliminarily eligible for enrollment and 
randomization, the same procedures as above will be followed until approximately 137 
compounds/vaccine units are enrolled into the study.  
 
Enrollment occurs with receipt of AL treatment. Randomization into the study occurs with first 
vaccination scheduled in the first eligible member of a family compound. Subjects who are 
screened but not vaccinated may be replaced.  
 
Early Termination Visit 
If a subject withdraws or is withdrawn from the study after receipt of the first vaccination but 
before their final study visit, then they will be encouraged to return to the clinic for an Early 
Termination Visit, at which they will complete as many procedures of the end-of-study visit as 
possible. 
12 Study Procedures/Evaluations 
 
Photographs of Rash or Injection Site Reactions 
If a subject develops a rash or injection site reaction, photographs may be taken by the 
investigators. These photographs will not include the subject’s face or any identifying scars, 
marks, or tattoos. 

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Blood Draw 
The amount of blood drawn will be within the limits allowed for adult and pediatric research 
subjects by the NIH (i.e., for children not exceeding 5 mL/kg in a single day or 9.5 mL/kg per 8-
week period). Blood will be used for evaluations and assays described below and may be stored 
for future research. 
 
Clinical Laboratory Testing 
Using standard techniques, the clinical laboratory will perform the following tests:  
1. 
CBC with differential and platelet count 
o The following CBC parameters will be assessed for safety throughout the 
trial: WBC count, absolute neutrophil count (ANC)/absolute granulocyte 
count (AGC), hemoglobin, and platelet count.  
o Absolute lymphocyte count (ALC) is collected for research purposes 
2. 
Serum Creatinine 
3. 
ALT 
4. 
HIV antibody test (can include rapid diagnostics, ELISA, western blot if 
indicated) 
5. 
Urine dipstick/urinalysis 
6. 
Urine and/or serum pregnancy testing (β-hCG) in females of childbearing 
potential 
 
Electrocardiogram 
Electrocardiograms (12-lead ECGs) will be performed during screening (Year 1 only given 
assessing genetic predisposition for prolonged QTc; can be repeated in Year 2 continuation if 
change in health or medication warranting re-evaluation) and as needed throughout the study by 
the study site team in Mali and read for QTc intervals by the site study team. Subjects with 
abnormally prolonged QTc (>450 milliseconds) will be excluded.  
 
Malaria Diagnostics  
 
Blood Smears (BS) 
The gold standard for malaria diagnosis is the detection of malaria parasites on Giemsa-stained 
thick blood films. Giemsa stained thick and thin films will be examined for asexual and sexual 
parasites in the MRTC clinical laboratory, as outlined in Appendix A. BS are prepared in 
duplicate according to standard procedures and evaluated by trained study microscopists.  
 

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For detection of gametocytemia, counts are reported per 1,000 WBCs. A positive gametocyte 
read is defined as a single, confirmed gametocyte seen by one reader and confirmed by the other 
microscopist per 1,000 WBCs. 
 
Thick BS may be prepared from the blood remaining in the venous cannula, or (at time points 
when no venous blood collection is planned) from a finger prick or venous blood sample at the 
subject’s request.  
 
Thin/thick BS will be used for detection of P. falciparum parasitemia as a primary efficacy 
endpoint for the main phase of the trial, and for malaria diagnosis throughout the study.  
12.5.1.1 Symptomatic Malaria 
Clinical or symptomatic malaria for this study is defined as the presence of asexual P. falciparum 
parasites at any parasitemia with at least one of the following symptoms: temperature of ≥37.5°C 
and/or one or more of the following symptoms: headache, myalgia, arthralgia, malaise, nausea, 
dizziness, or abdominal pain. Clinical or symptomatic malaria will be reported as an AE. 
 
Volunteers will be treated with either AL or another approved/licensed anti-malarial medication 
per Malian Government treatment guidelines.  
 
Rapid Diagnostics 
For unscheduled visits and clinical diagnostics, rapid diagnostic tests may be utilized for 
determination of an acute malarial illness; however, they cannot be used in place of microscopy 
for determination of parasitemia status.  
 
Malaria Polymerase Chain Reaction 
While detection of parasites on thick BS remains the most common primary endpoint in human 
challenge trials, both PCR- and nucleic acid sequence–based amplification methods have been 
increasingly used to support BS data in malaria vaccine trials.61,62 These research molecular 
assays have significantly increased sensitivity for detection of P. falciparum blood-stage 
infection approaching 20 parasites/mL, often resulting in diagnosis 2-4 days earlier than by 
paired thick BS. 63-65 Quantification of parasite density by these methods allows evaluation of 
parasite growth curves for assessing the utility of partially-effective vaccine candidates. LMIV 
has also developed a research quantitative PCR (qPCR) that detects 18s of P. falciparum with a 
detection limit of at least 20 parasites/mL; this technique will be used during the study for 
comparison to traditional thick BS. 
 
 
P. falciparum qPCR may be performed from all scheduled visits with a malaria BS noted (see 
Appendix A) to capture infections that remain below the detection limit for microscopy. For 
subject convenience, a finger-prick sample can be used for both preparation of the microscopy 

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slide and for deoxyribonucleic acid (DNA) preservation, but venous blood draws can also be 
used. 
 
Immunologic Laboratory Testing 
 
ELISA 
Anti-Pfs230 ELISAs will be performed on sera obtained from immunized subjects at MRTC in 
Bamako, Mali and may also be performed at collaborating laboratories.  
 
For Pfs230D1M ELISAs, microwell plates are first coated with antigen solution. Plates are 
washed with TRIS-buffered saline (TBS) containing Tween-20 (T-TBS) and blocked with TBS 
containing skim milk powder. After washing with T-TBS, diluted serum samples are added in 
triplicate and incubated at room temperature for 2 hours. After incubation, unbound antibodies 
are removed by washing the plates with T-TBS, and alkaline phosphatase-conjugated goat 
anti-human IgG solution is added to each well and incubated for 2 hours at room temperature. 
Plates are then washed with T-TBS, followed by adding phosphatase substrate solution to each 
well; the plates are then covered and incubated for 20 minutes at room temperature for color 
development. The plates are read immediately at 405 nm with a microplate reader. The optical 
density values are used to determine antibody levels by comparing to a standard curve generated 
from a known positive-control plasma included on each ELISA plate.  
 
Additionally, the magnitude and kinetics of IgG responses may be explored over time post-
vaccination. Quality of antibody responses may be explored via antibody avidity as well as 
assessment of antibody subclasses to provide useful insight into the humoral immune response. 
 
Transmission Assays 
The transmission-blocking assay which will be conducted is summarized in Table 14. 
 
Table 14. Transmission-Blocking Assay 
Assay 
Mosquitoes 
Test Samples 
Site 
Standard Membrane-
Feeding Assay 
Lab strain  
(Anopheles stephensi) 
Membrane feeds with lab cultured 
parasites mixed with test serum/plasma  
LMIV 
LMVR  
Direct Skin Feeding 
Assay 
Colony strain 
(Anopheles coluzzi) 
Mosquitoes feed directly on skin of study 
participants 
MRTC 
 
Feeding assays demonstrate biologic activity of transmission-blocking antibodies and are critical 
to selection of TBV candidates. Subjects will be screened periodically by BS (as seen in 
Appendix A) for the presence of asexual parasites and gametocytes.  

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Standard Membrane Feeding Assays 
Membrane-feeding assays demonstrate biologic activity of transmission-blocking antibody and 
are critical to selection of vaccine candidates. SMFAs will be performed on sera obtained at 
baseline and periodically after vaccination as outlined in Appendix A. In a SMFA, test serum 
obtained from immunized subjects is mixed with parasites from a laboratory culture and the 
mixture is placed in a feeding cup covered with an artificial membrane. Pre-starved mosquitoes 
from a laboratory colony are allowed to feed through the membrane. A similar procedure is 
carried out on a malaria-naïve control serum at the same time using mosquitoes raised from the 
same laboratory colony. One week after the feed, mosquitoes are dissected, and midguts are 
stained with mercurochrome for the oocyst form of the parasite. The reduction of the proportion 
of oocyst-laden mosquitoes or the reduction of average oocyst numbers per mosquito compared 
to mosquitoes fed on the control group demonstrate biologic function of the antibody, and may 
be predictive of efficacy in the field. SMFA results have been shown to correlate with ELISA 
antibody titers against Pfs25 in several species. 66 The SMFAs will be conducted at LMIV and 
Laboratory of Malaria and Vector Research (LMVR) in Rockville, Maryland. 
 
SMFA will be conducted at LMIV using laboratory-strain mosquitoes and parasites. Assays will 
compare feedings with the following: 
• Plasma/sera. 
• IgGs purified from the selected plasma/sera, mixed with a malaria-naïve human sera pool 
(to eliminate non-specific factors which may be present in plasma).  
 
To confirm anti-Pfs230-specific TBAs, SMFAs may also be conducted by the following 
methods: 
• Using Pfs230-specific IgG purified using affinity chromatography. 
• Using test plasma/sera that has been depleted of Pfs230-specific antibodies using 
recombinant Pfs230 proteins. 
 
Direct Skin Feeds 
While the SMFA is standardized and reproducible and has been shown to correlate with ELISA 
antibody titers in several species, the parasite strain, gametocyte load in the feeder, and lab 
colony mosquitoes are all significantly different from those found under field conditions.66 We 
believe DSFs, in which insectary-raised clean mosquitoes are directly fed on infected 
individuals, may be more likely to be predictive of an intervention’s impact on transmission than 
membrane feeds. Thousands of subjects in Bancoumana or neighboring areas have previously 
been enrolled in direct-feeding tests without significant adverse effects reported. In 1994 and 
1995, Toure and colleagues conducted direct feeds on 72 children 2 years of age or more without 
any risks identified.44 From 1996 to 1998, Diallo and colleagues conducted DSFs on 
372 children 4 to 18 years of age without any safety problems.45 In 2002 and 2003, DSFs were 

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carried out in Bancoumana and in a neighboring village on a total of 44 gametocyte carriers 6 to 
18 years of age,67 also without any reported safety problems. 
 
More recently, as described in detail in Section 4, the study team in Mali has performed over 
10,000 DSFs on subjects older than 5 years since June 2011. All DSF participants were actively 
followed 24 hours post feed and for as many as 24 DSFs. Except for 1 case of definitely related 
Grade 2 erythema that resolved within 48 hours and 5 episodes of definitely related Grade 1 
pruritus, there have been no other expected or unexpected AEs recorded as definitely, probably, 
or possibly related to the feeding procedures in either study.  
Additionally, under protocol #11-I-N143, multiple DSFs during a 24-hour period have been 
completed in 18 subjects to date. In these 18 subjects, DSFs were well-tolerated, and there have 
been no reported AEs resulting from having multiple direct skin feeds completed in a short 
period of time. 
 
For DSFs, 2 feeding pints with approximately 30 pre-starved female mosquitoes in each will be 
prepared. Each subject will be exposed to the feeding pints for 15-20 minutes. All subjects will 
be offered a topical antihistamine and/or topical antipruritic to use following the feeds. Systemic 
medications may also be prescribed for treatment of local or systemic symptoms following DSFs 
if clinically indicated.  
 
The total number of mosquitoes used for each DSF will be maintained at appropriately 
60 mosquitoes, and no subject will undergo more than 8 DSFs during the study period in Year 1 
and no more than 10 DSFs during the study period in Year 2. Note, if subjects 9-18 years of age 
are not willing to undergo a feed at the time of the DSF, they will not be withdrawn, but rather 
will be followed per protocol with periodic DSF or without undergoing further DSFs.  
 
In our previous TBV protocols, DSFs started either 1 or 2 weeks post vaccination in order to 
time the DSFs with expected peak antibody responses. As antibody responses have significantly 
improved, in peak levels and more importantly durability, the question of functional activity no 
longer is focused on peak time periods, but rather duration of activity. Thus, at this time it is 
planned that post Vaccination #3, DSFs will start 14 days post Vaccination #3 and continue 
every 14 days for 8 total DSF assays.  Post Vaccination #4, DSFs will also start 14 days post 
Vaccination #4 and continue every 14 days for 10 total DSF assays. 
 
Some experiments have already been completed under protocols #13-I-N109 and #11-I-N143 to 
try to help determine which time of day and body part will maximize positive DSFs. Although 
there was not a significant difference found between location and time of feed in this small 
experiment, we did find that feeding at dawn and on the arm were estimated to have the highest 
counts.  
 

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After the feed, surviving mosquitoes will be assessed for infectiousness by microscopy or 
molecular assays. 
12.6.4.1 Withdrawal from DSF 
The following criteria will be checked prior to each DSF and are contraindications to DSFs:  
• Severe local or systemic reaction to mosquito bites following a previous DSF. 
• Positive urine or serum β-hCG test (pregnancy testing results obtained within the last 7-
10 days before the DSF are acceptable; see Appendix A). 
• Acute illness with an oral temperature >37.5˚C at the time of the DSF. 
• Any other condition that in the opinion of the Investigator poses a threat to the individual if 
he/she undergoes DSF or that may complicate interpretation of the safety of DSF. 
 
Recent use of antimalarial medications is not a contraindication to participating in the DSFs. The 
medication used and time period the medication was taken should be recorded on the subject’s 
source documents and case report forms (CRFs) accordingly. 
 
Entomological Procedures and Insectary Used in DSF 
12.6.5.1 Mosquito Rearing at the Insectary 
A laboratory colony of A. gambiae (coluzzi) established from a local catch in 2008 will be used 
for the DSFs. This colony has been used in the current assay development study and was 
demonstrated to have similar susceptibility as the F1 progeny of the wild-caught mosquitoes. The 
colony has been maintained using blood meals collected under standard procedures of the blood 
transfusion center from local healthy donors. To ensure that the donor is free of potentially 
transovarial arbovirus in incubation, the donated blood is only used 5-7 days after the collection 
and after the donor is confirmed to remain healthy during this period. Potentially transovarial 
arbovirus in mosquitoes or in blood meals may be detected by ELISA and PCR methods. 
However, it is not feasible to conduct these tests routinely on blood donor volunteers due to 
highly restricted access to positive controls required in these tests, but blood samples from 
donors and mosquitoes from the colony are tested for Rift Valley fever (RVF) virus on a regular 
basis in the US.  
 
The insectary in which mosquitoes are reared in Bamako has been in use for more than 15 years. 
Security is ensured by the use of double doors, which prevents the escape of reared mosquitoes 
as well as the entry of non-insectary mosquitoes. Mosquitoes are transported to Bancoumana or 
Doneguebougou in net-sealed feeding cups secured in wooden holders inside a cooler, with wet 
towels to maintain adequate humidity. After arrival at the assigned insectary in Bancoumana or 
Doneguebougou, mosquitoes are secured within the transporting containment. The insectary is 
adequately equipped with rooms with humidifiers which are regularly monitored according to 
standard insectary procedures, and access to the insectary is limited to study personnel only. 

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After feeds, mosquitoes, still in net-sealed cups, are transported back to the insectary in Bamako. 
All subsequent handling of mosquitoes will take place in Bamako. 
12.6.5.2 Mosquito Dissection 
Mosquitoes are knocked down and then transferred into a Petri dish containing a slightly wet 
paper towel. Under a dissecting scope and on a slide, each individual specimen is placed in a 
drop mercurochrome solution. The midgut is pulled and covered with a coverslip. The oocysts 
are detected under a light microscope. The presence of oocyst and oocyst counts for each 
specimen will be recorded. A mosquito will be considered positive if ≥1 oocyst is present. 
 
If needed, the head and thorax of the mosquitoes may be processed for ELISA to detect 
sporozoites and for PCR to identify species and the molecular form of the mosquito. 
 
Additional details on mosquito rearing, feeds, and dissection will be described in study SOPs. 
 
Schistosomiasis and Helminthes Infection Testing 
Endemic helminthes infections in sub-Saharan Africa, such as helminth and schistosomiasis 
infections, are known to elicit a wide range of immunomodulating responses and may impact the 
efficacy of vaccinations.68-70 This potential impact of co-infections on vaccine immunogenicity 
will depend on the prevalence of local helminth and schistosomiasis infections. With the 
introduction of phase 1 malaria trials into Africa, consideration and initial exploration into the 
existence of co-infections in vaccinees and the impact on vaccine efficacy and immunogenicity 
needs to be explored. 
 
To assess the impact of co-infections on immune responses, schistosomiasis testing (may include 
urine microscopic examination for S. haematobium and stool PCR for S. mansoni; urine antigen 
detection) and helminthes testing71 (stool PCR for 8 common gastrointestinal pathogens) may be 
completed at the time of AL dosing until first vaccination.  Given that these tests are run for 
research purposes and retrospectively, if these results are available prior to the final study visit 
and parasites are detected, and if recommended, subjects will be treated with standard treatment 
and continued in the study. Urinary schistosomiasis testing may be completed as well at the time 
of screening if there is concern it may be the etiology of subject’s proteinuria and/or hematuria; 
these subjects will receive treatment at no cost if they test positive.  
 
Other Laboratory Assays 
If there is adequate remaining blood sample available to fulfill the laboratory objectives, other 
laboratory assays may be performed as follows: 
• Malaria rapid diagnostic tests may be used for clinical diagnostic purposes. 
• qPCR may be used to detect gametocytes using whole blood collected on the day of 
positive BS.  

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• Filter paper filled with whole blood or mosquitoes may be used to determine parasite 
genotype. 
• Antibodies against sporozoite, pre-erythrocytic, blood, and sexual stages may be 
determined by ELISA.  
• Mosquito species and molecular forms may be identified by qPCR. 72 
• Typing of hemoglobin variants may be assessed by gel electrophoresis  
• Hepatitis B and C status may be assessed by serology and/or PCR 
• Cytokine levels may be evaluated during and following vaccination. 
 
Study physicians may ask for additional laboratory exams related to subject care. 
 
Collection of Malaria Prevention Measures During the Transmission Season 
Following family compound/vaccine unit receipt of Vaccination #3 and #4, on a monthly basis 
enrolled subjects will be asked at one of their clinic visits about other malaria prevention 
measures being utilized by the individual or family over the last month, including use of bednets, 
indoor residual spraying, seasonal malaria chemoprophylaxis, personal use of insecticide, and/or 
intermittent preventive treatment.  
 Exploration of Human Movement During Transmission Season (Optional) 
To maintain malaria transmission, the human reservoir, competent vector population, and the 
malaria parasite must overlap in space and time.20,73-77 While there is evidence that this overlap is 
likely to take place in or near an individual’s residence, which shares an environment with the 
vector, the dynamics of onward transmission from this focal point to others living further from 
competent vector populations is not fully understood.78 Where individuals spend their time, how 
frequently they travel, and the duration of travel can inform where individuals are likely to 
acquire and transmit malaria.77 
 
Commercially available GPS data loggers have the potential to measure human movement to 
capture and describe both small-scale and large-scale movement patterns simultaneously. Their 
low cost, availability and ease of use makes them ideal for tracking human movement to infer 
exposure to infection.79 Commercially available GPS data loggers have previously been used to 
determine how human movement impacts other infectious diseases, such as schistosomiasis, 
hookworm, and dengue virus in various settings. 79-82 Commercially available GPS data loggers 
are able to track individual movements continuously over pre-determined period of time.80  
 
Use of GPS data loggers, piloted in randomized subpopulation our DSF cohort (Arms 1a/1b, 
3c/3d; 9-18 years of age) during the period of DSF assessments (starting 14 days post 
Vaccination #3 through 112 days post Vaccination #3) will allow for initial estimates of human 

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movement in rural areas of Doneguebougou, Mali and surrounding villages, which may aid in 
describing malaria transmission in this area.  
 
Our targeted enrollment population, children/young adults 9-18 years of age, will have the option 
to participate in this portion of the study. Each participant approached for participation, if willing 
to participate, will be asked to carry the logger daily for up to 4 months total.  Eligible 
participants will be provided information regarding their participation via an additional 
consent/assent forms which will include the use of GPS data loggers, how they can be worn or 
carried, and will have the opportunity to ask questions and raise concerns. Information about 
what the GPS logger can and cannot do will be provided for review prior to participation. 
Participation in this portion of the study will be optional and initial results descriptive during this 
pilot phase.  
 Human Movement (2019-2020) 
The pilot study to assess the feasibility of using small GPS devices (IgotU ® GT-600) to track the 
movement of study participants enrolled in the study in Arms 1a/1b or 3c/3d (9-18 year-olds) 
was successfully implemented in August 2019. VUs with both enrolled and vaccinated male and 
female participants in the targeted age range were ranked and approached for participation; one 
male and one female from each unit were selected to facilitate comparison by sex.  
 
Enrollment in the Optional Human Movement study is summarized in Figure 31.  
 
 
Figure 31. Summary of Human Movement Study Enrollment  
Table summarizing key participant characteristics of GPS cohort (human movement subgroup) and overall trial 
enrollment of 9-18 year old children/young adults. Map representative of vaccine unit perimeters (abbreviated on 
this map = UV, referenced elsewhere as VU) in Doneguebougou, Mali with units participating in the human 
movement study (GPS cohort) colored in purple.  
 

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As planned, data from the units had to be pre-processed to remove coordinates from before 
distribution and after collection. Erroneous points, defined as the difference between two 
consecutive coordinates that indicated a velocity greater 10 km/hour, were flagged for removal 
Trajectories of individuals were built connecting the points in consecutive time points in ArcMap 
10.3.1 Tracking Analyst. 
 
Key logistical parameters of interest in this pilot were determining the failure rates of the units 
and the quality of the data collection over time, with failure rates seen ~10%. GPS units were 
well accepted by the community, with only one refusal, which was secondary to planned travel 
during the evaluation period. Data from Year 1 identified distinct differences in 9-18 year old 
male and female movements that may be informative to transmission dynamics in the 
community. 
 
Given these preliminary results, the study team is proposing to continue GPS monitoring of the 
same children/young adults for Year 2. In addition to strengthening the analysis of movement’s 
impact on malaria transmission and subsequent parasitemia, with the ongoing COVID-19 
pandemic, any changes in movement from pre-COVID-19 and post COVID-19 will also be able 
to be assessed.  
13 Research Use of Stored Human Samples, Specimens, or Data 
Intended Use: Samples, specimens, and data collected under this protocol may be used to study 
malaria and related diseases as well as vaccination. Genetic testing will be limited to hemoglobin 
typing and/or HLA testing. NIH researchers will have access to personally identifiable 
information.  
 
Storage: Access to stored research samples will be limited using either a locked room or a 
locked freezer. Temporary storage of samples collected in Mali, prior to shipment to LMIV, may 
occur at the Core Immunology Laboratory or the MRTC College of American Pathologists 
(CAP)–certified laboratory. Samples will be stored at the LMIV in Rockville, MD, or at LMIV’s 
designated repository, Thermo Scientific, Rockville, MD, with the exception of retention 
specimens which may be kept at the MRTC in Mali for quality control. Samples and data will be 
stored using codes assigned by the investigators or their designees. Data will be kept in 
password-protected computers. Only investigators or their designees will have access to the 
samples and data. 
 
Tracking: Samples will be tracked using a sample-tracking software program (e.g., 
Freezerworks). Data will be tracked as described in Section 19.1. 
 
Disposition at the Completion of the Protocol: In the future, other investigators (both at NIH 
and outside) may wish to study these samples and/or data. Before any sharing of samples, data, 

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or clinical information, either IRB and EC approval must be obtained or the NIH Office of 
Human Subjects Research Protections (OHSRP) and FMPOS EC must determine that the 
research is exempt from IRB/EC oversight. OHSRP and the EC can make this determination for 
some research where the samples or data have no personal identifying information about the 
study subject and the researcher is not able to ascertain it. 
At the time of protocol termination, samples will be either destroyed or, after IRB/EC approval, 
transferred to another existing protocol. Data will be archived by the study team in compliance 
with requirements for retention of research records; alternatively, after IRB/EC and study 
sponsor approval, the data may be either destroyed or transferred to another repository. 
 
Reporting the Loss or Destruction of Samples/Specimens/Data to the IRB: 
Any loss or unanticipated destruction of samples (for example, due to freezer malfunction) or 
data (for example, misplacing a printout of data with identifiers) that meets the definition of a 
protocol deviation, unanticipated problem (UP), and/or compromises the scientific integrity of 
the data collected for the study, will be reported to the NIH IRB and FMPOS EC. 
Additionally, subjects may decide at any point not to have their samples stored. In this case, the 
PI will destroy all known remaining samples and report what was done to both the subject and to 
the IRB and EC. This decision will not affect the individual’s participation in any other protocols 
at NIH. 
14 Data Sharing Plan 
In NIH's and MRTC’s view, all data should be considered for data sharing. Data should be made 
as widely and freely available as possible while safeguarding the privacy of subjects, and 
protecting confidential and proprietary data. We recognize that the public dissemination of our 
scientific results can facilitate the creation of collaborative efforts with domestic and 
international collaborators. Furthermore, we recognize that the proposed project may result in 
novel ideas for new methods, technologies, and data that could benefit the entire research 
community. Therefore, final research data will be shared openly and timely in accordance with 
the most recent NIH guidelines (http://grants.nih.gov/grants/policy/data_sharing/) while being 
mindful that the confidentiality and privacy of subjects in research must be protected at all times. 
Timelines for distribution of data will vary depending on any required restrictions in accordance 
with federal and/or institutional policies and guidelines. In general, we expect de-identified data 
will be available through NIH-funded or approved public repository, speaking engagements and 
publications, presentations at scientific symposia and seminars. Effort will be made to publish 
our research findings in scientific journals. All final peer-reviewed manuscripts that arise from 
this proposal will be submitted to the digital archive PubMed Central. For tools, reagents, data 
and model organisms generated by the proposed study, pending third parties’ rights, 
LMIV/MRTC will transfer materials to outside researchers in both the private and public sectors 
under a Material Transfer Agreement or Research Collaboration Agreement. 

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15 Assessment of Safety 
 
Definitions 
Adverse Event: An AE is any untoward or unfavorable medical occurrence in a human 
participant, including any abnormal sign (e.g., abnormal physical exam or laboratory finding), 
symptom, or disease, temporally associated with the individual’s participation in the research, 
whether or not considered related to the research. 
 
Adverse Reaction (AR): An AE that is caused by an investigational agent (drug or biologic). 
 
Suspected Adverse Reaction (SAR): An AE for which there is a reasonable possibility that the 
investigational agent caused the AE. ‘Reasonable possibility’ means that there is evidence to 
suggest a causal relationship between the drug and the AE. An SAR implies a lesser degree of 
certainty about causality than AR, which implies a high degree of certainty. 
 
Serious Adverse Event (SAE): An SAE is an AE that results in one or more of the following 
outcomes: 
• death 
• a life-threatening event (places the participant at immediate risk of death from the event 
as it occurred) 
• an inpatient hospitalization or prolongation of an existing hospitalization 
• a persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions 
• a congenital anomaly/birth defect 
• a medically important event* 
 
*Medical and scientific judgment should be exercised in deciding events that may not be 
immediately life threatening or result in death or hospitalization but may jeopardize the subject 
or may require intervention to prevent one of the other outcomes listed above. 
 
Unexpected Adverse Event: An AE is unexpected if it is not listed in the investigator’s 
brochure or package insert (for marketed products) or is not listed at the specificity or severity 
that has been observed. It is the responsibility of the Investigational New Drug application (IND) 
sponsor to make this determination. 
 
Serious and Unexpected Suspected Adverse Reaction (SUSAR): A SUSAR is an SAR that is 
both serious and unexpected. 
 

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Unanticipated Problem: A UP is any event, incident, experience, or outcome that is 
• unexpected in terms of nature, severity, or frequency in relation to 
o the research risks that are described in the IRB-approved research protocol and 
informed consent document, investigator’s brochure, or other study documents; 
and 
o the characteristics of the subject population being studied; and 
• possibly, probably, or definitely related to participation in the research; and 
• places subjects or others at a greater risk of harm (including physical, psychological, 
economic, or social harm) than was previously known or recognized. (Per the IND 
sponsor, an AE with a serious outcome will be considered increased risk.) 
 
Serious Unanticipated Problem (UP): A UP that meets the definition of a Serious Adverse 
Event or compromises the safety, welfare or rights of subjects or others. 
 
Unanticipated Problem that is not an Adverse Event (UPnonAE): A UP that does not fit the 
definition of an AE, but which may, in the opinion of the investigator, involve risk to the 
participant, affect others in the research study, or significantly impact the integrity of research 
data. Such events would be considered non-serious UPs. For example, we will report occurrences 
of breaches of confidentiality, accidental destruction of study records, or unaccounted-for study 
drug.  
 
New Onset of Chronic Illness (NOCI) 
The new onset of chronic illness (NOCI) is defined as a diagnosis of a new medical condition 
that is chronic in nature, including those potentially controllable by medication (e.g., diabetes, 
asthma). Any NOCI will be recorded in the same manner as unsolicited AEs. 
 
 
 
Documenting, Recording, and Reporting Adverse Events 
At each contact with the participant, information regarding AEs will be elicited by appropriate 
questioning and examinations and will be: 
• immediately documented in the subject’s medical record/source document, 
• recorded on the Adverse Event Case Report Form (AE CRF) or electronic database, and 
• reported as outlined below (e.g., IND sponsor, NIH IRB, FMPOS EC, U.S. Food and 
Drug Administration [FDA]). 
 
A study clinician will be available during the study period and will be available to the study 
subjects at all times. Should a subject call a study clinician to report an AE, it will be discussed 
with the PI and documented, recorded, and reported appropriately. 

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All abnormal laboratory findings will be reviewed on a routine basis by the PI to identify 
potential safety signals. An abnormal lab not included on the toxicity table should be assessed in 
a similar fashion to the criteria above. 
 
Investigator Assessment of Adverse Events 
If a diagnosis is clinically evident (or subsequently determined), the diagnosis rather than the 
individual signs and symptoms or lab abnormalities will be recorded as the AE. 
 
All solicited (see Table 15 below) and unsolicited AEs will be recorded through 7 days after 
each vaccination, including injection site reactions, or until resolved. After that period only, 
unsolicited AEs (including symptomatic malaria), SAEs, UPs, and NOCIs will be recorded. Note 
that a positive BS without associated clinical symptoms will not be reported as an AE. 
 

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Table 15. Solicited Adverse Events 
Systemic adverse events 
Fever (temperature ≥38.0 °C) 
Headache  
Nausea/Vomiting  
Diarrhea 
Abdominal pain 
Fatigue 
Myalgia  
Arthralgia  
Urticaria  
Laboratory adverse events 
Hemoglobin – decreased hemoglobin 
WBC – leukopenia, leukocytosis 
ANC/AGC – decreased neutrophil/granulocyte count 
Platelet count – thrombocytopenia  
ALT – increased ALT  
Cr – increased Cr 
Local reactogenicity 
Injection pain/tenderness 
Injection erythema/redness 
Injection swelling 
Injection induration 
Injection pruritus 
Limitation of arm movement 
Abbreviations: ALT, alanine transaminase; ANC, absolute neutrophil count; AGC, absolute granulocyte count; CR, 
creatinine; WBC, white blood cell. 
Additional laboratory abnormalities other than those specified as safety labs in the protocol 
should be reported as AEs if they require intervention. Interventions include, but are not limited 
to, discontinuation of treatment, dose reduction/delay, additional assessments, or concomitant 
treatment. In addition, any medically important laboratory abnormality may be reported as an AE 
at the discretion of the investigator. This could include a laboratory result for which there is no 
intervention, but the abnormal value suggests a disease or organ toxicity. In addition, similar to 
solicited AEs, all laboratory AEs as defined in Appendix C, will be collected and graded 
through 7 days after each vaccination or until resolved. 
 
The investigator will assess all AEs with respect to seriousness (criteria listed above), severity 
(intensity or grade), and causality (relationship to study agent and relationship to research) 
according to the following guidelines. 

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Severity 
Severity of AEs will be assessed by the investigator as described in Appendix C. AEs not 
included in the Appendices will be graded for severity using the followings definitions as seen in 
Table 16. 
 
Table 16: Definitions for Severity of AE Grading 
Severity 
Definition 
Grade 1 (Mild) 
No interference with activity, may use 1 dose 
of an over the counter medication 
Grade 2 (Moderate) 
Repeated use of non-narcotic pain reliever 
>24 hours or some interference with activity 
Grade 3 (Severe) 
Activities of daily living limited to <50% of 
baseline, medical evaluation/therapy required 
Grade 4 (Potentially Life-Threatening) 
Extreme limitation in activity, significant 
assistance required; immediate medical 
intervention or therapy required to prevent 
death 
Grade 5 
Death 
 
Causality 
Causality (likelihood that the event is caused by the study agent(s)) will be assessed considering 
the factors listed under the following categories: 
 
Definitely Related 
• reasonable temporal relationship 
• follows a known response pattern 
• clear evidence to suggest a causal relationship 
• there is no alternative etiology 
 
Probably Related 
• reasonable temporal relationship 
• follows a suspected response pattern (based on similar agents) 
• no evidence of a more likely alternative etiology 
 
Possibly Related 
• reasonable temporal relationship 
• little evidence for a more likely alternative etiology 
 

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Unlikely Related 
• 
does not have a reasonable temporal relationship 
OR 
• 
good evidence for a more likely alternative etiology 
 
Not Related 
• 
does not have a temporal relationship 
OR 
• 
definitely due to an alternative etiology 
 
Note: Other factors (e.g., dechallenge, rechallenge) should also be considered for each causality 
category when appropriate. Causality assessment is based on available information at the time of 
the assessment of the AE. The investigator may revise the causality assessment as additional 
information becomes available. 
 
The degree of certainty with which an AE can be attributed to administration of the study 
vaccine will be determined by how well the event can be understood in terms of one or more of 
the following: 
• The event being temporally related with vaccination or reproduced on re-vaccination. 
• A reaction of similar nature having previously been observed with this type of vaccine 
and/or formulation. 
• The event having been reported in the literature for similar types of vaccines. 
• Whether or not there is another identifiable cause. 
 
All local (injection site) reactions will be considered causally related to vaccination. All malaria 
cases will be reported as not related to vaccination. Asymptomatic parasitemia (positive BS 
without related malaria clinical symptoms) will not be reported as an AE. Clinical malaria will 
be reported as an AE. 
 
Reports will further classify AEs as follows: 
• Related - all AEs that are assessed as definitely, probably, or possibly related. 
• Unrelated - all AEs assessed as unlikely or definitely not related. 
 
When reporting to regulatory authorities and IRB/EC is needed, AE relationship will be 
determined as noted above.  

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Follow-up of Adverse Events and Serious Adverse Events 
AEs that occur following receipt of a single vaccination are followed until the final outcome is 
known or until the end of the study follow-up period.  
 
SAEs that have not resolved by the end of the follow-up period will be followed until final 
outcome is known. If it is not possible to obtain a final outcome for an SAE (e.g., the subject is 
lost to follow-up), the reason a final outcome could not be obtained will be recorded by the 
investigator on the AE CRF (if the CRF is still open) and the Safety Expedited Report Form 
(SERF).  
 
SAEs that occur after the study follow-up period (e.g., after 3 months following the last 
vaccination) that are reported to and assessed by the Investigator to be possibly, probably, or 
definitely related must be reported to the Clinical Safety Office (CSO) as described below. 
 
Investigator Reporting Responsibilities to the Sponsor 
 
Adverse Events 
Line listings, frequency tables and other summary AE data will be submitted to the IND sponsor 
when requested for periodic safety assessments, review of IND Annual Reports, review of IND 
Safety Reports, and preparation of final study reports. 
 
Serious Adverse Events 
All SAEs (regardless of relationship and whether or not they are also UPs) must be reported on 
the SERF and sent to the CSO by fax or email attachment. Deaths and immediately life-
threatening SAEs must be reported to the CSO within 1 business day after the site becomes 
aware of the event. All other SAEs must be reported within 3 business days of site awareness. 
 
CSO CONTACT INFORMATION: 
Clinical Safety Office 
5705 Industry Lane 
Frederick, MD 21704 
Phone: 301-846-5301 
Fax: 301-846-6224 
E-mail: rchspsafety@mail.nih.gov  
 
SAEs that have not resolved by the end of the follow-up period are followed until final outcome 
is known. If it is not possible to obtain a final outcome for an SAE (e.g., the participant is lost to 
follow-up), the reason a final outcome could not be obtained will be recorded by the investigator 
on the AE CRF and the SERF. 
 

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SAEs that occur after the final study visit that are reported to and are assessed by the investigator 
to be possibly, probably, or definitely related to study drug must be reported to the CSO. 
 
The clinical site investigator in Mali will also notify LMIV PI and the site medical monitor in 
Mali by email, fax, or telephone within 1 working day of notification of an SAE occurrence. 
 
LMIV Contact Information: 
Patrick Duffy, MD 
Tel: (301-761-5089 
Fax: (301) 480-1962 
Email: patrick.duffy@nih.gov 
 
Unanticipated Problems 
UPs that are also AEs must be reported to the CSO by fax or e-mail attachment using the NIH 
Reportable Events Form no later than 7 calendar days of site awareness of the event. UPs that are 
not AEs are not reported to the CSO. 
 
Pregnancy 
Pregnancy itself is not an AE. However, complications of pregnancies are AEs and may be 
SAEs. Pertinent obstetrical information for all pregnancies will be reported to the CSO and 
LMIV via fax or email within 3 business days from the site awareness of the pregnancy. All 
pregnancies will be reported on the Pregnancy Notification/Outcome Form to the CSO within 
1 business day from site awareness. 
 
Pregnancy outcome data (e.g., delivery outcome, spontaneous or elective termination of the 
pregnancy) will be reported to the CSO and LMIV within 3 business days of the site’s 
awareness. 
 
In the event of pregnancy, the following steps will be taken: 
• Discontinue the study agent. 
• Discuss with women who become pregnant continued participation in the study, 
including continued safety/research labs (at investigator’s discretion given blood 
volumes), direct skin feeds, and clinical visits.  
• Report to FMPOS EC as an informational item. 
• Report to the DSMB, Sponsor Medical Monitor, and Malian Independent Safety Monitor 
(ISM). 
• Advise research participant to notify the obstetrician of study participation and potential 
study agent exposure. 

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Investigator Reporting Procedures to the NIH IRB 
 
Definitions 
Protocol Deviation: Any change, divergence, or departure from the IRB-approved research 
protocol.  
Major Deviations: Deviations from the IRB approved protocol that have, or may have 
the potential to, negatively impact, the rights, welfare or safety of the subject, or to 
substantially negatively impact the scientific integrity or validity of the study.   
 
Minor Deviations: Deviations that do not have the potential to negatively impact the 
rights, safety, or welfare of subjects or others, or the scientific integrity or validity of the 
study.  
 
Non-Compliance: Failure of investigator(s) to follow the applicable laws, regulations, or 
institutional policies governing the protection of human subjects in research, or the requirements 
or determinations of the IRB, whether intentional or not. Failure of subjects to comply with the 
research protocol does not represent non-compliance unless that failure is due to an action or 
omission of a member of the research team, for example, the failure to give adequate instruction 
to the subject. 
 
Serious non-compliance: Non-compliance, whether intentional or not, that results in 
harm or otherwise materially compromises the rights, welfare and/or safety of the subject. 
Non-compliance that materially effects the scientific integrity or validity of the research 
may be considered serious non-compliance, even if it does not result in direct harm to 
research subjects.  
 
Continuing non-compliance: A pattern of recurring non-compliance that either has 
resulted, or, if continued, may result in harm to subjects or otherwise materially 
compromise the rights, welfare and/or safety of subjects, affect the scientific integrity of 
the study or validity of the results. The pattern may comprise repetition of the same non-
compliant action(s), or different noncompliant events. Such non-compliance may be 
unintentional (e.g. due to lack of understanding, knowledge, or commitment), or 
intentional (e.g. due to deliberate choice to ignore or compromise the requirements of any 
applicable regulation, organizational policy, or determination of the IRB).  
15.6.1.1 Expedited Reporting to the NIH IRB and FMPOS EC 
Non-compliance: Any actual or suspected non-compliance by any investigator or entity 
associated with the protocol must be reported by the NIH PI/designee within 7 calendar days of 
any investigator or individual associated with the protocol first becoming aware, unless 
otherwise indicated in this policy.  

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Non-NIH IRB determinations of serious and/or continuing noncompliance about an NIH 
investigator: If the NIH is relying on a non-NIH IRB and the Reviewing IRB makes a 
determination of serious and/or continuing non-compliance regarding an NIH 
investigator, then, even if the determination has already been provided to OHSRP either 
directly or via the NIH Institutional Official (IO)/designee, the NIH PI/designee must 
report this in iRIS within 7 calendar days of any member of the research team being 
notified of the determination by the Reviewing IRB. The NIH PI must provide the 
OHSRP office of Compliance and Training with documentation from the Reviewing IRB 
unless this documentation has already been provided directly to the office by the 
Reviewing IRB or via the IO.  
 
Major Deviation: A deviation must be reported within 7 calendar days of an investigator 
becoming aware of an actual or suspected deviation. Although protocol deviations are also non-
compliance, these should only be reported once as deviations.  
 
Unanticipated Problem (UP): A UP must be reported within 7 calendar days of an investigator 
becoming aware of the actual or suspected UP.  
 
Death: Any death of a research subject that is possibly, probably or definitely related to the 
research must be reported within 24 hours of an investigator becoming aware of the death.  
 
New information that might affect the willingness of a subject to enroll or remain in the study 
should be reported within 7 calendar days of an investigator first becoming aware.  
 
Any suspension or termination of research activities, including holds on new enrollment, placed 
upon the research by the study sponsor, NIH or IC leadership, or any regulatory agency must be 
reported within 7 calendar days of an investigator becoming aware.  
 
Investigators must provide the following information to the IRB in summary format at the time 
of continuing review: Minor protocol deviations; Adverse events and Serious Adverse Events 
that do not meet the definition of an UP.  
 
Investigators are required to immediately (i.e., no longer than 10 days) report SAEs or UADEs to 
the study sponsor and, if also an actual or suspected UP, to the IRB within 7 calendar days of an 
investigator becoming aware. 
15.6.1.2 Annual Reporting to the NIH IRB and FMPOS EC 
All events will be reported to the NIH IRB and FMPOS EC in summary format at the time of 
Continuing Review according to NIH policy 801. 

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Investigator Reporting Responsibilities to the Local IRB 
Investigators are responsible for submitting IND Safety Reports and UP summaries that are 
received from the IND sponsor to the FMPOS EC. Investigators must also comply with all 
FMPOS EC reporting requirements, including expedited and annual reporting requirements. 
 
Sponsor’s Reporting Responsibilities 
SUSARs, as defined in Title 21 of the United States Code of Federal Regulations (CFR) 
Part 312.32 and determined by the IND sponsor, will be reported to FDA and all participating 
investigators as IND Safety Reports. 
 
The IND sponsor will also submit an IND Annual Report of the progress of the investigation to 
the FDA as defined in 21 CFR 312.33. 
 
Pausing Rules for the Protocol 
Pausing is the suspension of administration of study agent to a single subject or specified group 
until a decision is made whether or not to resume administration of the study agent. 
 
Pausing Rules for an Individual Subject 
The pausing criteria for a SINGLE subject in this study include any of the following; note Year 1 
and Year 2 will be evaluated independently given time between dosing: 
• A subject experiences one SAE that is determined as possibly, probably, or definitely 
related to a study agent 
• A subject experiences ≥2 Grade 3 or greater AEs that are deemed not to be a SAE 
(solicited local/systemic or unsolicited; lasting 48 hours or more) that are possibly, 
probably, or definitely related to a study agent, within the 7 days post vaccination  
• A subject experiences ≥2 Grade 2 or greater laboratory abnormality (see Appendix C) or 
any Grade 3 or higher laboratory abnormality that are not deemed to be a SAE and are 
possibly, probably, or definitely related to the study agent, within the 7 days post 
vaccination 
• ≥1 subject experiences any local or solicited AE leading to hospitalization, or fever 
>40°C (104°F) that is possibly, probably, or definitely related to the vaccine, or necrosis 
at the injection site, within the 7 days post vaccination 
• Any safety issue that the site investigator determines should pause administration of a 
study agent to a single subject. 
 
The CSO, in collaboration with the PI, may also pause for an individual subject if a safety 
concern is identified during routine aggregate data analysis. 

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Pausing Rules for a Particular Age Group 
The pausing criteria for a SPECIFIC AGE group in this study include any of the following; note 
Year 1 and Year 2 will be evaluated independently given time between dosing: 
• An age group enrolled (e.g. 5-8 years of age, 9-18 years of age) experiences one SAE that 
is determined as possibly, probably, or definitely related to a study agent 
• ≥5% of a specific age group experiences Grade 3 or greater AEs (solicited local/systemic 
or unsolicited; lasting 48 hours or more) that are possibly, probably, or definitely related 
to a study agent, within the 7 days post vaccination 
• ≥10% of a specific age group experiences Grade 2 or greater laboratory abnormality (see 
Appendix C) or ≥5% Grade 3 or higher laboratory abnormality that are possibly, 
probably, or definitely related to the study agent, within the 7 days post vaccination  
• Any safety issue or identifiable trend in AE reporting that the site investigator determines 
should pause administration of a study agent to a specific age group 
 
The CSO, in collaboration with the PI, may also pause a specific age group if a safety concern is 
identified during routine aggregate data analysis. 
 
 
Reporting a Pause 
If a pausing criterion is met, a description of the AE(s) or safety issue must be reported by the PI 
within 2 business days to the CSO, the IRB/EC, and the DSMB by fax or email. 
 
Resumption of a Paused Study 
The CSO, in collaboration with the PI and DSMB, will determine whether or not it is safe to 
resume administration of the study agent to the subject or a specific age group. The PI will notify 
the IRB/EC of the decision on resumption of the study agent. A subject who does not resume to 
receive the study agent will continue to be followed for safety.  
 Halting Rules for the Protocol 
Halting the study requires immediate discontinuation of study agent administered for all subjects 
and suspension of enrollment until a decision is made whether or not to continue enrollment and 
study agent administration. 
 
The following criteria will be used to define unacceptable reactogenicity of the malaria vaccine 
(AEs that are possibly, probably, or definitely related to the vaccine will be considered “related” 
and will be summarized as such); note Year 1 and Year 2 will be evaluated independently given 
time between dosing: 
• One or more subjects experience death or any life-threatening SAE, or 
 

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• ≥2 subjects experience an SAE as defined in Section 15.1 of this protocol that is 
determined to be possibly, probably, or definitely related to the vaccine, or 
• ≥1 subjects are withdrawn from the study (by investigator or subject request) following a 
Grade 3 AE that is determined to be possibly, probably, or definitely related to the 
vaccine, or 
• ≥1 subjects experience a hypersensitivity reaction (e.g. anaphylaxis, diffuse urticaria) that 
is determined to be possibly, probably, or definitely related to the vaccine, or 
• ≥1 subjects experience any local or solicited AE leading to hospitalization, or fever 
>40°C (104°F) that is possibly, probably, or definitely related to the vaccine, or necrosis 
at the injection site, within the 7 days post vaccination, or 
• ≥10% subjects enrolled experience any Grade 3 solicited local AE (lasting 48 hours or 
more) that is determined to be possibly, probably, or definitely related to the vaccine as 
defined in this protocol, within the 7 days post vaccination, or  
• ≥10% subjects enrolled experience any Grade 3 solicited systemic AE (lasting 48 hours 
or more) that is determined to be possibly, probably, or definitely related to the vaccine 
as defined in this protocol, within the 7 days post vaccination, or  
• ≥30% subjects enrolled experience the same Grade 2 or higher laboratory abnormality 
(see Appendix C) or ≥10% subjects enrolled experience the same Grade 3 or higher 
laboratory abnormality regardless of vaccine relationship, within the 7 days post 
vaccination, or  
• ≥10% subjects enrolled experience the same or similar Grade 3 unsolicited AE that is 
determined to be possibly, probably, or definitely related to the vaccine as defined in this 
protocol, or 
• Any safety issue that the study PI or IND sponsor determines should halt the study. 
 
The PI and/or CSO will determine if the study should be halted. In addition, the FDA may halt 
the study at any time following review of any safety concerns. The NIH IRB or FMPOS EC may 
also halt the study. 
 
Reporting a Study Halt 
If a halting rule is met, a description of the AE(s) or safety issue must be reported by the PI 
within 1 business day to the CSO, the IRB/EC, and the DSMB by fax or email. 
 
Resumption of a Halted Study 
The IND sponsor, in collaboration with the PI and the DSMB, will determine if it is safe to 
resume the study. The conditions for resumption of the study will be defined in this notification. 
The PI will notify the IRB/EC of the decision on resumption of the study. 

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 Discontinuation of Study 
The NIAID/Office of Clinical Research Policy and Regulatory Operations (OCRPRO) as the 
study sponsor, the NIH IRB, FMPOS EC, and the FDA may terminate this study at any time. 
Reasons for terminating the study may include, but are not limited to, the following: 
• The incidence or severity of an AE in this or other studies indicates a potential health 
hazard to subjects. 
• Subject enrollment is unsatisfactory. 
• Data recording is inaccurate or incomplete. 
• Investigators do not adhere to the protocol or applicable regulatory guidelines in 
conducting the study. 
 Withdrawal Criteria for an Individual Participant 
A subject will not be considered to have completed the trial if any of the following reasons 
apply; note Year 1 and Year 2 will be evaluated independently given time between dosing: 
1. 
Research terminated by Sponsor or Investigator – applies to the situation 
where the entire study is terminated by the Sponsor or Investigator, or other 
regulatory authority for any reason. 
2. 
Withdrawal of consent – applies to a subject who withdraws consent to 
participate in the study for any reason. The investigator will attempt to 
determine the reason for the subject’s decision and document it in the study 
chart.  
3. 
Non-compliance with protocol – applies to a subject who does not comply 
with protocol-specific visits or evaluations on a consistent basis, and to the 
extent that it is potentially harmful to the subject or to the integrity of the 
study data. This also applies to a subject who is lost to follow-up and is not 
reachable by telephone or other means of communication and cannot be 
located. 
4. 
Developed an AE – applies to a subject who is withdrawn from study due to 
an AE, serious or otherwise. Any grade 3 or greater AE that is assessed as 
possibly, probably, or definitely related to vaccination (other than local 
reactions lasting <72 hours, or systemic reactions lasting <24 hours) will 
result in withdrawal of the subject from further vaccinations. Subjects may 
also be withdrawn for ANY AE that would cause continued participation in 
the study to not be in the best interest of the subject, as per the investigator’s 
judgment. Any subject who is withdrawn from the study because of an AE 
related to study agent will be followed for safety until at least resolution of 
that AE and will be encouraged to remain in the safety evaluation for the 
duration of the study. 

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5. 
Other – is used when previous categories do not apply and a written 
explanation is required. 
 
If a subject withdraws or is withdrawn prior to completion of the study, the reason for this 
decision will be recorded in the source documents and CRFs. Any subject who has received at 
least 1 dose of vaccine will be encouraged to remain in the safety evaluation for the duration of 
the study. The subject’s data will be included in the safety and immunogenicity analysis. If a 
subject fails to complete all planned vaccinations because of an AE or SAE, the subject will be 
followed until resolution or stabilization of the event. If a subject withdraws, the investigator will 
make a reasonable effort to determine the reason.  
 
Replacement of Withdrawn Participants or Participants Who Discontinue Study 
Agent 
Subjects who have received at least 1 vaccination and who withdraw or are terminated from the 
study prior to completion will not be replaced. Subjects withdrawn before the first vaccination 
may be replaced. 
 Safety Oversight 
 
Safety Review and Communications Plan 
A Safety Review And Communications Plan (SRCP) has been developed for the protocol. The 
SRCP is an internal communications document between the PI and the CSO, which delineates 
the safety oversight responsibilities of the PI, the CSO, and other stakeholders. The SRCP also 
includes the overall plan for conducting periodic safety surveillance assessments. 
 
Sponsor Medical Monitor 
A medical monitor, representing the IND sponsor (OCRPRO), has been appointed for oversight 
of safety in this clinical study. The sponsor medical monitor will be responsible for performing 
safety assessments as outlined in the SRCP. 
 
Independent Safety Monitor (ISM)  
An ISM in Mali will review the study prior to initiation and will be available to advise the 
Investigators on study-related medical issues and to act as a representative for the welfare of the 
subjects. The ISM will conduct independent safety monitoring and recommend appropriate 
action regarding AEs and other safety issues. The ISM is an expert in the field of oversight of 
clinical trials conducted in Mali and internal medicine, specifically in the population under study 
in Mali. The ISM does not have direct involvement in the conduct of the study and does not have 
other interests with any collaborating pharmaceutical firms or their competitors. 
 

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Prior to each ISM review (including DSMB meeting safety reports and at least twice yearly 
reviews), the PI will provide a safety summary report (similar to the DSMB safety reports). After 
each ISM review, a recommendation as to whether the study is to be modified or terminated will 
be provided in a summary report to the study PI. If the study is to continue as is, no report will 
need to be submitted by the ISM except for communication to the PI that the review has been 
completed (via in-person communication, phone, or email). All SAEs, all UPs, and all IND 
Safety Reports will be reported by the PI to the ISM at the same time they are submitted to the 
IRB/EC or IND sponsor. The ISM will be notified immediately if any pausing or halting rule is 
met and the ISM will provide recommendation for continuation, modification, or termination of 
the study. The PI will submit the written ISM summary report with the recommendations to their 
IRB/EC on a biannual basis or more frequently if a safety concern is raised.  
 
Data and Safety Monitoring Board (DSMB) 
The NIAID intramural DSMB includes independent experts that do not have direct involvement 
in the conduct of the study and have no significant conflicts of interests as defined by NIAID 
policy. The DSMB will review the study prior to initiation and at the end of the study, and may 
convene additional reviews as necessary. The board will review the study data to evaluate the 
safety, efficacy, study progress, and conduct of the study as denoted in Figure 23 and Figure 24. 
All SAEs, all UPs, and all IND Safety Reports will be reported by the PI to the DSMB at the 
same time they are submitted to the IRB or IND sponsor. The PI will notify the board at the time 
pausing or halting criteria are met and obtain a recommendation concerning continuation, 
modification, or termination of the study. The PI will submit the written DSMB summary reports 
with recommendations to the IRB/EC. 
16 Site Monitoring Plan 
According to the International Conference on Harmonisation (ICH) E6(R2) Good Clinical 
Practice (GCP) guidelines, section 5.18, and FDA 21 CFR 312.50, clinical protocols are required 
to be adequately monitored by the study sponsor. This study monitoring will be conducted 
according to the “NIAID Intramural Clinical Monitoring Guidelines.” Monitors under contract to 
the NIAID/OCRPRO will visit the clinical research site to monitor aspects of the study in 
accordance with the appropriate regulations and the approved protocol. The objectives of a 
monitoring visit will be: 1) to verify the existence of signed informed consent documents and 
documentation of the consent process for each monitored subject; 2) to verify the prompt and 
accurate recording of all monitored data points and prompt reporting of all SAEs; 3) to compare 
abstracted information with individual subjects’ records and source documents (subjects’ charts, 
laboratory analyses and test results, physicians’ progress notes, nurses’ notes, and any other 
relevant original subject information); and 4) to help ensure investigators are in compliance with 
the protocol. The monitors also will inspect the clinical site regulatory files to ensure that 
regulatory requirements (Office for Human Research Protections [OHRP], FDA) and applicable 
guidelines (ICH GCP) are being followed. During the monitoring visits, the investigator (and/or 

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designee) and other study personnel will be available to discuss the study progress and 
monitoring visit. 
 
The investigator (and/or designee) will make study documents (e.g., consent forms, DataFax data 
abstracts) and pertinent hospital or clinical records readily available for inspection by the 
FMPOS EC, FDA, the site monitors, and the NIAID staff for confirmation of the study data. 
 
A specific protocol monitoring plan will be discussed with the PI and study staff prior to 
enrollment. The plan will outline the frequency of monitoring visits based on such factors as 
study enrollment, data collection status, and regulatory obligations. 
17 Statistical Considerations 
 
Pilot Study 
 
Primary Objective 
The primary objective of the pilot study is to assess the safety and reactogenicity of Pfs230D1M-
EPA/AS01 in Malian children (Arms 1a, 2a) compared to comparator vaccine (Arms 1b, 2b). 
• The frequency of systemic and local AEs will be summarized. 
• A line listing of each clinical and laboratory AE classified as local, solicited, or other will 
be displayed in tables stratified by vaccine allocation. 
• AEs will be summarized by severity and relationship to vaccine. 
• The proportion of subjects with at least 1 AE will be compared by study group, and tests 
will be performed to assess whether these groups differ with respect to these proportions. 
To evaluate the difference in AEs between the initial vaccination and the subsequent 
vaccinations, a Wilcoxon signed rank test (WSRT) will be performed, where the response 
for each subject is the difference between the numbers of AEs in the 7 days following 
each vaccination. To compare number and severity of AEs between control and vaccine 
arms, the Wilcoxon-Mann-Whitney test (WMW, also known as Wilcoxon rank sum test 
and Mann-Whitney U test) as well as linear regression may be used. 
• SAEs occurring within the study period will be listed by relationship to vaccine. 
 
 
Secondary Objective 
The secondary objectives of this study are: 
• To assess ELISA titer response to Pfs230D1M-EPA/AS01 in Malian children (Arms 1a, 
2a) 
• To assess functional antibody response by SMFA to Pfs230D1M-EPA/AS01 in Malian 
children (Arms 1a, 2a) 

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Anti-Pfs230 antibody will be measured by ELISA on vaccination days, 2 weeks after each 
vaccination, and periodically until study completion. Antibody responses over time will be 
shown graphically with geometric mean antibodies and the associated confidence intervals based 
on the t-distribution applied to the log-transformed responses. We will compare antibody and 
functional assay responses within arms over time. We will also look at differences in antibody 
decline over time in the vaccination groups. Graphs will display transmission-blocking assays as 
a function of ELISA antibody levels. 
 
Exploratory Objective 
The exploratory objective of this study is: 
• To explore cellular responses and antibody repertoire of functional antibody responses to 
vaccination (all arms) 
 
We will characterize and compare host immune responses and antibody repertoires to malarial 
antigens and to vaccine antigens in African children prior to and after vaccination with 
Pfs230D1M-EPA/AS01 by mixed effects model or WSRT depending on the type of immune 
response. We will also make figures depicting immune response based on the study arm. 
 
Should the study be terminated early, the investigative team will discuss with the DSMB the 
reason for termination and determine which study questions can be addressed in an unbiased 
manner with the available data. The available data will be analyzed and interpreted in light of 
early termination. 
 
Main Study 
 
Primary Objective 
To assess vaccine activity of Pfs230D1M-EPA/AS01 against P. falciparum transmission by 
direct skin feeding assay (DSF) (Arms 1a/1b and 3c/3d only) 
 
A Poisson type model with compound level clustering with offset for the number 
of DSF tests that were performed will be used. The primary analysis of activity measured by 
DSF will be the Poisson GEE, which will be performed on the data once with Pm/Po midguts, 
and once without. The proportion of subjects infecting at least 1 mosquito in at least 1 DSF over 
the primary analysis period following dose 3 will be compared by use of a GEE model for 
binary response with compound level clustering. 
 
Each of the above analyses will be performed on the subsample of subjects that had a 
concurrent positive BS (any parasite sexual and/or asexual) or rapid test positive with their DSF 
results. 
 

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Secondary Objectives 
To assess vaccine efficacy of Pfs230D1M-EPA/AS01 against P. falciparum infection measured 
by BS (All Arms except Arms 3e/3f) 
 
The vaccine efficacy will be assessed by comparing the rates of positive BS in children 5-18 
years of age receiving Pfs230D1M-EPA/AS01vs those receiving the comparator vaccines. An 
additional measure of vaccine protection will be assessed in the participants 1-4 years of age in 
Arms 4a/4b by comparing the rates of positive BS in the compounds receiving Pfs230D1M-
EPA/AS01vs those receiving the comparator vaccines.  
 
To assess safety and reactogenicity of administration of Pfs230D1M-EPA/AS01 (All Arms)  
 
• The frequency of systemic and local AEs will be summarized. 
• A line listing of each clinical and laboratory AE classified as local, solicited, or other will 
be displayed in tables stratified by vaccine allocation. 
• AEs will be summarized by severity and relationship to vaccine. 
• The proportion of subjects with at least 1 AE will be compared by study group, and tests 
will be performed to assess whether these groups differ with respect to these proportions. 
To evaluate the difference in AEs between the initial vaccination and the subsequent 
vaccinations, a WSRT will be performed, where the response for each subject is the 
difference between the numbers of AEs in the 7 days following each vaccination. To 
compare number and severity of AEs between control and vaccine arms, the Wilcoxon-
Mann-Whitney test (WMW, also known as Wilcoxon rank sum test and Mann-Whitney 
U test) as well as linear regression may be used. 
• SAEs occurring within the study period will be listed by relationship to vaccine. 
 
 
ELISA and SMFA assays will be conducted on a subset of samples deemed to be of scientific 
interest by DSF or BS assays due to the intensive laboratory analysis required to generate the 
data. Analysis of these data will take into account the compound randomization.  
 
 
Exploratory Objectives 
To explore cellular and humoral responses to Pfs230D1M (All Arms except Arms 4a/4b) 
To identify host and parasite factors associated with transmission (All Arms) 
To track transmission of parasites from human to human using highly variant gene fragments 
(All Arms) 
 
GEE analyses with compound level clustering will be used to analyze ELISA, SMFA and safety 
endpoints. 
 

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Sample Size and Power Calculations 
 
Pilot Study 
17.3.1.1 Safety 
The arms are sufficiently sized for safety. For each dose level (n=15) in Arms 1a, 2a, vaccination 
of 15 subjects gives a probability of at least 0.90 for detecting 1 or more serious or severe AEs 
that occur with a probability of 0.142 or more per subject. 
 
If we combine all treated groups (arms 1a and 2a), 30 subjects, we have 95% power to detect 1 
or more serious or severe AEs that occur with a probability of 0.095 or more per subject. Due to 
arms having different age ranges, this analysis would only be done to catch an AE that 
transcended these discrepancies. 
 
We will compare all AE event proportions between the control arm and treated arm by Fisher’s 
exact test. 
17.3.1.2 ELISA Analysis 
There are several questions of interest based on the antibody response information to include the 
change in ELISA values from baseline after a given number of doses of vaccine and the change 
in ELISA values between doses. For this we will use age-group-specific WSRT within the 
15 subjects receiving a given vaccine regimen.  
 
The data from Protocol #17-I-N006 on subjects who had Pfs230 ELISA measurements after 
receiving two doses allow us to estimate the standard deviation (SD) of the log transformed 
Pfs230 ELISA responses at baseline (mean 3.688401 sd 0.5490291) and 3 months post 
vaccination 2 (mean 5.356736 sd 0.8868963). 
 
Assuming similar values in this trial, there would be over 0.99 power to detect the difference 
between baseline and 3 months post-vaccination 2. This approximate power is calculated 
counting the number of significant comparisons out of a thousand comparisons between random 
normal draws from N(3.68,0.55) and N(5.36, 0.88) using the Wilcoxon test. 
 
Using the background information from #17-I-N006, we have greater than 80% power to reject a 
2-sided 0.05 level WMW test if the geometric mean Pfs230 ELISA baseline level was 2.3-fold 
higher geometric mean than the level of detection in the vaccinated group post vaccination 2 
(Note: 2.3-fold is lower than the 5.31-fold observed in #17-I-N006, so we treat these numbers as 
conservative). This approximate power is calculated counting the number of significant 
comparisons out of a thousand comparisons between random normal draws from N(3.68,0.55) 
and N(3.68+log(2.3), 0.88) using the Wilcoxon test.  

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17.3.1.3 SMFA Analysis 
 
For a group of 15 participants we anticipate at least 60% power to detect a difference in TRAs of 
32% (the observed difference in #17-I-N006) and 80% power for a 40% difference. These Power 
calculations come from a simulation assuming values similar to the SMFA data from Protocol 
#17-I-N006, where the control group had an average TRA of 38 (sd 39) and a treatment group 
with sd 34. Thus counting the number of significant comparisons out of a thousand comparisons 
between random normal draws from N(38,39) and N(38+40, 34) using the Wilcoxon test can 
give approximate power. 
 
If TBAs are used, we will standardize the TBAs to a common target control mean first using the 
methods of Swihart, Fay, & Miura (2018). A nice feature of the standardized TBA is that its 
power is identical to those of the TRA. 
 
 
Main Study 
17.3.2.1 Vaccine Activity 
Vaccine activity will be measured by DSFs in children 9-18 years of age. The Bancoumana 
Community Study assessed monthly DSFs in children 10-18 years of age from March to August 
with a seasonal DSF positivity rate (i.e. at least 1 positive DSF) of 6%. We anticipate that this 
seasonal DSF rate will be increased to 12-16% for children 9-18 years of age during the rainy 
season with twice-monthly DSF testing. Doneguebougou has 560 children 9-18 years of age. We 
conservatively estimate that about 400 of these will be randomized and not drop out of the study. 
 
Since the study is randomized by compound, clustering due to compound needs to be addressed 
in the power calculations. The Bancoumana Community Study provided data on seasonal 
parasitemia positivity which we use as a proxy for seasonal DSF positivity. For parasitemia, the 
intra-compound-correlation (ICC) was estimated to be .01 using a beta-binomial model. This low 
ICC coupled with the large number of compounds and relatively small number of subjects 9-
18 years of age per compound (median of 4) indicates that clustering will be negligible. Power 
was simulated for a study with 100 compounds and 400 subjects with monthly DSF testing for 
4 months. Since testing will be twice-monthly, simulating monthly testing will be conservative. 
The intra-subject correlation (ISC) for monthly serial DSFs was estimated to be .14 using a beta-
binomial model for children 10-18 years of age from the Bancoumana Community Study. Four 
months of DSFs per child were generated using a beta-binomial model with ISC=.14 and 
parameters chosen to achieve seasonal DSF positivity rates of 12-16% and seasonal vaccine 
activities (VAs) for the probability of a positive DSF of .50 or .60. A GEE model with compound 
level clustering and the number of positive DSFs as outcome was used for analysis. Ten 

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thousand clinical trials were simulated. Powers are provided in Table 17 and Table 18 which 
indicates we have power greater than .80 for scenarios with a seasonal DSF rate of 12-16%.  
 
Data collection for the Age DE study during the 2019 season was encouraging for achievement 
of the primary endpoint, positive Direct Skin Feeds.  As of March, there were 45 unique 
individuals with a positive DSF of the 369 participants monitored for this endpoint in the 9-
18 year old cohort.   
 
Our initial sample size for the power calculations for the VA in the Main Study was based on an 
assumption about seasonal infection rates ranging from 0.12-0.16 and power to reject a null 
hypothesis of no difference in rate of DSF positivity by arm is ultimately a function of the 
overall rate of DSF positivity and the true vaccine activity (Table 17). The current data indicates 
that the seasonal infection rate for the period monitored by DSF was 12% which was below the 
desired power threshold of 0.8.  
 
Table 17. Sample Size Power Calculations Based on Seasonal DSF Infection Rates and 
Seasonal Vaccine Activity 
 
Number of DSF+ Individuals Needed 
Power 
VA = 0.4 
VA = 0.5 
VA = 0.6 
0.8 
115 
64 
40 
0.9 
153 
87 
53 
 
As we remain blinded, we simulate power under three different scenarios for vaccine activity 
(Figure 32).  In the first scenario, we assume that vaccine activity is 0.4. In this simulation, 
115 unique individuals would need to experience a DSF+ in order to reject the null hypothesis of 
no difference at alpha= 0.5 with a type 1 error rate of 0.8.  In the scenario of 0.5 vaccine activity, 
64 unique individuals would need to experience a positive feed.  In the scenario with 0.6 VA, 40 
unique individuals would need to experience a positive feed to achieve 0.8 power. 
 

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Figure 32. Simulated power under assumptions of 0.4, 0.5, and 0.6 vaccine activity. 
These simulations would indicate that a second year of study is required in order to capture more 
of the seasonal infections, as the DSFs were monitored only from September – March.  To guard 
against scenarios in which the VA falls in the lower ranges, we intend to combine the endpoints 
from Year 1 and Year 2 to achieve the needed power to distinguish vaccine activity.  With 
additional follow up time, it appears feasible to have power for the all above scenarios.   
 
17.3.2.2 Vaccine Efficacy 
Vaccine efficacy will be measured by blood smear (BS) and detection of asexual Pf parasitemia. 
The background infection rate in the population is measured as the proportion of individuals 
from a recent PfSPZ trial (NIAID Protocol #16-I-N004) with any BS+ during follow up. Using 
this as the rate of background infection we can calculate power under various scenarios using as 
endpoint parasitemia positivity over the season. We use simple binomial tests with alpha =.05 
two-sided and ignore the anticipated negligible effects of compound level clustering. Sample 
sizes are multiplied by an inflation factor of two to adjust for potential compound level 
contamination of the vaccine effects due to mixing of mosquitoes or movement of vaccinees. We 
see that with a minimum of 350 total subjects 1-8 years of age that are randomized and do not 
drop out, we have power to detect VEs of 30% or greater within this age group alone based on 
the risk of first infection. We will analyze subjects 9-18 years of age based on the proportion of 
positive blood smears.   
 
 
 

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Table 18. Vaccine Efficacy Sample Size Power Calculations Based on Predicted Vaccine 
Efficacy 
Background 
Infection Rate 
VE 
Power 
Total 
Sample Size 
Total Sample Size per Arm 
After Adjustment 
for mosquito-human contamination 
70% 
20% 
80% 
375 
750 
70% 
25% 
80% 
242 
484 
70% 
30% 
80% 
172 
343 
70% 
35% 
80% 
128 
256 
70% 
40% 
80% 
98 
196 
17.3.2.3 Analysis 
The primary DSF analysis will use a GEE model with compound level clustering and the number 
of positive DSFs as outcome will be used for analysis with an offset for the number DSF tests. 
The primary parasitemia analysis will be analogous. The primary analysis population will follow 
the intention-to-treat principle. Additional analyses will explore the effects of different covariates 
such as time since start of the rainy season, SMC treatment, compound-level vaccine coverage, 
or proximity to a control compound on vaccine efficacy.  
 
The primary safety and immunogenicity analyses will compare the rates of AEs or levels of 
antibody/activity between vaccinated and control individuals using standard statistical methods 
that respect compound-based randomization, as appropriate.  
 
Randomization  
See Section 11.2 for more information on individual study schedule. 
 
This study will be cluster-randomized with family compounds, or groups of compounds (vaccine 
units) serving as the unit of comparison. A listing of the compounds in the study area with the 
numbers of individuals residing in their compounds will be created and used for randomization. 
The number of vaccine units will be the similar for each group. Enumerated compounds should 
have a target of approximately 80% participation rate of eligible participants for inclusion as a 
study unit, but consideration will be given to the number of children to finalize the decision for 
inclusion. Randomization for the full trial needs to be completed prior to the age de-escalation so 
that the randomization for vaccinated participants in the pilot phase study holds through the main 
phase. 
 

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18 Ethics/Protection of Human Subjects 
This research will be conducted in compliance with the protocol, GCP, and all applicable 
regulatory requirements. 
 
Institutional Review Board (IRB) and Ethics Committee (EC) 
A copy of the protocol, informed consent forms, and other study related information to be 
completed by subjects, such as questionnaires, diary cards, medical history forms, and any 
proposed advertising/recruitment materials or letters to the subjects will be submitted to the 
reviewing IRB and EC for written approval. The investigator must submit and obtain approval 
from the IRB/EC for all subsequent amendments to the protocol, informed consent documents, 
and other study documentation referenced above. The investigator will be responsible for 
obtaining IRB/EC approval of the annual continuing review throughout the duration of the study. 
The investigators will notify the reviewing IRB/EC of protocol violations and SAEs as specified 
in the relevant sections of the protocol. 
 
Informed Consent Process 
Informed consent is a process where information is presented to enable persons to voluntarily 
decide whether or not to participate as a research subject. It is an ongoing conversation between 
the human research subject and the researchers which begins before consent is given and 
continues until the end of the subject’s involvement in the research. Discussions about the 
research will provide essential information about the study and include purpose, duration, 
experimental procedures, alternatives, risks, and benefits. Subjects will be given the opportunity 
to ask questions and have them answered. 
 
Consent and assent forms will be approved by the participating IRB/EC. The subject’s 
parent/guardian will sign the informed consent document prior to undergoing any research 
procedures. Children aged 7-17 years old (unless emancipated per Malian law, then will sign 
consent) will sign the assent document prior to undergoing any research procedures. The subjects 
or their parents/guardians may withdraw assent or consent at any time throughout the course of 
the trial. The informed consent process will be documented in the subject’s research chart, as 
required by 21 CFR 312.62. The informed consent form will be signed (or fingerprinted) and 
personally dated by the subject’s parent/guardian or the subject and the person who conducted 
the informed consent discussion. The original signed informed consent form, and if applicable 
the assent, will be retained in the subject’s chart and a signed and dated copy will be provided to 
the subject and/or their parent/guardian. The rights and welfare of the subjects will be protected 
by emphasizing to them that the quality of their medical care will not be adversely affected if 
they decline to participate in this study. 

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Mali Site Community Permission and Individual Informed Consent Process 
18.2.1.1 Community Permission 
Community permission will be obtained from village elders, family heads, and other community 
members after explanation and discussion of the study. 83 The community permission process 
goes through the following steps: 
• Study investigators/personnel explain the study to village leaders, including the village 
chief, family heads, women association, and elders. 
• The village leaders then discuss the study with family heads and community members 
and relay any additional questions or concerns they may have to study personnel. 
• The study and the informed consent process are explained in detail to heads of families 
by study investigators/personnel. 
 
At the time of community permission, the need for both husband and wife to agree to avoid 
pregnancy for the specified period if a wife chooses to volunteer will also be addressed. 
 
The individual informed consent process and forms will be translated into French. The study 
team conducts careful word-for-word review of the study consent form, and will translate the 
consent orally into local languages, as the majority of potential study subjects do not read or 
speak French. Verification that the oral translations are accurate and that the potential subjects 
understand the contents of the informed consent form will be done by an independent witness 
who is not a member of the study team. An evaluation checklist is performed to make sure that 
the study is understood by the subjects before enrollment. 
18.2.1.2 Individual Informed Consent 
Local households and families will be invited to come to the study clinic for review of the 
informed consent. Potential subjects will be included along with their parent(s)/legal guardian(s) 
in discussions about the study. Written assent will be obtained if the child is at least 7 years old 
and has the maturity and psychological ability to give assent, as judged by the study member 
obtaining consent. Two parents/legal guardians will provide permission for the minor subject to 
participate by signing/fingerprinting the consent form.  
 
At the consenting visit, the potential subject and their parent/guardian will read the consent or 
assent form, or have it explained in cases of illiteracy. Individuals in each family will be 
separately consented and not all individuals from a household need to participate. 
 
Subjects and/or subjects’ parents will be encouraged to ask questions and then will take a 
multiple-choice questionnaire (true/false; Malaria Comprehension Exam) to evaluate consent 
comprehension. All incorrect responses will be reviewed with the subject, and he or she must 

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verbalize understanding of all incorrect responses. A score of ≥80% correct is required for 
enrollment. For subjects scoring less than 80%, study staff may choose to review study details 
again with subject and reassess comprehension with a repeat Malaria Comprehension Exam. At 
the discretion of the investigator, any subject whose comprehension is questionable, regardless of 
score, may be excluded from enrollment.  
 
The Malaria Comprehension Exam will be translated into French and administered orally in the 
native dialect in the case of potential subjects who cannot read. Study staff will use incorrect 
answers from the questionnaire to identify those areas of the informed consent that need further 
review with subject. This will help ensure that the subject has sufficient understanding before the 
consent form is signed. The subject may either sign the consent form immediately or later after 
further consideration. Subjects unable to read will place a fingerprint in the place of a signature. 
In addition, an independent witness will sign the consent form to attest that the consent was fully 
explained, and all questions were answered.  
 
Informed Consent Process for Minors Who Reach the Age of Consent 
Legally effective informed consent will be obtained from subjects who initially enrolled on the 
study as a minor and remain on study when they reach the age of consent, even if the research 
does not involve any ongoing interactions or interventions with the subject but continues to meet 
the regulatory definition of “human subjects research” (e.g., it involves the continued analysis of 
identifiable specimens or data). Subjects and their families will be told of this requirement at the 
time of initial enrollment. We will make a reasonable attempt to reach subjects who have 
completed study participation or are lost to follow-up; however, if we are unable to contact the 
subject for reconsent, we request that the requirements for obtaining informed consent are 
waived, provided the IRB/EC agrees that the following conditions are met: 
1. 
The research involves no more than minimal risk to the subjects. 
o The research samples and data will have already been collected and thus 
the ongoing analysis will not require additional contact with the former 
subject. 
2. 
The waiver will not adversely affect the rights and welfare of the subjects. 
o This study focuses on a better understanding of malaria, vaccination, and 
immunology. Participation in this study should in no way affect the rights 
and welfare of subjects. 
3. 
The research could not be practicably carried out without the waiver. 
o Ongoing analysis of the samples and data collected in this study is needed 
to address the study objectives. Thus, research would be hindered if 
analysis were halted. 
4. 
Whenever appropriate, the subjects must be provided with additional pertinent 
information after participation. 

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o If former subjects are recontacted in the future, any new results obtained 
since the last contact will be provided to them. 
 
Informed Consent Process for Emancipated Minors 
A legally married minor female 16 years old and above is considered emancipated according to 
Malian law. Therefore, married female minors 16 years old and above will be able to provide 
legally effective informed consent to volunteer in this study.  
 
Subject Confidentiality 
All records will be kept confidential to the extent provided by federal, state, and local law. The 
study monitors and other authorized representatives of the sponsor may inspect all documents 
and records required to be maintained by the investigator, including but not limited to, medical 
records. Records will be kept locked and data will be coded. Any personally identifiable 
information maintained for this study will be kept on restricted-access computers and networks. 
Personally identifiable information will only be shared with individuals authorized to receive it 
under this protocol. Individuals not authorized to receive personally identifiable information will 
be provided with coded information only, as needed. Clinical information will not be released 
without written permission of the subject, except as necessary for monitoring by NIH IRB, 
FMPOS EC, the FDA, NIAID, OHRP, or the sponsor’s designee.  
 
Potential Risks 
Risks to the subjects are associated with vaccination and venipuncture. These risks are outlined 
below: 
 
Study Vaccine 
18.4.1.1 Intramuscular (IM) Vaccinations 
Possible local vaccine reactions resulting from IM injection include pain, swelling, erythema, 
induration, limitation of limb movement for several days, lymphadenopathy, or pruritus at the 
injection site. Systemic reactions such as fever, chills, headache, fatigue, malaise, myalgia, and 
joint pain may also occur, and may range from mild to severe. These side effects will be 
monitored but are generally mild and self-limiting. 
18.4.1.2 Immunization with Pfs230D1M-EPA/AS01 
18.4.1.3 Pfs230D1M 
Common solicited AEs during past immunization with Pfs230D1M-EPA/Alhydrogel and 
Pfs230D1M-EPA/AS01 include injection site pain, redness, induration, and swelling. Other 
commonly reported solicited AEs include headache, malaise, diarrhea, nausea, and fever. 
 

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While several subjects in the US and the Mali studies experienced changes in hematologic 
parameters (such as anemia, leukopenia, and neutropenia), some even moderate (Grade 2) in 
severity, an examination of trends in the US study and the unblinded portion of the Mali study 
(Group 1) has not shown any pattern deemed related to vaccination. In addition, no neutropenia 
events were associated with fever and/or subsequent acute infection attributable to the drop in 
neutrophil counts. 
 
A single subject in Protocol #15-I-0044, a healthy 51-year-old woman, presented 6 days 
following receipt of her fourth study vaccination with an acute onset of hemiplegia. She was 
diagnosed as having a cerebrovascular accident via computerized tomography (CT) scan and 
neurological assessment at the hospital in Bamako, Mali and was hospitalized. Her symptoms 
worsened overnight, and she died the following day. This SAE was reviewed at length by the 
study team, ISM, sponsor medical monitor, NIH IRB, FMPOS EC, and the study’s DSMB and 
was determined unrelated to vaccination. In addition, the SAE was submitted to the FDA for 
review as an informational item. At this time, we do not believe that arterial or venous occlusion 
are possible risk factors associated with vaccination, but we will provide as much available 
information to future study subjects as possible.  
18.4.1.4 AS01 
Overall, with the exception of local reactogenicity (pain, swelling, erythema, tenderness at the 
site of injection), and systemic symptoms such as low-grade fever and short-term flu-like 
symptoms (fatigue, myalgia, headache, malaise), MPL-based adjuvants (AS01) have been safe 
and well tolerated. 
18.4.1.5 EPA 
EPA has been studied in both malaria transmission vaccine studies (as noted with Pfs230D1M) 
and other vaccination studies. 11-13,84,85 The use of EPA has identified no safety issues to date. 
 
As with any vaccine, immediate hypersensitivity reactions including urticaria, anaphylaxis, or 
other IgE-mediated responses are possible. There is a theoretical possibility of risks about which 
we have no present knowledge. Subjects will be informed of any such risks should further data 
become available. 
18.4.1.6 Other Risks of the Study Vaccine 
The study vaccine can cause non-specific inflammation and may be harmful while pregnant 
(Section 9.8). Women of reproductive potential will be required to agree to use birth control as 
outlined in Section 9.4. Because this is a research study, women of reproductive potential will be 
asked to notify the site immediately upon learning of pregnancy during this study and will be 
tested for pregnancy prior to administration of the vaccine and periodically throughout the course 
of the study as outlined in Appendix A. 

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18.4.1.7 Comparator vaccine 
18.4.1.7.1 HAVRIX 
In studies of adults and children 2 years of age and older, the most common solicited adverse 
events were injection-site soreness (56% of adults and 21% of children) and headache (14% of 
adults and less than 9% of children). Syncope (fainting) can occur in association with 
administration of injectable vaccines. 
 
Data are limited regarding use of the hepatitis A vaccine during breast feeding and its excretion 
in human milk is unknown. The manufacturer recommends caution when administering to 
nursing mothers; however according to the ACIP, inactivated vaccines pose no risk to breast-
feeding mothers or their infants and is not contraindicated for administration. 
 
For complete safety details, please refer to the package insert provided. If for some unexplained 
reason, HAVRIX is not available, another Hepatitis A inactivated vaccine approved, either by 
FDA and/or WHO, in all enrolled age groups will be used instead. The package insert for that 
particular Hepatitis A vaccine will be submitted to NIH IRB/FMPOS EC, FDA, and Sponsor 
prior to use. 
18.4.1.7.2 TYPHIM Vi 
TYPHIM Vi can commonly (≥10%) cause fevers, malaise, headache, myalgias and local 
injection site reactions, in particular during the first 48 hours post vaccination. Less commonly 
(1-10%), it can cause additional systemic symptoms, including abdominal pain, nausea, 
vomiting, diarrhea, rash, pruritus.  
 
Data are limited regarding use of the TYPHIM Vi and/or inactivated, subunit vaccines in general 
during breast feeding and its excretion in human milk is unknown. The manufacturer 
recommends caution when administering to nursing mothers; however, according to the ACIP, 
inactivated vaccines pose no risk to breast-feeding mothers or their infants and are not 
contraindicated for administration. 
 
For complete safety details, please refer to the package insert provided. If for some unexplained 
reason, TYPHIM Vi is not available, another typhoid inactivated vaccine approved, either by 
FDA and/or WHO, in all enrolled age groups will be used instead. The package insert for that 
particular typhoid vaccine will be submitted to NIH IRB/FMPOS EC, FDA, and Sponsor prior to 
use. 
18.4.1.7.3 Menactra® 
For Menactra® the most common (≥10%) solicited adverse events include injection site pain, 
redness, induration, and swelling; anorexia and diarrhea.  Other common reported solicited 

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adverse events in adults includes headache, fatigue, malaise, and arthralgia.   Brief fainting spells 
and related symptoms have been reported following vaccination, most often in adolescents, that 
may result in falls and/or injuries.   
 
Data are limited regarding use of the Menactra® and/or inactivated, subunit vaccines in general 
during breast feeding and its excretion in human milk is unknown. The manufacturer 
recommends caution when administering to nursing mothers; however, according to the ACIP, 
inactivated vaccines pose no risk to breast-feeding mothers or their infants and are not 
contraindicated for administration. 
 
For complete safety details, please refer to the package insert provided. If for some unexplained 
reason, Menactra® is not available, another meningococcal vaccine approved, either by FDA 
and/or WHO, in all enrolled age groups will be used instead. The package insert for that 
particular meningococcal vaccine will be submitted to NIH IRB/FMPOS EC, FDA, and Sponsor 
prior to use. 
 
 
Artemether/Lumefantrine (AL) 
All enrolled subjects will receive either one or two treatment courses with AL (e.g. Coartem) 
during the study regardless of their malaria status. A treatment course of AL for all ages is daily 
administration of AL over 3 consecutive days. AL is a registered, licensed, proven, and highly 
efficacious oral treatment. AL will be provided by the study team or designee for directly 
observed treatment by all study participants. This may occur outside of the clinical research site, 
but dosing and timing of dosing will be documented by a trained study team member. 
 
AL has an acceptable safety profile. Individuals who may have any contraindication for the use 
of this drug (e.g. prolonged QTc or taking other medications that can prolong QTc, history of 
myocardial infarction) will be excluded at screening. The most common side effects (i.e., >30%) 
are: headache, anorexia, dizziness, asthenia, arthralgia, and myalgia. Discontinuation of AL due 
to AE is rare (0.2%). Rare but serious hypersensitivity reactions (urticarial and angioedema) and 
skin reactions (bullous eruption) have been reported post marketing. AL should be taken with 
milk or another fatty food. 
 
AL is a Category C pregnancy drug. Thus, all female participants will undergo pregnancy testing 
prior to receipt of the investigational dose of AL.  
 
For complete safety details, please refer to the package insert provided for AL. 

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Venipuncture 
Risks occasionally associated with venipuncture include pain, bruising, bleeding, and infection at 
the site of venipuncture, lightheadedness, and rarely, syncope. 
 
Direct Skin Feeds 
Risks associated with mosquito bites include mild to moderate swelling and itching at the site. 
Participants will be offered symptomatic treatment (i.e., local application of antipruritic agent) if 
these occur. While malaria and filarial parasites are transmitted through Anopheline mosquito 
bites as part of the parasite life cycle, mosquitoes raised in an insectary that are used for DSFs 
have never been exposed to human blood (breeding stocks are maintained on human blood as 
noted in Section 12.6.5) and since these parasites are not transovarially (vertically) transmitted in 
mosquitoes, there is no risk of transmission to participants. O’nyong’nyong virus is transmitted 
to humans by anopheles mosquitoes, but is not vertically transmitted so does not pose a risk of 
infection to subjects or the mosquito colony. Anopheline mosquitoes can rarely transmit RVF 
virus, and RVF has been reported to transovarially infect anopheles.86 RVF is primarily 
transmitted to humans by direct contact with infected animals, although transmission by 
mosquitoes can occur, with Aedes mosquitoes being the primary vector. The incubation period is 
2-6 days.87 No outbreaks of RVF have been reported in Mali, and transovarial infection is rare 
and extremely unlikely to persist for generations (for example, in a lab colony maintained at 
MRTC or elsewhere). To document the lack of viral contamination, the lab strain and field 
caught mosquitoes were tested for RVF virus using molecular methods. Testing conducted over 
the last 2 years in Mali on LMIV/MRTC clinical trials have all been negative for RVF virus. 
Because routine tests for this virus are not feasible, we installed a procedure to ensure that blood 
meals used to rear the mosquitoes are free of the virus (see Section 12.6.5). 
 
This study involves direct feeds with Anopheles mosquitoes only. Aedes and Culex mosquitoes 
transmit viral diseases such as dengue, West Nile, and yellow fever, which may be vertically 
transmitted in mosquitoes; however, no Aedes and Culex mosquitoes are raised in the MRTC 
insectary, thus transmission of these viral agents to subjects is not possible through feeds 
conducted as part of this study. As previously described (see Section 4 and 12.6.4), hundreds of 
subjects in Bancoumana or neighboring areas have been enrolled in DSFs. Except for 1 case of 
definitely related Grade 2 erythema that was resolved within 48 hours and 5 cases of definitely 
related local site pruritus at the DSF site (in the same individual), there have been no other 
expected or unexpected AEs recorded as related (definitely, probably, possibly) to the feeding 
procedures. 
 
Other Risks 
Women of reproductive potential will be required to agree to use birth control as outlined in 
Section 9.4. Because this is a research study, women of reproductive potential will be asked to 
notify the site immediately upon learning of pregnancy during this study and will be tested for 

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pregnancy prior to administration of the vaccine(s) and periodically throughout the course of the 
study as outlined in Appendix A.  
 
 
Potential Benefits 
Given the family compound design of the study, broad family compound/vaccine unit coverage 
(approximately 80%), and supporting evidence that malaria transmission is local, there is the 
potential benefit to all enrolled in regard to decreased malaria infection incidence where they 
live, including their own risk of future infection in the same transmission season.  
 
Compensation 
Subjects (adults or parents) will be given in kind (such as rice and/or millet) or cash equivalent, 
in multiple installments as outlined in Table 19 and Table 20, to compensate for the time taken 
to come to the study clinic for study-related visits. Preferred compensation is in kind, such as rice 
and/or millet, rather than cash, which had been decided in consultation with village elders, but 
case-by-case exceptions to receive the cash equivalent have been considered acceptable.  
 
The FMPOS EC recommends compensating the study subject for their time lost for study 
procedures. The amount equivalent to $6 in United States dollars (USD) for each scheduled visit 
with laboratory procedures/feeding procedures and equivalent to USD $3 for each scheduled 
visit without laboratory procedures. 
 
 

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Table 19: Estimated Compensation Schedule (Year 1)1 
Study Activity 
Number of Visits 
US Dollar 
Local Currency (CFA)2 
1-4 years old 
Screening 
1 
$3 
1500 
Study Visits 
17 
$102 
51000 
Extra 9, 12 month 
visits4 
1 
$6 
3000 
Total 
19 
$111 
55500 
5-8 years old 
Screening 
1 
$6 
3000 
Vaccination #1 
6 
$30 
15000 
Vaccination #2 
5 
$24 
12000 
Vaccination #3 
16 
$84 
42000 
Extra Pilot Arm 
Visits 
3 
$12 
6500 
Extra 9, 12 month 
visit4 
1 
$6 
3000 
Total 
32/29 
$162/$150 
81000/75000 
9-18 years old 
Screening 
1 
$6 
3000 
Vaccination #1 
6 
$30 
15000 
Vaccination #2 
5 
$24 
12000 
Vaccination #3 
16 
$93 
46500 
Extra Pilot Arm 
Visits 
3 
$12 
6500 
Extra 9, 12 month 
visit4 
1 
$6 
3000 
Total 
32/29 
$171/$159 
85500/79500 
19 years of age and older 
Screening 
1 
$6 
3000 
Vaccination #1 
6 
$30 
15000 
Vaccination #2 
5 
$24 
12000 
Vaccination #3 
11 
$54 
27000 
Extra 9, 12 month 
visit4 
1 
$6 
3000 
Total 
24 
$120 
60000 
Unscheduled visits (all ages) 
Unscheduled visits3 
TBD 
$3-6 
1500-3000 
GPS Participation 
Per month 
6 
$36 
18000 

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1 Compensation installments may be paid out in 3-6 installments at the clinic’s discretion to the subjects 
during these specified time periods. 
2 Assuming currency exchange rate of USD $1 = 500 CFA 
3 Compensation for unscheduled visits will be dependent if blood draw is indicated for research purposes 
4 12 month follow-up visit = rescreening visit, so compensation included on Year 2 schedule now. 
 
Table 20: Estimated Compensation Schedule (Year 2)1 
Study Activity 
Number of Visits 
US Dollar 
Local Currency (CFA)2 
1-4 years old (Arms 4a/4b) 
Rescreening 
1 
$3 
1500 
Study Visits 
18 
$108 
54000 
Total 
19 
$111 
55500 
5-8 years old (Arms 2a/2b, 3a/3b) 
Rescreening 
1 
$6 
3000 
Study Visits 
18 
$108 
54000 
Total 
19 
$114 
57000 
9-18 years old (Arms 1a/1b, 3c/3d) 
Rescreening 
1 
$6 
3000 
Study Visits 
17 
$102 
51000 
Total 
18 
$108 
54000 
19 years of age and older (Arms 3e/3f) 
Rescreening 
1 
$6 
3000 
Study Visits 
10 
$57 
28500 
Total 
11 
$63 
31500 
Unscheduled visits (all ages) 
Unscheduled visits3 
TBD 
$3-6 
1500-3000 
GPS Participation 
Per month 
5 
$30 
15000 
1 Compensation installments may be paid out in 3-6 installments at the clinic’s discretion to the subjects 
during these specified time periods. 
2 Assuming currency exchange rate of USD $1 = 500 CFA 
3 Compensation for unscheduled visits will be dependent if blood draw is indicated for research purposes 
 
 
19 Data Handling and Record Keeping 
 
Data Capture and Management  
In Mali, study data will be entered directly into a study-specific DataFax electronic database. 
Data from electronic CRFs will be collected directly from subjects during study visits and 
telephone calls or will be abstracted from subjects’ medical records. Electronic CRFs and 
supporting laboratory, entomology documentation will be used as source. Any type of 

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corrections to the electronics CRFs will be documented and tracked. All CRFs should be 
reviewed by the Investigator and signed as required with written signature. 
 
Data entry will be performed by authorized individuals. Corrections to the electronic data 
systems will be tracked electronically (password protected or through an audit trail) with time, 
date, individual making the correction, and what was changed. The investigator is responsible for 
assuring that the data collected are complete, accurate, and recorded in a timely manner. NIH 
researchers will have access to personally identifiable information.  
 
Record Retention 
The investigator is responsible for retaining all essential documents listed in the ICH GCP 
guidelines. Study records will be maintained by the PI according to the timelines specified in 21 
CFR 312.62 or a minimum of 5 to 7 years, and in compliance with institutional, IRB/EC, state, 
and federal medical records retention requirements, whichever is longest. All stored records will 
be kept confidential to the extent required by federal, state, and local law. 
 
Should the investigator wish to assign the study records to another party and/or move them to 
another location, the investigator will provide written notification of such intent to 
OCRPRO/NIAID and the FMPOS EC with the name of the person who will accept responsibility 
for the transferred records and/or their new location. NIAID and the FMPOS EC will be notified 
in writing and written OCRPRO/NIAID and FMPOS EC permission shall be obtained by the site 
prior to destruction or relocation of research records. 
 Protocol Revisions 
No revisions to this protocol will be permitted without documented approval from the NIH 
IRB/FMPOS EC that granted the original approval for the study. Any change to the protocol will 
be submitted to the Sponsor and to the participating IRB/EC as a protocol amendment; changes 
not affecting risk to subjects may request an expedited review. In the event of a medical 
emergency, the Investigator shall perform any medical procedures that are deemed medically 
appropriate and will notify the IND sponsor of all such occurrences.

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Appendix A: Schedule of Procedures/Evaluations 
Year 1: Arms 1a/1b (9-18 years of age; PILOT): 
 
 
 
 
 
 
 
Months
0
1
2
3
4
5
6
7
8
9
10
13
Visits
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Procedures
Blood Volume
Study Day
-7
0
1
3
7
14
28
29
31
35
42
56
84
112
126
127
129
133
140
154
168
182
196
210
224
238
252
266
280
294
378
Days post Vac 
-7
0
1
3
7
14
0
1
3
7
14
28
56
84
0
1
3
7
14
28
42
56
70
84
98
112
126
140
154
168
252
Visit Windows (days) (1)
±7
0
±1
±2
±3
±7
0
±1
±2
±3
±7
±14
±28
±56
0
±1
±2
±7
±7
±7
±7
±7
±7
±7
±7
±7
±7
±7
±28
±56
Complete medical history/ physical
X
Informed consent
X
EKG
X
Pre-test/Post-test HIV counseling
X
Interim clinical evaluation
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
AE/SAE assessment
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Pregnancy Prevention
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Conmed review
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
VACCINATION
X
X
X
Artemether/Lumefantrine
X
Unblinding
Feeding Evaluation (1)
X
X
X
X
X
X
X
X
CBC with differential
EDTA
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
ALT/ Creatinine
SST
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
HIV testing
SST
5.0
Urine dipstick or Urinalysis
Urine Container
X
Hemoglobin Typing
EDTA
X
Urine/Serum pregnancy test 
(females only)
Urine Container or SST
X
X
X
X
X
X
X
X
X
Schistosomiasis +/- Helminth 
Testing
Urine +/- Stool Sample
Malaria Blood Smear
CAP/EDTA
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Malaria PCR/Genotyping/ 
Gametocyte
CAP/EDTA
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1
1
1
1
1
1
1
1
1.0
1.0
1.0
1.0
1.0
 Pfs230 ELISA
SMFA (2)
DSF (3)
X
X
X
X
X
X
X
X
X
Transcriptional analysis
PAXGene
1
1
1
1
1
1
1
10.0
7.5
17.5
0.0
6.0
0.0
11.5
11.5
0.0
6.0
0.0
11.5
0.0
0.0
11.5
11.5
0.0
6.0
5.0
17.5
1.5
1.5
6.5
1.5
1.5
1.5
6.5
1.5
1.5
1.5
7.5
6.5
10.0
17.5
35.0
35.0
41.0
41.0
52.5
64.0
64.0
70.0
70.0
81.5
81.5
81.5
93.0
104.5
104.5
110.5
115.5
133.0
134.5
136.0
142.5
144.0
145.5
147.0
153.5
155.0
156.5
158.0
165.5
172.0
Arms 1a/1b (9-18 year olds)
Screening      
(-56)
X
SST
5
10
5
Daily blood draw volume in mL
Cumulative blood volume in mL
Clinical Procedures
Laboratory Procedures
5
5
5
5
5
2 SMFA may be completed on subjects at Study Day 0 and 182; other SMFA time points indicated above will be completed depending on associated ELISA and DSF results at that time point.
3 DSF will be completed twice monthly post Vaccination #3 through day 112 post Vaccination #3 (total = 8 feeds).   
5
5
5
10
1 Visit windows are based off timing of days post the preceding vaccination

Pfs230D1M Age De-escalation and Family Compound Trial 
Protocol Version 7.0 
September 24, 2021 
Page 161 of 223 
 
 
Year 1: Arms 3c/3d (9-18 years of age, MAIN): 
 
 
 
 
  
Months
0
1
2
3
4
5
6
7
8
11
Visits
1
2
3
4
5
6
7
8
9
10
11 (4)
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Procedures
Blood Volume
Study Day
-7
0
1
3
7
14
28
29
31
35
42
56
57
59
63
70
84
98
112
126
140
154
168
182
196
210
224
308
Days post Vac 
-7
0
1
3
7
14
0
1
3
7
14
0
1
3
7
14
28
42
56
70
84
98
112
126
140
154
168
252
Visit Windows (days) (1)
±7
0
±1
±2
±3
±7
0
±1
±2
±28
±56
0
±1
±2
±7
±7
±7
±7
±7
±7
±7
±7
±7
±7
±7
±28
±56
Complete medical history/ physical
X
Informed consent
X
EKG
X
Pre-test/Post-test HIV counseling
X
Interim clinical evaluation
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
AE/SAE assessment
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Pregnancy Prevention
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Conmed review
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
VACCINATION
X
X
X
Artemether/Lumefantrine
X
Unblinding
Feeding Evaluation (1)
X
X
X
X
X
X
X
X
CBC with differential
EDTA
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
ALT/ Creatinine
SST
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
HIV testing
SST
5.0
Urine dipstick or Urinalysis
Urine Container
X
Hemoglobin Typing
EDTA
X
Urine/Serum pregnancy test 
(females only)
Urine Container or SST
X
X
X
X
X
X
X
X
X
Schistosomiasis +/- Helminth 
Testing
Urine +/- Stool Sample
Malaria Blood Smear
CAP/EDTA
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Malaria PCR/Genotyping/ 
Gametocyte
CAP/EDTA
1.0
1.0
1.0
1.0
1.0
1.0
1
1
1
1
1
1
1
1
1.0
1.0
1.0
1.0
1.0
 Pfs230 ELISA
SMFA (2)
DSF (3)
X
X
X
X
X
X
X
X
X
Transcriptional analysis
PAXGene
1
1
1
1
1
1
1
10.0
7.5
17.5
0.0
6.0
0.0
11.5
11.5
0.0
6.0
0.0
11.5
11.5
0.0
6.0
5.0
17.5
1.5
1.5
6.5
1.5
1.5
1.5
6.5
1.5
1.5
1.5
7.5
6.5
10.0
17.5
35.0
35.0
41.0
41.0
52.5
64.0
64.0
70.0
70.0
81.5
93.0
93.0
99.0
104.0
121.5
123.0
124.5
131.0
132.5
134.0
135.5
142.0
143.5
145.0
146.5
154.0
160.5
5
Clinical Procedures
Laboratory Procedures
Arms 3c/3d (9-18 year olds)
5
X
5
5
5
Cumulative blood volume in mL
5
Screening      
(-56)
SST
10
5
1 Visit windows are based off timing of days post the preceding vaccination
2 SMFA may be completed on subjects at Study Day 0 and 182; other SMFA time points indicated above will be completed depending on associated ELISA and DSF results at that time point.
4 Note this visit may occur twice depending on the approval of the V3.0 protocol; the first D42 visit (D42A) will include all D42 procedures EXCEPT AL dosing. This will visit will occur to maintain safety labs and assessments are completed on ALL subjects approximately 14 days post Vaccination #2 (±3 days). If this visit occurs, the next visit will be D42B for AL dosing and completed of ALL 
procedures as outlined in the visit (if D42A completed >14 days from D42B). This is to ensure the AL dosing in participants occurs approximately 14 days prior to Vaccination #3. If both visits are completed, subjects will receive an additional 3000 CFA ($6 USD). 
3 DSF will be completed twice monthly post Vaccination #3 through day 112 post Vaccination #3 (total = 8 feeds).   
5
10
5
Daily blood draw volume in mL

Pfs230D1M Age De-escalation and Family Compound Trial 
Protocol Version 7.0 
September 24, 2021 
Page 162 of 223 
Year 1: Arms 2a/2b (5-8 years of age; PILOT):  
 
 
 
 
 
 
 
 
 
 
 
Months
0
1
2
3
4
5
6
7
8
9
10
13
Visits
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Procedures
Study Day
-7
0
1
3
7
14
28
29
31
35
42
56
84
112
126
127
129
133
140
154
168
182
196
210
224
238
252
266
280
294
378
Days Post Vac
-7
0
1
3
7
14
0
1
3
7
14
28
56
84
0
1
3
7
14
28
42
56
70
84
98
112
126
140
154
168
252
Visit Windows (days) 
(1)
±7
0
±1
±2
±3
±7
0
±1
±2
±3
±7
±14
±28
±56
0
±1
±2
±7
±7
±7
±7
±7
±7
±7
±7
±7
±7
±7
±28
±56
Complete medical history/ physical
X
Informed consent
X
EKG
X
Pre-test/Post-test HIV counseling
X
Interim clinical evaluation
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
AE/SAE assessment
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Conmed review
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Artemether/Lumefantrine
X
X
VACCINATION
X
X
X
Unblinding
CBC with differential
EDTA
2
2
2
2
2
2
2
2
2
2
2
2
ALT/ Creatinine
SST
3
3
3
3
3
3
3
3
3
3
3
3
HIV testing
SST
5
Urine dipstick or Urinalysis
Urine Container
X
Hemoglobin Typing (2)
EDTA
(X)
Schistomiasis testing
Urine Container
Malaria Blood Smear
CAP/EDTA
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Malaria PCR/Genotyping/ Gametocyte
CAP/EDTA
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Pfs230 ELISA
SMFA (3)
Transcriptional Analysis
PaxGene
1
1
1
1
1
1
10
7.5
17.5
0
6
0
11.5
11.5
0
6
0
11.5
0
0
11.5
11.5
0
6
0
11.5
1.5
1.5
6.5
1.5
1.5
1.5
6.5
1.5
1.5
1.5
7.5
6.5
10
17.5
35
35
41
41
52.5
64
64
70
70
81.5
81.5
81.5
93
104.5
104.5
110.5
110.5
122
123.5
125
131.5
133
134.5
136
142.5
144
145.5
147
154.5
161
Cumulative blood volume in mL
2 Hemoglobin typing, if not completed at screening, given completed retrospectively, can be completed at any time when there's a scheduled blood draw with an EDTA tube
3 SMFA may be completed on subjects at Study Day 0 and 182; other SMFA time points indicated above will be completed depending on associated ELISA results at that time point.
5
5
5
5
Arms 2a/2b (5-8 year olds)
Screening      (-
56)
Blood Volume
X
SST
10
5
5
Laboratory Procedures
Clinical Procedures
1 Visit windows are based off timing of days post the preceding vaccination
5
5
5
5
Daily blood draw volume in mL

Pfs230D1M Age De-escalation and Family Compound Trial 
Protocol Version 7.0 
September 24, 2021 
Page 163 of 223 
Year 1: Arms 3a/3b (5-8 years of age; MAIN):  
 
  
 
Months
0
1
2
3
4
5
6
7
8
11
Visits
1
2
3
4
5
6
7
8
9
10
11 (4)
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Procedures
Study Day
-7
0
1
3
7
14
28
29
31
35
42
56
57
59
63
70
84
98
112
126
140
154
168
182
196
210
224
308
Days Post Vac
-7
0
1
3
7
14
0
1
3
7
14
0
1
3
7
14
28
42
56
70
84
98
112
126
140
154
168
252
Visit Windows (days) 
(1)
±7
0
±1
±2
±3
±7
0
±1
±2
±28
±56
0
±1
±2
±7
±7
±7
±7
±7
±7
±7
±7
±7
±7
±7
±28
±56
Complete medical history/ physical
X
Informed consent
X
EKG
X
Pre-test/Post-test HIV counseling
X
Interim clinical evaluation
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
AE/SAE assessment
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Conmed review
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Artemether/Lumefantrine
X
X (4)
VACCINATION
X
X
X
Unblinding
CBC with differential
EDTA
2
2
2
2
2
2
2
2
2
2
2
ALT/ Creatinine
SST
3
3
3
3
3
3
3
3
3
3
3
HIV testing
SST
5
Urine dipstick or Urinalysis
Urine Container
X
Hemoglobin Typing (2)
EDTA
(X)
Schistomiasis testing
Urine Container
Malaria Blood Smear
CAP/EDTA
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Malaria PCR/Genotyping/ Gametocyte
CAP/EDTA
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Pfs230 ELISA
SMFA (3)
Transcriptional Analysis
PaxGene
1
1
1
1
1
1
10
7.5
17.5
0
6
0
11.5
11.5
0
6
0
11.5
11.5
0
6
0
11.5
1.5
1.5
6.5
1.5
1.5
1.5
6.5
1.5
1.5
1.5
7.5
6.5
10
17.5
35
35
41
41
52.5
64
64
70
70
81.5
93
93
99
99
110.5
112
113.5
120
121.5
123
124.5
131
132.5
134
135.5
143
149.5
Arms 3a/3b (5-8 year olds)
Screening      (-
56)
Blood Volume
Clinical Procedures
Laboratory Procedures
5
X
SST
10
Cumulative blood volume in mL
5
5
5
5
5
2 Hemoglobin typing, if not completed at screening, given completed retrospectively, can be completed at any time when there's a scheduled blood draw with an EDTA tube
3 SMFA may be completed on subjects at Study Day 0 and 182; other SMFA time points indicated above will be completed depending on associated ELISA results at that time point.
4 Note this visit may occur twice depending on the approval of the V3.0 protocol; the first D42 visit (D42A) will include all D42 procedures EXCEPT AL dosing. This will visit will occur to maintain safety labs and assessments are completed on ALL subjects approximately 14 days post Vaccination #2 (±3 days). If this visit occurs, the next visit will be D42B for AL dosing and completed of ALL procedures as outlined in 
the visit (if D42A completed >14 days from D42B). This is to ensure the AL dosing in participants occurs approximately 14 days prior to Vaccination #3. If both visits are completed, subjects will receive an additional 3000 CFA ($6 USD). 
5
Daily blood draw volume in mL
5
5
1 Visit windows are based off timing of days post the preceding vaccination

Pfs230D1M Age De-escalation and Family Compound Trial 
Protocol Version 7.0 
September 24, 2021 
Page 164 of 223 
Year 1: Arms 3e/3f (Adults 19 years of age and older): 
  
 
 
Months
0
1
2
3
4
5
6
7
8
11
Visits
1
2
3
4
5
6
7
8
9
10
11 (3)
12
13
14
15
16
17
18
19
20
21
22
23
Procedures
Blood Volume
Study Day
-7
0
1
3
7
14
28
29
31
35
42
56
57
59
63
70
84
112
140
168
196
224
308
Days post Vac 
-7
0
1
3
7
14
0
1
3
7
14
0
1
3
7
14
28
56
84
112
140
168
252
Visit Windows (days) (1)
±7
0
±1
±2
±3
±7
0
±1
±2
±28
±56
0
±1
±2
±7
±7
±7
±7
±7
±7
±28
±56
Complete medical history/ physical
X
Informed consent
X
EKG
X
Pre-test/Post-test HIV counseling
X
Interim clinical evaluation
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
AE/SAE assessment
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Pregnancy Prevention
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Conmed review
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
VACCINATION
X
X
X
Artemether/Lumefantrine
X
Unblinding
CBC with differential
EDTA
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
ALT/ Creatinine
SST
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
HIV testing
SST
5.0
Urine dipstick or Urinalysis
Urine Container
X
Hemoglobin Typing
EDTA
X
Urine/Serum pregnancy test 
(females only)
Urine Container or SST
X
X
X
X
X
Malaria Blood Smear
CAP/EDTA
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Malaria PCR/Genotyping/ 
Gametocyte
CAP/EDTA
1.0
1.0
1.0
1.0
1.0
1.0
1
1
1
1.0
1.0
Pfs230 ELISA
SMFA (2)
Transcriptional analysis
PAXGene
1
1
1
1
1
1
1
10.0
7.5
17.5
0.0
6.0
0.0
11.5
11.5
0.0
6.0
0.0
11.5
11.5
0.0
6.0
5.0
17.5
0.0
6.5
0.0
6.5
0.0
7.5
6.5
10.0
17.5
35.0
35.0
41.0
41.0
52.5
64.0
64.0
70.0
70.0
81.5
93.0
93.0
99.0
104.0
121.5
121.5
128.0
128.0
134.5
134.5
142.0
148.5
10
5
5
5
Arms 3e/3f (Adults 19 years of age and older)
Screening      (-
56)
SST
10
5
Clinical Procedures
4 Note this visit may occur twice depending on the approval of the V3.0 protocol; the first D42 visit (D42A) will include all D42 procedures EXCEPT AL dosing. This will visit will occur to maintain safety labs and assessments are completed on ALL subjects approximately 14 days post Vaccination #2 (±3 days). If this visit occurs, the next visit 
will be D42B for AL dosing and completed of ALL procedures as outlined in the visit (if D42A completed >14 days from D42B). This is to ensure the AL dosing in participants occurs approximately 14 days prior to Vaccination #3. If both visits are completed, subjects will receive an additional 3000 CFA ($6 USD). 
5
Laboratory Procedures
1 Visit windows are based off timing of days post the preceding vaccination
2 SMFA may be completed on subjects at Study Day 0 and 182; other SMFA time points indicated above will be completed depending on associated ELISA and DSF results at that time point.
5
5
Daily blood draw volume in mL
Cumulative blood volume in mL
5
5

Pfs230D1M Age De-escalation and Family Compound Trial 
Protocol Version 7.0 
September 24, 2021 
Page 165 of 223 
Year 1: Arms 4a/4b (1-4 years of age): 
 
 
 
 
Months
(1)
0
1
2
3
4
5
6
9
12
Visits
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Procedures
Study Day
-14
0
1
3
7
14
28
42
56
70
84
98
112
126
140
154
168
252
336
Visit Windows (days)
0
±7
0
±1
±2
±7
±7
±7
±7
±7
±7
±7
±7
±7
±7
±7
±28
±56
±56
Complete medical history/ physical
X
Informed consent
X
EKG
X
Interim clinical evaluation
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
AE/SAE assessment
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Conmed review
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Artemether/Lumefantrine
X
Malaria Blood Smear
CAP/EDTA
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Malaria PCR/Genotyping/ Gametocyte
CAP/EDTA
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
0
1.5
3
4.5
6
7.5
9
10.5
12
13.5
15
16.5
18
19.5
21
22.5
24
25.5
27
28.5
1 Note this visit will occur when the majority of the child's VU present for their study day 42 visit (14 days prior to vaccination #3)
Cumulative blood volume in mL
Daily blood draw volume in mL
Arms 4a/4b (1-4 year olds)
Screening (-56)
Blood Volume
Clinical Procedures
Laboratory Procedures

Pfs230D1M Age De-escalation and Family Compound Trial 
Protocol Version 7.0 
September 24, 2021 
Page 166 of 223 
 
Year 2: Arms 4a/4b (1-4 years of age) 
 
 
 
 
 
 
Months
13.5
14
15
16
17
18
19
20
23
26
Visits
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
Procedures
Study Day
378 (Rescreening)
385
392
406
420
434
448
462
476
490
504
518
532
546
560
644
728
Days Post Vac
322
329
336/0
14
28
42
56
70
84
98
112
126
140
154
168
252
336
Visit Windows (days) 
(1)
±84
±14
±112
±14
±14
±14
±14
±14
±14
±14
±14
±14
±14
±14
±14
±56
±56
Updated medical history/ physical
X
Reconsent
X
Interim clinical evaluation
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
AE/SAE assessment
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Conmed review
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Artemether/Lumefantrine
X
Unblinding (VU)
X
Malaria Blood Smear
CAP/EDTA
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Malaria PCR/Genotyping/ Gametocyte
CAP/EDTA
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
150.5
151.5
152.5
153.5
154.5
155.5
156.5
157.5
158.5
159.5
160.5
161.5
162.5
163.5
164.5
165.5
166.5
Arms 2a/2b (5-8 year olds)
Daily blood draw volume in mL
Cumulative blood volume in mL
1 Visit windows are based off timing of days post the preceding vaccination; broad visit window in place for Booster dose in attempt to group VU together and provide flexibility given the ongoing COVID pandemic; increased visit windows for visits to align with parental visits
Blood Volume
Clinical Procedures
Laboratory Procedures

Pfs230D1M Age De-escalation and Family Compound Trial 
Protocol Version 7.0 
September 24, 2021 
Page 167 of 223 
Year 2: Arms 2a/2b (5-8 years of age; Pilot Phase) 
 
 
 
 
 
 
Months
15.5
16
17
18
19
20
21
22
25
28
Visits
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
Procedures
Study Day
434 
(Rescreening)
441
448
451
455
462
476
490
504
518
532
546
560
574
588
602
616
700
784
Days Post Vac
322
329
336/0
3
7
14
28
42
56
70
84
98
112
126
140
154
168
252
336
Visit Windows (days) 
(1)
±84
±7
±112
±1
±2
±7
±7
±7
±7
±7
±7
±7
±7
±7
±7
±7
±7
±56
±56
Updated medical history/ physical
X
Reconsent
X
Pre-test/Post-test HIV counseling
X
Interim clinical evaluation
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
AE/SAE assessment
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Conmed review
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Artemether/Lumefantrine
X
VACCINATION
X
Unblinding
X
CBC with differential
EDTA
2
2
2
2
ALT/ Creatinine
SST
3
3
3
HIV testing
SST
Malaria Blood Smear
CAP/EDTA
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Malaria PCR/Genotyping/ Gametocyte
CAP/EDTA
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Pfs230 ELISA
SMFA (3)
Transcriptional Analysis
PaxGene
1
1
1
1
9
0
11
6
0
11
1
1
6
1
1
1
6
1
1
1
7
6
6
170
170
181
187
187
198
199
200
206
207
208
209
215
216
217
218
225
231
237
SST
Arms 2a/2b (5-8 year olds)
Blood Volume
Clinical Procedures
Laboratory Procedures
5
5
5
Daily blood draw volume in mL
Cumulative blood volume in mL
1 Visit windows are based off timing of days post the preceding vaccination; broad visit window in place for Booster dose in attempt to group VU together and provide flexibility given the ongoing COVID pandemic
2 SMFA may be completed on subjects at Study Day 448 and 504; other SMFA time points indicated above will be completed depending on associated ELISA results at that time point.
5
5
5
5
5

Pfs230D1M Age De-escalation and Family Compound Trial 
Protocol Version 7.0 
September 24, 2021 
Page 168 of 223 
Year 2: Arms 3a/3b (5-8 years of age; Main Phase) 
 
 
 
 
 
 
 
Months
13.5
14
15
16
17
18
19
20
23
26
Visits
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
Procedures
Study Day
378 (Rescreening)
385
392
395
399
406
420
434
448
462
476
490
504
518
532
546
560
644
728
Days Post Vac
322
329
336/0
3
7
14
28
42
56
70
84
98
112
126
140
154
168
252
336
Visit Windows (days) 
(1)
±84
±7
±112
±1
±2
±7
±7
±7
±7
±7
±7
±7
±7
±7
±7
±7
±7
±56
±56
Updated medical history/ physical
X
Reconsent
X
Pre-test/Post-test HIV counseling
X
Interim clinical evaluation
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
AE/SAE assessment
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Conmed review
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Artemether/Lumefantrine
X
VACCINATION
X
Unblinding
X
CBC with differential
EDTA
2
2
2
2
ALT/ Creatinine
SST
3
3
3
HIV testing
SST
Malaria Blood Smear
CAP/EDTA
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Malaria PCR/Genotyping/ Gametocyte
CAP/EDTA
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Pfs230 ELISA
SMFA (3)
Transcriptional Analysis
PaxGene
1
1
1
1
9
0
11
6
0
11
1
1
6
1
1
1
6
1
1
1
7
6
6
158.5
158.5
169.5
175.5
175.5
186.5
187.5
188.5
194.5
195.5
196.5
197.5
203.5
204.5
205.5
206.5
213.5
219.5
225.5
1 Visit windows are based off timing of days post the preceding vaccination; broad visit window in place for Booster dose in attempt to group VU together and provide flexibility given the ongoing COVID pandemic
2 SMFA may be completed on subjects at Study Day 336 and 448; other SMFA time points indicated above will be completed depending on associated ELISA results at that time point.
5
5
5
Daily blood draw volume in mL
Cumulative blood volume in mL
5
5
5
Arms 3a/3b (5-8 year olds)
Blood Volume
Clinical Procedures
Laboratory Procedures
SST
5
5

Pfs230D1M Age De-escalation and Family Compound Trial 
Protocol Version 7.0 
September 24, 2021 
Page 169 of 223 
Year 2: Arms 1a/1b (9-18 years of age; Pilot Phase) 
 
 
 
 
 
 
Months
15.5
16
17
18
19
20
21
22
25
28
Visits
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
Procedures
Blood Volume
Study Day
434 
(Rescreening)
448
451
455
462
476
490
504
518
532
546
560
574
588
602
616
700
784
Days post Vac 
322
336/0
3
7
14
28
42
56
70
84
98
112
126
140
154
168
252
336
Visit Windows (days) (1)
±84
±112
±1
±2
±7
±7
±7
±7
±7
±7
±7
±7
±7
±7
±7
±7
±56
±56
Updated medical history/ physical
Reconsent
X
Pre-test/Post-test HIV counseling
X
Interim clinical evaluation
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
AE/SAE assessment
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Pregnancy Prevention
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Conmed review
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
VACCINATION
X
Unblinding
X
Feeding Evaluation (1)
CBC with differential
EDTA
2.0
2.0
2.0
2.0
ALT/ Creatinine
SST
3.0
3.0
3.0
HIV testing
SST
Urine/Serum pregnancy test 
(females only)
Urine Container or SST
X
X
X
X
X
X
X
Malaria Blood Smear
CAP/EDTA
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Malaria PCR/Genotyping/ 
Gametocyte
CAP/EDTA
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
 Pfs230 ELISA
SMFA (2)
DSF (3)
X
X
X
X
X
X
X
X
X
X
X
Transcriptional analysis
PAXGene
1
1
1
1
14.0
11.0
6.0
5.0
17.0
1.0
1.0
6.0
1.0
1.0
1.0
6.0
1.0
1.0
1.0
7.0
6.0
6.0
186.0
197.0
203.0
208.0
225.0
226.0
227.0
233.0
234.0
235.0
236.0
242.0
243.0
244.0
245.0
252.0
258.0
264.0
Arms 1a/1b (9-18 year olds)
Clinical Procedures
Laboratory Procedures
SST
5
5
5
5
10
X
5.0
5
Daily blood draw volume in mL
Cumulative blood volume in mL
1 Visit windows are based off timing of days post the preceding vaccination; broad visit window in place for Booster dose in attempt to group VU together and provide flexibility given the ongoing COVID pandemic
2 SMFA may be completed on subjects at Study Day 448 and 504; other SMFA time points indicated above will be completed depending on associated ELISA and DSF results at that time point.
3 DSF will be completed twice monthly post Vaccination #4 from day 463 through 588 (total = 10 feeds).   
5
5
5

Pfs230D1M Age De-escalation and Family Compound Trial 
Protocol Version 7.0 
September 24, 2021 
Page 170 of 223 
Year 2: Arms 3c/3d (9-18 years of age; Main Phase) 
 
 
 
 
 
Months
13.5
14
17
18
19
20
21
22
25
28
Visits
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
Procedures
Blood Volume
Study Day
378 
(Rescreening)
392
395
399
406
420
434
448
462
476
490
504
518
532
546
560
644
728
Days post Vac 
322
336/0
3
7
14
28
42
56
70
84
98
112
126
140
154
168
252
336
Visit Windows (days) (1)
±84
±112
±1
±2
±7
±7
±7
±7
±7
±7
±7
±7
±7
±7
±7
±7
±56
±56
Updated medical history/ physical
Reconsent
X
Pre-test/Post-test HIV counseling
X
Interim clinical evaluation
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
AE/SAE assessment
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Pregnancy Prevention
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Conmed review
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
VACCINATION
X
Unblinding
X
Feeding Evaluation (1)
CBC with differential
EDTA
2.0
2.0
2.0
2.0
ALT/ Creatinine
SST
3.0
3.0
3.0
HIV testing
SST
Urine/Serum pregnancy test 
(females only)
Urine Container or SST
X
X
X
X
X
X
X
Malaria Blood Smear
CAP/EDTA
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Malaria PCR/Genotyping/ 
Gametocyte
CAP/EDTA
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
 Pfs230 ELISA
SMFA (2)
DSF (3)
X
X
X
X
X
X
X
X
X
X
X
Transcriptional analysis
PAXGene
1
1
1
1
14.0
11.0
6.0
5.0
17.0
1.0
1.0
6.0
1.0
1.0
1.0
6.0
1.0
1.0
1.0
7.0
6.0
6.0
174.5
185.5
191.5
196.5
213.5
214.5
215.5
221.5
222.5
223.5
224.5
230.5
231.5
232.5
233.5
240.5
246.5
252.5
2 SMFA may be completed on subjects at Study Day 336 and 448; other SMFA time points indicated above will be completed depending on associated ELISA and DSF results at that time point.
3 DSF will be completed twice monthly post Vaccination #4 from day 406 through 532 (total = 10 feeds).   
5
5
5
Daily blood draw volume in mL
Cumulative blood volume in mL
1 Visit windows are based off timing of days post the preceding vaccination; broad visit window in place for Booster dose in attempt to group VU together and provide flexibility given the ongoing COVID pandemic
5
5
10
5
Arms 3c/3d (9-18 year olds)
Clinical Procedures
Laboratory Procedures
X
SST
5
5
5.0

Pfs230D1M Age De-escalation and Family Compound Trial 
Protocol Version 7.0 
September 24, 2021 
Page 171 of 223 
Year 2: Arms 3e/3f (≥19 years of age) 
 
Months
13.5
14
15
16
18
20
23
26
Visits
24
25
26
27
28
29
30
31
32
33
34
Procedures
Blood Volume
Study Day
378 (Rescreening)
392
395
399
406
420
448
504
560
644
728
Days post Vac 
322
336/0
3
7
14
28
56
112
168
252
336
Visit Windows (days) (1)
±84
±112
±1
±2
±7
±7
±7
±7
±28
±56
±56
Updated medical history/ physical
Reconsent
X
Pre-test/Post-test HIV counseling
X
Interim clinical evaluation
X
X
X
X
X
X
X
X
X
X
AE/SAE assessment
X
X
X
X
X
X
X
X
X
X
X
Pregnancy Prevention
X
X
X
X
X
X
X
X
X
X
X
Conmed review
X
X
X
X
X
X
X
X
X
X
X
VACCINATION
X
Unblinding
X
CBC with differential
EDTA
2
2
2
2
ALT/ Creatinine
SST
3
3
3
HIV testing
SST
Urine/Serum pregnancy test 
(females only)
Urine Container or SST
0.5
X
Malaria Blood Smear
CAP/EDTA
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Malaria PCR/Genotyping/ 
Gametocyte
CAP/EDTA
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Pfs230 ELISA
SMFA (2)
Transcriptional analysis
PAXGene
1
1
1
1
12.5
11.0
6.0
5.0
17.0
0.0
6.0
6.0
7.0
6.0
6.0
161.0
172.0
178.0
183.0
200.0
200.0
206.0
212.0
219.0
225.0
231.0
1 Visit windows are based off timing of days post the preceding vaccination; broad visit window in place for Booster dose in attempt to group VU together and provide flexibility given the ongoing COVID pandemic
2 SMFA may be completed on subjects at Study Day 392 and 448; other SMFA time points indicated above will be completed depending on associated ELISA results at that time point.
5
5
5
5
5
10
5
5
Arms 3e/3f (Adults 19 years of age and older)
Clinical Procedures
Laboratory Procedures
SST
Daily blood draw volume in mL
Cumulative blood volume in mL
5
5

Pfs230D1M Age De-escalation and Family Compound Trial 
Protocol Version 7.0 
September 24, 2021 
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Appendix B: Day to Day Schedule by Arms 
Arms 4a/4b (1-4 year olds): No Vaccination 
 
Study Day -14 (±0 days; Drug Treatment Visit; should align with Vaccine Unit visit prior 
to Vaccination #3) 
The following evaluations/procedures will be completed before vaccination: 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear and malaria PCR. 
5. Administer first dose of artemether/lumefantrine (AL). 
a. Additional doses of AL will occur on study days -13, -12 
 Study Day 0 (±7 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear and malaria PCR. 
Study Day 1 (±0 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear and malaria PCR. 
Study Day 3 (±1 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear and malaria PCR. 
Study Day 7 (±2 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear and malaria PCR. 
Study Day 14 (±7 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear and malaria PCR. 

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Study Day 28 (±7 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear and malaria PCR. 
Study Day 42 (±7 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear and malaria PCR. 
Study Day 56 (±7 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear and malaria PCR. 
Study Day 70 (±7 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear and malaria PCR. 
Study Day 84 (±7 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear and malaria PCR. 
Study Day 98 (±7 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear and malaria PCR. 
Study Day 112 (±7 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear and malaria PCR. 

Pfs230D1M Age De-escalation and Family Compound Trial 
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September 24, 2021 
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Study Day 126 (±7 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear and malaria PCR. 
Study Day 140 (±7 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear and malaria PCR. 
Study Day 154 (±7 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear and malaria PCR. 
Study Day 168 (±28 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear and malaria PCR. 
Study Day 252 (±56 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear and malaria PCR. 
Study Day 378 (±84 day) 
1. Reconsent for continuation on study. 
2. Perform updated medical history and physical examination, emphasizing examination of 
any acute complaints.  
3. Record vital signs (weight, length, blood pressure, temperature, and heart rate). 
4. Record AEs and concomitant medications, if applicable. 
5. Obtain approximately 1 mL of blood for malaria blood smear and malaria PCR. 
6. Administer first dose of artemether/lumefantrine (AL). 
a. Additional doses of AL will occur on study days -13, -12. 
 Study Day 385 (±14 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 

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3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1 mL of blood for malaria blood smear and malaria PCR. 
5. Administer first dose of artemether/lumefantrine (AL). 
6. Additional doses of AL will occur on study days 386, 387. 
Study Day 392 (±112 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1 mL of blood for malaria blood smear and malaria PCR. 
Study Day 406 (±14 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1 mL of blood for malaria blood smear and malaria PCR. 
Study Day 420 (±14 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1 mL of blood for malaria blood smear and malaria PCR. 
Study Day 434 (±14 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1 mL of blood for malaria blood smear and malaria PCR. 
Study Day 448 (±14 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1 mL of blood for malaria blood smear and malaria PCR. 
Study Day 462 (±14 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1 mL of blood for malaria blood smear and malaria PCR. 

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September 24, 2021 
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Study Day 476 (±14 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1 mL of blood for malaria blood smear and malaria PCR. 
Study Day 490 (±14 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1 mL of blood for malaria blood smear and malaria PCR. 
Study Day 504 (±14 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1 mL of blood for malaria blood smear and malaria PCR. 
Study Day 518 (±14 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1 mL of blood for malaria blood smear and malaria PCR. 
Study Day 532 (±14 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1 mL of blood for malaria blood smear and malaria PCR. 
Study Day 546 (±28 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1 mL of blood for malaria blood smear and malaria PCR. 
5. Scheduled unblinding of VU 
 
Study Day 560 (±14 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 

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September 24, 2021 
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4. Obtain approximately 1 mL of blood for malaria blood smear and malaria PCR. 
Study Day 644 (±56 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1 mL of blood for malaria blood smear and malaria PCR. 
Study Day 728 (±56 day) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1 mL of blood for malaria blood smear and malaria PCR. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Protocol Version 7.0 
September 24, 2021 
Page 178 of 223 
Arms 2a/2b (PILOT), 3a/3b (MAIN) (5-8 year olds):  
3 Vaccinations (0, 1, 4, 16 months—PILOT; 0, 1, 2, 14 months—MAIN) 
 
Study Day -7 (±7 days; Drug Treatment Visit) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 7.5 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, hemoglobin typing, malaria blood smear, malaria PCR, and transcriptional 
analysis (NOTE: if hemoglobin typing not obtained, can be obtained later during study at 
anytime when an EDTA tube is drawn). If safety labs were obtained for screening within 
≤ 2 days prior to day -7, can use for study day -7 visit and do not need to repeat.  
5. Obtain urine for Schistosomiasis testing (permittable to be collected any day between 
start of AL dosing and first vaccination; Main Phase only) 
6. Administer first dose of artemether/lumefantrine (AL). 
a. Additional doses of AL will occur on study days -6, -5 
Study Day 0 (day of Vaccination #1) 
The following evaluations/procedures will be completed before vaccination: 
1. Verify that informed consent was obtained.  
2. Ensure that all inclusion/exclusion criteria are met.  
3. Ensure that CBC, ALT, creatinine, HIV, and urine results from screening are within 
protocol-defined limits (see Exclusion Criteria above) before vaccinating 
4. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
5. Record vital signs (blood pressure, temperature, and heart rate). 
6. During the physical examination, study staff will educate the subject regarding signs and 
symptoms of potential adverse events (AEs) and indications for use of antipyretics if 
fever, headache, or malaise occurs. 
7. Confirm that all criteria for vaccination have been met according to inclusion/exclusion 
criteria 
8. Confirm continued eligibility to receive vaccination  
9. Obtain approximately 17.5 mL of blood for CBC with differential and platelet count, 
ALT, creatinine, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA and 
standard membrane feeding assay (SMFA), and transcriptional analysis. 
10. Obtain urine for Schistosomiasis testing (permittable to be collected any day between 
start of AL dosing and first vaccination; Main Phase only) 
11. Administer the vaccine 
The following evaluations/procedures will be completed following vaccination: 
1. Observe the subject for at least 30 minutes after vaccination to evaluate immediate adverse 
reactions.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Perform interim history and physical examination, focusing on any acute complaints.  
4. Record AEs and concomitant medications, if applicable. 
 

Pfs230D1M Age De-escalation and Family Compound Trial 
Protocol Version 7.0 
September 24, 2021 
Page 179 of 223 
Study Day 1 (±0 days; 1 day after Vaccination #1)  
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 3 (±1 day; 3 days after Vaccination #1) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 6 mL of blood for CBC with differential and platelet count, ALT, and 
creatinine, and transcriptional analysis. 
 
Study Day 7 (±2 days; 7 days after Vaccination #1) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 14 (±3 days; 14 days after Vaccination #1) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 11.5 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA and standard 
membrane feeding assay (SMFA). 
 
Study Day 28 (±7 days; day of Vaccination #2) 
The following evaluations/procedures will be completed before vaccination: 
1. Perform interim history and physical examination, focusing on any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. During the physical examination, study staff will educate the subject regarding signs and 
symptoms of potential adverse events (AEs) and indications for use of antipyretics if fever, 
headache, or malaise occurs. 
4. Obtain approximately 11.5 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA, and standard 
membrane feeding assay (SMFA).  
5. Confirm continued eligibility to receive vaccination  
6. Administer the vaccine.  
 
The following evaluations/procedures will be completed following vaccination: 
1. Observe the subject for at least 30 minutes after vaccination to evaluate immediate adverse 
reactions.  
2. Record vital signs (blood pressure, temperature, and heart rate). 

Pfs230D1M Age De-escalation and Family Compound Trial 
Protocol Version 7.0 
September 24, 2021 
Page 180 of 223 
3. Perform interim history and physical examination, focusing on any acute complaints.  
4. Record AEs and concomitant medications, if applicable. 
 
Study Day 29 (±0 days; 1 day after Vaccination #2)  
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 31 (±1 day; 3 days after Vaccination #2) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 6 mL of blood for CBC with differential and platelet count, ALT and 
creatinine, and transcriptional analysis. 
 
Study Day 35 (±2 days; 7 days after Vaccination #2) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
PILOT ONLY 
 
Study Day 42 (±3 days; 14 days after Vaccination #2) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 11.5 mL of blood for CBC with differential and platelet count, 
ALT, creatinine, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA, and 
standard membrane feeding assay (SMFA).  
 
Study Day 56 (±7 days; 28 days after Vaccination #2)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 84 (±14 days; 56 days after Vaccination #2)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 

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September 24, 2021 
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PILOT AND MAIN PHASE JOIN SCHEDULES (STUDY DAYS 
OFF BY 70 DAYS) 
 
Study Day 112 (PILOT) + 42 (MAIN) (±28 days; 84 days after Vaccination #2 in Pilot; 14 
days after Vaccination #2 in Main) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 11.5 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear and malaria PCR, anti-Pfs230 antibody ELISA, and standard 
membrane feeding assay (SMFA). 
5. Administer first dose of artemether/lumefantrine (AL). (NOTE if D42A, no AL dosing; if 
D42B AL dosing to be completed) 
a. Pilot: Additional doses of AL will occur on study days 113, 114 
b. Pilot: Additional doses of AL will occur on study days 43, 44 
 
Study Day 126 (PILOT) + 56 (MAIN) (±56 days; day of Vaccination #3) 
The following evaluations/procedures will be completed before vaccination: 
1. Perform interim history and physical examination, focusing on any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. During the physical examination, study staff will educate the subject regarding signs and 
symptoms of potential adverse events (AEs) and indications for use of antipyretics if fever, 
headache, or malaise occurs. 
4. Obtain approximately 11.5 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA and standard 
membrane feeding assay (SMFA). 
5. Confirm continued eligibility to receive vaccination  
6. Administer the vaccine.  
 
The following evaluations/procedures will be completed following vaccination: 
1. Observe the subject for at least 30 minutes after vaccination to evaluate immediate adverse 
reactions.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Perform interim history and physical examination, focusing on any acute complaints.  
4. Record AEs and concomitant medications, if applicable. 
 
Study Day 127 (PILOT) + 57 (MAIN) (±0 days; 1 day after Vaccination #3)  
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 

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Protocol Version 7.0 
September 24, 2021 
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Study Day 129 (PILOT) + 59 (MAIN) (±1 day; 3 days after Vaccination #3) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 6 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, transcriptional analysis. 
 
Study Day 133 (PILOT) + 63 (MAIN)  (±2 days; 7 days after Vaccination #3) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 140 (PILOT) + 70 (MAIN) (±7 days; 14 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 11.5 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA and standard 
membrane feeding assay (SMFA). 
 
Study Day 154 (PILOT) + 84 (MAIN) (±7 days; 28 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear and malaria PCR. 
 
Study Day 168 (PILOT) + 98 (MAIN) (±7 days; 42 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear and malaria PCR. 
 
Study Day 182 (PILOT) + 112 (MAIN) (±7 days; 56 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 6.5 mL of blood for malaria blood smear, malaria PCR, anti-Pfs230 
antibody ELISA and standard membrane feeding assay (SMFA). 
 

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September 24, 2021 
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Study Day 196 (PILOT) + 126 (MAIN) (±7 days; 70 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear and malaria PCR. 
 
Study Day 210 (PILOT) + 140 (MAIN) (±7 days; 84 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear and malaria PCR. 
 
Study Day 224 (PILOT) + 154 (MAIN) (±7 days; 98 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear, malaria PCR. 
 
Study Day 238 (PILOT) + 168 (MAIN) (±7 days; 112 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable.  
4. Obtain approximately 6.5 mL of blood for malaria blood smear, malaria PCR, anti-Pfs230 
antibody ELISA and standard membrane feeding assay (SMFA). 
 
Study Day 252 (PILOT) + 182 (MAIN) (±7 days; 126 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear and malaria PCR. 
 
Study Day 266 (PILOT) + 196 (MAIN) (±7 days; 140 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear and malaria PCR. 
 
Study Day 280 (PILOT) + 210 (MAIN) (±7 days; 154 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  

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2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5mL of blood for malaria blood smear and malaria PCR. 
 
Study Day 294 (PILOT) + 224 (MAIN) (±28 days; 168 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable.  
4. Obtain approximately 7.5 mL of blood for malaria blood smear, malaria PCR, anti-Pfs230 
antibody ELISA and standard membrane feeding assay (SMFA), and transcriptional analysis. 
 
Study Day 378 (PILOT) + 308 (MAIN) (±56 days; 252 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable.  
4. Obtain approximately 6.5 mL of blood for malaria blood smear, malaria PCR, anti-Pfs230 
antibody ELISA and standard membrane feeding assay (SMFA). 
 
Study Day 434 (PILOT- 308 days post Vaccination #3) + 378 (MAIN) 322 days after 
Vaccination #3) (±84 days); 
1. Reconsent for continuation on study. 
2. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
3. Record vital signs (blood pressure, temperature, and heart rate). 
4. Record AEs and concomitant medications, if applicable. 
5. Obtain approximately 9 mL of blood for HIV, CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear and malaria PCR and transcriptional analysis.  
 
Study Day 441 (PILOT 315 days post Vaccination #3) + 385 (MAIN 329 days post 
Vaccination #3) (±7 days; day of Vaccination #4) 
The following evaluations/procedures will be completed before vaccination: 
1. Perform interim history and physical examination, focusing on any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Administer first dose of artemether/lumefantrine (AL).  
 
a) Pilot: Additional doses of AL will occur on study days 442, 443 
b) Pilot: Additional doses of AL will occur on study days 386, 387 
 
Study Day 448 (PILOT) + 392 (MAIN) (±112 days; day of Vaccination #4)  
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 

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3. During the physical examination, study staff will educate the subject regarding signs and 
symptoms of potential adverse events (AEs) and indications for use of antipyretics if fever, 
headache, or malaise occurs. 
4. Confirm continued eligibility to receive vaccination. 
5. Obtain approximately 11 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA and standard 
membrane feeding assay (SMFA). 
6. Administer the vaccine.  
 
The following evaluations/procedures will be completed following vaccination: 
1. Observe the subject for at least 30 minutes after vaccination to evaluate immediate adverse 
reactions.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Perform interim history and physical examination, focusing on any acute complaints.  
4. Record AEs and concomitant medications, if applicable. 
 
Study Day 451 (PILOT) + 395 (MAIN) (±1 day; 3 days after Vaccination #4) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 6 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, transcriptional analysis. 
 
Study Day 455 (PILOT) + 399 (MAIN) (±2 days; 7 days after Vaccination #4) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 462 (PILOT) + 406 (MAIN) (±7 days; 14 days after Vaccination #4) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 11 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA, standard 
membrane feeding assay (SMFA) and transcriptional analysis. 
 
Study Day 476 (PILOT) + 420 (MAIN) (±7 days; 28 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1 mL of blood for malaria blood smear and malaria PCR. 
 

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Study Day 490 (PILOT) + 434 (MAIN) (±7 days; 42 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1 mL of blood for malaria blood smear and malaria PCR. 
 
Study Day 504 (PILOT) + 448 (MAIN) (±7 days; 56 days after Vaccination #4  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 6 mL of blood for malaria blood smear, malaria PCR, anti-Pfs230 
antibody ELISA and standard membrane feeding assay (SMFA). 
 
Study Day 518 (PILOT) + 462 (MAIN) (±7 days; 70 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1 mL of blood for malaria blood smear and malaria PCR. 
 
Study Day 532 (PILOT) + 476 (MAIN) (±7 days; 84 days after Vaccination #4) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1 mL of blood for malaria blood smear and malaria PCR. 
 
Study Day 546 (PILOT) + 490 (MAIN) (±7 days; 98 days after Vaccination #4) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1 mL of blood for malaria blood smear, malaria PCR. 
 
Study Day 560 (PILOT) + 504 (MAIN) (±7 days; 112 days after Vaccination #4) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable.  
4. Obtain approximately 6 mL of blood for malaria blood smear, malaria PCR, anti-Pfs230 
antibody ELISA and standard membrane feeding assay (SMFA). 
 

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Study Day 574 (PILOT) + 518 (MAIN) (±7 days; 126 days after Vaccination #4) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1 mL of blood for malaria blood smear and malaria PCR. 
 
Study Day 588 (PILOT) + 532 (MAIN) (±7 days; 140 days after Vaccination #4) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1 mL of blood for malaria blood smear and malaria PCR. 
 
Study Day 602 (PILOT) + 546(MAIN) (±28 days; 154 days after Vaccination #4) 
1.  Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1 mL of blood for malaria blood smear and malaria PCR. 
5. Scheduled unblinding of VU 
a. If received Pfs230D1M-EPA/ASO1 offer meningitis comparator vaccination 
 
Study Day 616 (PILOT) + 560 (MAIN) (±28 days; 168 days after Vaccination #4) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable.  
4. Obtain approximately 7 mL of blood for malaria blood smear, malaria PCR, anti-Pfs230 
antibody ELISA and standard membrane feeding assay (SMFA), and transcriptional analysis. 
 
Study Day 700 (PILOT) + 644 (MAIN) (±56 days; 252 days after Vaccination #4) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable.  
4. Obtain approximately 6 mL of blood for malaria blood smear, malaria PCR, anti-Pfs230 
antibody ELISA and standard membrane feeding assay (SMFA). 
 
Study Day 784 (PILOT) + 728 (MAIN) (±56 days; 336 days after Vaccination #4) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable.  
4. Obtain approximately 6 mL of blood for malaria blood smear, malaria PCR, anti-Pfs230 
antibody ELISA and standard membrane feeding assay (SMFA). 

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Arms 1a/1b (PILOT), 3c/3d (MAIN) (9-18 year olds):  
3 Vaccinations (0, 1, 4 months—PILOT; 0, 1, 2 months—MAIN) 
 
Study Day -7 (±7 days; Drug Treatment Visit) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
1. Record vital signs (blood pressure, temperature, and heart rate). 
2. Record AEs and concomitant medications, if applicable. 
3. Obtain approximately 7.5 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, hemoglobin typing. malaria blood smear and malaria PCR, and transcriptional 
analysis. (NOTE: if hemoglobin typing not obtained, can be obtained later during study at 
anytime when an EDTA tube is drawn). If safety labs were obtained for screening within 
≤ 2 days prior to day -7, can use for study day -7 visit and do not need to repeat. 
4. Obtain Urine +/- Stool Sample for Schistosomiasis +/- Helminth testing (permittable to 
be collected any day between start of AL dosing and first vaccination) 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before proceeding. 
6. For females, ensure agreement and compliance with pregnancy prevention before AL 
dosing. 
7. Administer first dose of artemether/lumefantrine (AL). 
a. Additional doses of AL will occur on study days -6, -5 
Study Day 0 (day of Vaccination #1) 
The following evaluations/procedures will be completed before vaccination: 
 
1. Ensure that all inclusion/exclusion criteria are met.  
2. Ensure that CBC, ALT, creatinine, HIV, and urine results from screening are within 
protocol-defined limits (see Exclusion Criteria above) before vaccinating 
3. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
4. Record vital signs (blood pressure, temperature, and heart rate). 
5. During the physical examination, study staff will educate the subject regarding signs and 
symptoms of potential adverse events (AEs) and indications for use of antipyretics if 
fever, headache, or malaise occurs. 
6. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before vaccinating.  
7. For females, ensure agreement and compliance with pregnancy prevention before 
vaccinating. 
8. Confirm that all criteria for vaccination have been met according to inclusion/exclusion 
criteria 
9. Confirm continued eligibility to receive vaccination  
10. Obtain approximately 17.5 mL of blood for CBC with differential and platelet count, 
ALT, creatinine, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA, 
standard membrane feeding assay (SMFA), and transcriptional analysis. 
11. Obtain Urine +/- Stool Sample for Schistosomiasis +/- Helminth testing (permittable to 
be collected prior to day 0; any day between start of AL dosing and first vaccination) 

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12. Administer the vaccine. 
The following evaluations/procedures will be completed following vaccination: 
1. Observe the subject for at least 30 minutes after vaccination to evaluate immediate adverse 
reactions.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Perform interim history and physical examination, focusing on any acute complaints.  
4. Record AEs and concomitant medications, if applicable. 
 
Study Day 1 (±0 days; 1 day after Vaccination #1)  
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 3 (±1 day; 3 days after Vaccination #1) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 6.0 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, and transcriptional analysis. 
 
Study Day 7 (±2 days; 7 days after Vaccination #1) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 14 (±3 days; 14 days after Vaccination #1) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 11.5 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA and standard 
membrane feeding assay (SMFA). 
 
Study Day 28 (±7 day of Vaccination #2) 
The following evaluations/procedures will be completed before vaccination: 
1. Perform interim history and physical examination, focusing on any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. During the physical examination, study staff will educate the subject regarding signs and 
symptoms of potential adverse events (AEs) and indications for use of antipyretics if fever, 
headache, or malaise occurs. 
4. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before vaccinating. 

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5. For females, ensure agreement and compliance with pregnancy prevention before vaccinating 
6. Obtain approximately 11.5 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA, and standard 
membrane feeding assay (SMFA). 
7. Confirm continued eligibility to receive vaccination.  
8. Administer the vaccine.  
 
The following evaluations/procedures will be completed following vaccination: 
1. Observe the subject for at least 30 minutes after vaccination to evaluate immediate adverse 
reactions.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Perform interim history and physical examination, focusing on any acute complaints.  
4. Record AEs and concomitant medications, if applicable. 
 
Study Day 29 (±0 days; 1 day after Vaccination #2)  
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 31 (±1 day; 3 days after Vaccination #2) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 6.0 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, and transcriptional analysis.  
 
Study Day 35 (±2 days; 7 days after Vaccination #2) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
PILOT ONLY 
 
Study Day 42 (±3 days; 14 days after Vaccination #2) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
5. Obtain approximately 11.5 mL of blood for CBC with differential and platelet count, 
ALT, creatinine, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA and 
standard membrane feeding assay (SMFA). 
 

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Study Day 56 (±7 days; 28 days after Vaccination #2)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 84 (±14 days; 56 days after Vaccination #2)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
PILOT AND MAIN PHASE JOIN SCHEDULES (STUDY DAYS 
OFF BY 70 DAYS) 
 
Study Day 112 (PILOT) + 42 (MAIN) (±28 days; 84 days after Vaccination #2 in Pilot; 
14 days after Vaccination #2 in Main) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 11.5 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA, and standard 
membrane feeding assay (SMFA).  
 
Study Day 126 (PILOT) + 56 (MAIN) (±56 days; day of Vaccination #3) 
The following evaluations/procedures will be completed before vaccination: 
1. Perform interim history and physical examination, focusing on any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. During the physical examination, study staff will educate the subject regarding signs and 
symptoms of potential adverse events (AEs) and indications for use of antipyretics if fever, 
headache, or malaise occurs. 
4. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before vaccinating.  
5. For females, ensure agreement and compliance with pregnancy prevention before 
vaccinating. 
6. Obtain approximately 11.5 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA, and standard 
membrane feeding assay (SMFA). 
7. Confirm continued eligibility to receive vaccination  
8. Administer the vaccine.  
 
The following evaluations/procedures will be completed following vaccination: 
1. Observe the subject for at least 30 minutes after vaccination to evaluate immediate adverse 
reactions.  
2. Record vital signs (blood pressure, temperature, and heart rate). 

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3. Perform interim history and physical examination, focusing on any acute complaints.  
4. Record AEs and concomitant medications, if applicable. 
 
Study Day 127 (PILOT) + 57 (MAIN) (±0 days; 1 day after Vaccination #3)  
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 129 (PILOT) + 59 (MAIN) (±1 day; 3 days after Vaccination #3) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 6.0 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, and transcriptional analysis. 
 
Study Day 133 (PILOT) + 63 (MAIN) (±2 days; 7 days after Vaccination #3) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 5.0 mL of blood for anti-Pfs230 antibody ELISA, and standard 
membrane feeding assay (SMFA). 
 
Study Day 140 (PILOT) + 70 (MAIN) (±7 days; 14 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before proceeding.  
5. Obtain approximately 17.5 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA, standard 
membrane feeding assay (SMFA) and Transcriptional analysis. 
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 154 (PILOT) + 84 (MAIN) (±7 days; 28 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear, and malaria PCR 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  

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Study Day 168 (PILOT) + 98 (MAIN) (±7 days; 42 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear, malaria PCR 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before proceeding.  
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 182 (PILOT) + 112 (MAIN) (±7 days; 56 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 6.5 mL of blood for malaria blood smear, malaria PCR, anti-Pfs230 
antibody ELISA, standard membrane feeding assay (SMFA). 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 196 (PILOT) + 126 (MAIN) (±7 days; 70 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear, malaria PCR 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before proceeding. 
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 210 (PILOT) + 140 (MAIN) (±7 days; 84 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 224 (PILOT) + 154 (MAIN) (±7 days; 98 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 

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3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear and malaria PCR. 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before proceeding.  
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 238 (PILOT) + 168 (MAIN) (±7 days; 112 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable.  
4. Obtain approximately 6.5 mL of blood for malaria blood smear, malaria PCR, anti-Pfs230 
antibody ELISA, standard membrane feeding assay (SMFA). 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 252 (PILOT) + 182 (MAIN) (±7 days; 126 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear and malaria PCR. 
 
Study Day 266 (PILOT) + 196 (MAIN) (±7 days; 140 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear and malaria PCR. 
 
Study Day 280 (PILOT) + 210 (MAIN) (±7 days; 154 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1.5 mL of blood for malaria blood smear and malaria PCR. 
 
Study Day 294 (PILOT) + 224 (MAIN) (±28 days; 168 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable.  
4. Obtain approximately 7.5 mL of blood for malaria blood smear, malaria PCR, anti-Pfs230 
antibody ELISA, standard membrane feeding assay (SMFA), and transcriptional analysis. 
 

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Study Day 378 (PILOT) + 308(MAIN) (±56 days; 252 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable.  
4. Obtain approximately 6.5 mL of blood for malaria blood smear, malaria PCR, anti-Pfs230 
antibody ELISA, standard membrane feeding assay (SMFA). 
 
Study Day 462 (PILOT) + 392 (MAIN) (±56 days; 336 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable.  
4. Obtain approximately 6.5 mL of blood for malaria blood smear, malaria PCR, anti-Pfs230 
antibody ELISA, standard membrane feeding assay (SMFA). 
 
Study Day 434 (PILOT 308 days post Vaccination #3) + 378 (MAIN 322 days after 
Vaccination #3) (±84 days) 
1. Reconsent for continuation on study. 
2. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
3. Record vital signs (blood pressure, temperature, and heart rate). 
4. Record AEs and concomitant medications, if applicable. 
5. Obtain approximately 14 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, HIV, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA, standard 
membrane feeding assay (SMFA) and transcriptional analysis.  
6. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before proceeding. 
 
Study Day 448 (PILOT) + 392 (MAIN) (±112 days; day of Vaccination #4) 
The following evaluations/procedures will be completed before vaccination: 
1. Perform interim history and physical examination, focusing on any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. During the physical examination, study staff will educate the subject regarding signs and 
symptoms of potential adverse events (AEs) and indications for use of antipyretics if fever, 
headache, or malaise occurs. 
4. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before vaccinating.  
5. For females, ensure agreement and compliance with pregnancy prevention before 
vaccinating. 
6. Obtain approximately 11 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA, and standard 
membrane feeding assay (SMFA). 
7. Confirm continued eligibility to receive vaccination.  
8. Administer the vaccine.  

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The following evaluations/procedures will be completed following vaccination: 
1. Observe the subject for at least 30 minutes after vaccination to evaluate immediate adverse 
reactions.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Perform interim history and physical examination, focusing on any acute complaints.  
4. Record AEs and concomitant medications, if applicable. 
 
Study Day 451 (PILOT) + 395 (MAIN) (±1 day; 3 days after Vaccination #4)  
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 6.0 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, and transcriptional analysis. 
 
Study Day 455 (PILOT) + 399 (MAIN) (±2 day; 7 days after Vaccination #4) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 5.0 mL of blood for standard membrane feeding assay (SMFA). 
 
Study Day 462 (PILOT) + 406 (MAIN) (±7 days; 14 days after Vaccination #4) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before proceeding.  
5. Obtain approximately 17 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA, standard 
membrane feeding assay (SMFA) and Transcriptional analysis. 
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 476 (PILOT) + 420 (MAIN) (±7 days; 28 days after Vaccination #4) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1 mL of blood for malaria blood smear, and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 490 (PILOT) + 434(MAIN) (±7 days; 42 days after Vaccination #4)  

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1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before proceeding.  
5. Obtain approximately 1 mL of blood for malaria blood smear, and malaria PCR. 
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 504 (PILOT) + 448 (MAIN) (±7 days; 56 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 6 mL of blood for malaria blood smear, malaria PCR, anti-Pfs230 
antibody ELISA, standard membrane feeding assay (SMFA). 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 518 (PILOT) + 462 (MAIN) (±7 days; 70 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before proceeding.  
5. Obtain approximately 1 mL of blood for malaria blood smear, and malaria PCR. 
6. Feeding evaluations (DSF) will also be performed at this visit. 
b. Confirm eligible for DSF  
 
Study Day 532 (PILOT) + 476 (MAIN) (±7 days; 84 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1 mL of blood for malaria blood smear, malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 546 (PILOT) + 490 (MAIN) (±7 days; 98 days after Vaccination #4) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 

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4. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before proceeding. 
5. Obtain approximately 1 mL of blood for malaria blood smear and malaria PCR. 
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 560 (PILOT) + 504 (MAIN) (±7 days; 112 days after Vaccination #4) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 6 mL of blood for malaria blood smear, malaria PCR, anti-Pfs230 
antibody ELISA, standard membrane feeding assay (SMFA). 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 574 (PILOT) + 518(MAIN) (±7 days; 126 days after Vaccination #4) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable.  
4. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before proceeding.  
5. Obtain approximately 1 mL of blood for malaria blood smear and malaria PCR. 
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 588 (PILOT) + 532 (MAIN) (±7 days; 140 days after Vaccination #4) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1 mL of blood for malaria blood smear and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 602 (PILOT) + 546 (MAIN) (±28 days; 154 days after Vaccination #4) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1 mL of blood for malaria blood smear and malaria PCR. 
5. Scheduled unblinding of VU 
a. If received Pfs230D1M-EPA/ASO1 offer meningitis comparator vaccination 
 
Study Day 616 (PILOT) + 560 (MAIN) (±7 days; 168 days after Vaccination #4) 

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1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 7 mL of blood for malaria blood smear, malaria PCR, anti-Pfs230 
antibody ELISA, standard membrane feeding assay (SMFA), and transcriptional analysis. 
 
Study Day 700 (PILOT) + 644 (MAIN) (±56 days; 252 days after Vaccination #4) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable.  
4. Obtain approximately 6 mL of blood for malaria blood smear, malaria PCR and, anti-Pfs230 
antibody ELISA, standard membrane feeding assay (SMFA). 
 
 
Study Day 588 (PILOT) + 518 (MAIN) (±56 days; 336 days after Vaccination #4) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable.  
4. Obtain approximately 6 mL of blood for malaria blood smear, malaria PCR, anti-Pfs230 
antibody ELISA, standard membrane feeding assay (SMFA). 
 
 

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Arms 3e/3f (Adults 19 years of age and older): 3 Vaccinations (0, 1, 2 months) 
Study Day -7 (±7 days; Drug Treatment Visit) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 7.5 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, hemoglobin typing, malaria blood smear and malaria PCR, and transcriptional 
analysis. 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before proceeding. 
6. For females, ensure agreement and compliance with pregnancy prevention before AL 
dosing. 
7. Administer first dose of artemether/lumefantrine (AL). 
a. Additional doses of AL will occur on study days -6, -5 
Study Day 0 (day of Vaccination #1) 
The following evaluations/procedures will be completed before vaccination: 
1. Verify that informed consent was obtained.  
2. Ensure that all inclusion/exclusion criteria are met.  
3. Ensure that CBC, ALT, creatinine, HIV, and urine results from screening are within 
protocol-defined limits (see Exclusion Criteria above) before vaccinating 
4. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
5. Record vital signs (blood pressure, temperature, and heart rate). 
6. During the physical examination, study staff will educate the subject regarding signs and 
symptoms of potential adverse events (AEs) and indications for use of antipyretics if 
fever, headache, or malaise occurs. 
7. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before vaccinating.  
8. For females, ensure agreement and compliance with pregnancy prevention before 
vaccinating. 
9. Confirm that all criteria for vaccination have been met according to inclusion/exclusion 
criteria 
10. Confirm continued eligibility to receive vaccination  
11. Obtain approximately 17.5 mL of blood for CBC with differential and platelet count, 
ALT, creatinine, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA, and 
standard membrane feeding assay (SMFA), and transcriptional analysis. 
12. Administer the vaccine. 
The following evaluations/procedures will be completed following vaccination: 
1. Observe the subject for at least 30 minutes after vaccination to evaluate immediate adverse 
reactions.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Perform interim history and physical examination, focusing on any acute complaints.  
4. Record AEs and concomitant medications, if applicable. 

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Study Day 1 (±0 days; 1 day after Vaccination #1)  
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 3 (±1 day; 3 days after Vaccination #1) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 6.0 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, and transcriptional analysis. 
 
Study Day 7 (±2 days; 7 days after Vaccination #1) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 14 (±3 days; 14 days after Vaccination #1) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 11.5 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA and standard 
membrane feeding assay (SMFA). 
 
Study Day 28 (±7 days; day of Vaccination #2) 
The following evaluations/procedures will be completed before vaccination: 
1. Perform interim history and physical examination, focusing on any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. During the physical examination, study staff will educate the subject regarding signs and 
symptoms of potential adverse events (AEs) and indications for use of antipyretics if fever, 
headache, or malaise occurs. 
4. Obtain approximately 11.5 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA, and standard 
membrane feeding assay (SMFA). 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before vaccinating.  
6. For females, ensure agreement and compliance with pregnancy prevention before 
vaccinating. 
7. Confirm continued eligibility to receive vaccination. 
8. Administer the vaccine.  

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The following evaluations/procedures will be completed following vaccination: 
1. Observe the subject for at least 30 minutes after vaccination to evaluate immediate adverse 
reactions.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Perform interim history and physical examination, focusing on any acute complaints.  
4. Record AEs and concomitant medications, if applicable. 
 
Study Day 29 (±0 days; 1 day after Vaccination #2)  
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 31 (±1 day; 3 days after Vaccination #2) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 6.0 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, and transcriptional analysis. 
 
Study Day 35 (±2 days; 7 days after Vaccination #2) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 42 (±28 days; 14 days after Vaccination #2) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 11.5 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA and standard 
membrane feeding assay (SMFA). 
 
Day 56 (±56 days; day of Vaccination #3) 
The following evaluations/procedures will be completed before vaccination: 
1. Perform interim history and physical examination, focusing on any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. During the physical examination, study staff will educate the subject regarding signs and 
symptoms of potential adverse events (AEs) and indications for use of antipyretics if fever, 
headache, or malaise occurs. 
4. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before vaccinating.  

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5. For females, ensure agreement and compliance with pregnancy prevention before 
vaccinating. 
6. Obtain approximately 11.5 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA, and standard 
membrane feeding assay (SMFA). 
7. Confirm continued eligibility to receive vaccination  
8. Administer the vaccine.  
 
The following evaluations/procedures will be completed following vaccination: 
1. Observe the subject for at least 30 minutes after vaccination to evaluate immediate adverse 
reactions.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Perform interim history and physical examination, focusing on any acute complaints.  
4. Record AEs and concomitant medications, if applicable. 
 
Study Day 57 (±0 days; 1 day after Vaccination #3)  
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 59 (±1 day; 3 days after Vaccination #3) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 6.0 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, and transcriptional analysis. 
 
Study Day 63 (±2 days; 7 days after Vaccination #3) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 5.0 mL of blood for anti-Pfs230 antibody ELISA, and standard 
membrane feeding assay (SMFA). 
 
Study Day 70 (±7 days; 14 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 17.5 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA, standard 
membrane feeding assay (SMFA) and transcriptional analysis. 
 

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Study Day 84 (±7 days; 28 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 112 (±7 days; 56 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 6.5 mL of blood for malaria blood smear, malaria PCR, anti-Pfs230 
antibody ELISA, standard membrane feeding assay (SMFA). 
 
Study Day 140 (±7 days; 84 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 168 (±7 days; 112 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable.  
4. Obtain approximately 6.5 mL of blood for malaria blood smear, malaria PCR, anti-Pfs230 
antibody ELISA, standard membrane feeding assay (SMFA). 
 
Study Day 196 (±7 days; 140 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 224 (±28 days; 168 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable.  
4. Obtain approximately 7.5 mL of blood for malaria blood smear, malaria PCR, anti-Pfs230 
antibody ELISA, standard membrane feeding assay (SMFA), and transcriptional analysis. 
 
Study Day 308 (±56 days; 252 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 

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3. Record AEs and concomitant medications, if applicable.  
4. Obtain approximately 6.5 mL of blood for malaria blood smear, malaria PCR, anti-Pfs230 
antibody ELISA, standard membrane feeding assay (SMFA). 
 
Study Day 392 (±56 days; 336 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable.  
4. Obtain approximately 6.5 mL of blood for malaria blood smear, malaria PCR, anti-Pfs230 
antibody ELISA, standard membrane feeding assay (SMFA). 
 
Study Day 378 (±84 days; 322 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before vaccinating.  
5. Obtain approximately 12.5 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA and standard 
membrane feeding assay (SMFA) and transcriptional analysis. 
 
Day 392 (±112 days; day of Vaccination #4) 
The following evaluations/procedures will be completed before vaccination: 
1. Perform interim history and physical examination, focusing on any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. During the physical examination, study staff will educate the subject regarding signs and 
symptoms of potential adverse events (AEs) and indications for use of antipyretics if fever, 
headache, or malaise occurs. 
4. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before vaccinating.  
5. For females, ensure agreement and compliance with pregnancy prevention before 
vaccinating. 
6. Obtain approximately 11.0 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA, and standard 
membrane feeding assay (SMFA). 
7. Confirm continued eligibility to receive vaccination.  
8. Administer the vaccine.  
 
The following evaluations/procedures will be completed following vaccination: 
1. Observe the subject for at least 30 minutes after vaccination to evaluate immediate adverse 
reactions.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Perform interim history and physical examination, focusing on any acute complaints.  
4. Record AEs and concomitant medications, if applicable. 

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Study Day 395 (±1 day; 3 day after Vaccination #4)  
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 6.0 mL of blood for CBC with differential and platelet count, ALT and 
transcriptional analysis. 
 
Study Day 399 (±2 days; 7 day after Vaccination #4)  
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 5.0 mL of blood anti-Pfs230 antibody ELISA, and standard membrane 
feeding assay (SMFA). 
 
Study Day406 (±7 day; 14 days after Vaccination #4) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 17.0 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA, standard 
membrane feeding assay (SMFA) and transcriptional analysis. 
 
Study Day 420 (±7 days; 28 days after Vaccination #4) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 448 (±7 days; 56 days after Vaccination #4) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 6.0 mL of blood for malaria blood smear, malaria PCR, anti-Pfs230 
antibody ELISA, standard membrane feeding assay (SMFA). 
 
Study Day 504 (±7 days; 112 days after Vaccination #4) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 

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4. Obtain approximately 6.0 mL of blood for malaria blood smear, malaria PCR, anti-Pfs230 
antibody ELISA, standard membrane feeding assay (SMFA). 
 
Study Day 546  (±28 days; 154 days after Vaccination #4) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 7.0 mL of blood for malaria blood smear, malaria PCR, anti-Pfs230 
antibody ELISA, standard membrane feeding assay (SMFA) and transcriptional analysis. 
5. Scheduled unblinding of VU 
a. If received Pfs230D1M-EPA/ASO1 offer meningitis comparator vaccination 
 
Study Day 644 (±56 days; 252 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable.  
4. Obtain approximately 6.0 mL of blood for malaria blood smear, malaria PCR, anti-Pfs230 
antibody ELISA, standard membrane feeding assay (SMFA). 
 
Study Day 728 (±56 days; 336 days after Vaccination #4) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 6.0 mL of blood for malaria blood smear, malaria PCR, anti-Pfs230 
antibody ELISA, standard membrane feeding assay (SMFA). 
` 
 
 

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Appendix C: Toxicity Tables 
Local Reactogenicity Grading1 
Local Reaction to 
Injectable Product 
Mild  
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially Life 
Threatening 
(Grade 4) 
Pain at site 
Does not 
interfere 
with activity 
Repeated use of 
non-narcotic 
pain reliever 
> 24 hours or 
interferes with 
activity 
Any use of 
narcotic pain 
reliever or 
prevents daily 
activity 
ER visit or 
hospitalization 
Erythema/Redness at site2          
>15 years of age 
2.5 – 5 cm 
5.1 – 10 cm 
> 10 cm 
Necrosis or 
exfoliative 
dermatitis 
≤ 15 years of age 
≤ 2.5 cm in 
diameter 
> 2.5 cm in 
diameter with < 
50% surface 
area of the 
extremity 
segment 
involved (e.g., 
upper arm or 
thigh) 
≥ 50% surface area 
of the extremity 
segment involved 
(e.g., upper arm or 
thigh) 
Potentially life- 
threatening 
consequences (e.g., 
abscess, exfoliative 
dermatitis, necrosis 
involving dermis or 
deeper tissue) 
Induration/Swelling at site3                
>15 years of age 
2.5 – 5 cm and 
does not 
interfere 
with activity 
5.1 – 10 cm or 
interferes with 
activity 
> 10 cm or 
prevents daily 
activity 
Necrosis 
≤ 15 years of age 
≤ 2.5 cm in 
diameter 
> 2.5 cm in 
diameter with < 
50% surface 
area of the 
extremity 
segment 
involved (e.g., 
upper arm or 
thigh) 
≥ 50% surface area 
of the extremity 
segment involved 
(e.g., upper arm or 
thigh) 
Potentially life- 
threatening 
consequences (e.g., 
abscess, exfoliative 
dermatitis, necrosis 
involving dermis or 
deeper tissue) 
Bruising at site2                                        

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>15 years of age 
2.5 – 5 cm 
5.1 – 10 cm 
> 10 cm 
Necrosis or 
exfoliative 
dermatitis 
≤ 15 years of age 
≤ 2.5 cm in diameter 
> 2.5 cm in diameter 
with < 50% surface 
area of the extremity 
segment involved 
(e.g., upper arm or 
thigh) 
≥ 50% surface area of the 
extremity segment 
involved (e.g., upper arm 
or thigh) 
Potentially life- 
threatening consequences 
(e.g., abscess, exfoliative 
dermatitis, necrosis 
involving dermis or 
deeper tissue) 
Pruritus at Site 
Does not 
interfere 
with activity 
Repeated use of 
medication > 24 
hours or 
interferes with 
activity 
Prevents daily 
activity 
ER visit or 
hospitalization 
Limitation of Arm 
Movement 
Does not 
interfere 
with activity 
Repeated use of 
medication > 24 
hours or 
interferes with 
activity 
Prevents daily 
activity 
ER visit or 
hospitalization 
Abbreviations: ER, emergency room. 
1 The definitions provided in the table are modified versions taken from the FDA Guidance for Industry “Toxicity 
Grading Scale for Healthy Adults and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials” dated 
September 2007 and DAIDS Table for Grading the Severity of Adult and Pediatric Adverse Events version 2.1 July 
2017. 
2 In addition to grading the measured local reaction at the greatest single diameter, the measurement should be 
recorded as a continuous variable.  
3 Induration/Swelling should be evaluated and graded using the functional scale as well as the actual measurement. 
. 
 
 

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Vital Sign AE Grading1 
Vital Signs2  
Mild  
(Grade 1) 
Moderate 
(Grade 2) 
Severe  
(Grade 3) 
Potentially Life 
Threatening 
(Grade 4) 
Fever3 (°C) 
            (°F) 
38.0 – 38.4 
100.4 – 101.1 
38.5 – 38.9 
101.2 – 102.0 
39.0 – 40 
102.1 – 104 
> 40 
> 104 
Tachycardia - beats 
per minute; at rest + 
calm 
 
 
 
 
Adult (≥13 yo) 101 – 115 
116 – 130 
> 130 
ER visit or 
hospitalization 
for 
arrhythmia 
Pediatric (≤ 12 yo)  121 – 135 
136 -- 150 
>150 
ER visit or 
hospitalization 
for 
arrhythmia 
Bradycardia - beats 
per minute4; at rest + 
calm 
 
 
 
 
Adult (≥13 yo) 50 – 54 
45 – 49 
< 45 
ER visit or 
hospitalization 
for 
arrhythmia 
Pediatric (≤ 12 yo)  55 -- 59 
50 – 54 
< 50 
ER visit or 
hospitalization 
for 
arrhythmia 
Hypertension (systolic) 
-mm Hg; at rest + calm  
 
 
 
Adult (≥13 yo) 141 – 150 
151 – 155 
> 155 
ER visit or 
hospitalization 
for 
malignant 
hypertension 
Pediatric (≤ 12 yo) 131 -- 140 
141 -- 150 
> 150 
ER visit or 
hospitalization 
for 
malignant 
hypertension 

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Hypertension 
(diastolic) -mm Hg; at 
rest + calm 
 
 
 
 
Adult (≥13 yo) 91 – 95 
96 – 100 
> 100 
ER visit or 
hospitalization 
for 
malignant 
hypertension 
Pediatric (≤ 12 yo) 81 -- 90 
91 – 95 
> 95 
ER visit or 
hospitalization 
for 
malignant 
hypertension 
Hypotension (systolic) 
-mm Hg; at rest + calm  
 
 
 
Adult (≥13 yo) 85 – 89 
80 – 84 
< 80 
ER visit or 
hospitalization 
for 
hypotensive 
shock 
Pediatric (≤ 12 yo) 80 – 84 
75 – 79 
< 75 
ER visit or 
hospitalization 
for 
hypotensive 
shock 
Abbreviations: ER, emergency room. 
1 The definitions provided in the table are taken from the FDA Guidance for Industry “Toxicity Grading Scale for 
Healthy Adults and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials” dated September 2007. 
2 Subject should be at rest for all vital sign measurements.  
2 Oral temperature; no recent hot or cold beverages or smoking.  
3 When resting heart rate is between 60 – 100 beats per minute. Use clinical judgment when characterizing  
bradycardia among some healthy subject populations, for example, conditioned athletes. 
 
 

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Systemic AE Grading1 
Systemic 
AEs 
Mild  
(Grade 1) 
Moderate 
(Grade 2) 
Severe  
(Grade 3) 
Potentially 
Life 
Threatening 
(Grade 4) 
Diarrhea 
2 – 3 loose 
stools or 
< 400 gms/24 
hours 
4 – 5 stools or 
400 – 800 gms/24 
hours 
6 or more watery 
stools or 
> 800 gms/24 hours 
or requires outpatient 
IV hydration 
ER visit or 
hospitalization 
Headache 
No 
interference 
with activity 
Repeated use of non- 
narcotic pain 
reliever > 24 hours or 
some interference 
with activity 
Significant; any use 
of narcotic 
pain reliever or 
prevents daily 
activity 
ER visit or 
hospitalization 
Malaise 
No 
interference 
with activity 
Some interference 
with activity 
Significant; prevents 
daily activity 
ER visit or 
hospitalization 
Myalgia 
No 
interference 
with activity 
Some interference 
with activity 
Significant; prevents 
daily activity 
ER visit or 
hospitalization 
Arthralgia 
No 
interference 
with activity 
Some interference 
with activity 
Significant; prevents 
daily activity 
ER visit or 
hospitalization 
Nausea/ 
Vomiting 
No 
interference 
with activity 
or 1 – 2 
episodes/24 
hours 
Some interference 
with activity or > 2 
episodes/24 hours 
Prevents daily 
activity, requires 
outpatient IV 
hydration 
ER visit or 
hospitalization 
for 
hypotensive 
shock 

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Systemic 
AEs 
Mild  
(Grade 1) 
Moderate 
(Grade 2) 
Severe  
(Grade 3) 
Potentially 
Life 
Threatening 
(Grade 4) 
Seizures- 
<18 years 
of age; 
includes 
new or pre-
existing 
febrile 
seizures 
Seizure 
lasting < 5 
minutes with 
< 24 hours 
postictal state 
Seizure lasting 5 to < 
20 minutes with 
< 24 hours postictal 
state 
Seizure lasting ≥ 20 
minutes OR > 24 
hours postictal state 
 
Prolonged and 
repetitive 
seizures (e.g., 
status 
epilepticus) 
OR Difficult to 
control (e.g., 
refractory 
epilepsy) 
Abbreviations: ER, emergency room; IV, intravenous; PO, “per os” or oral administration. 
1 The definitions provided in the table are modified versions taken from the FDA Guidance for Industry “Toxicity 
Grading Scale for Healthy Adults and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials” dated 
September 2007 and DAIDS Table for Grading the Severity of Adult and Pediatric Adverse Events version 2.1 July 
2017. 
 

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Mali Laboratory AE Grading ≥15 years of age 
Hematology and 
Biochemistry Values1, 2 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially Life 
Threatening (Grade 
4) 
Hemoglobin (Male) - 
gm/dL  
9.5 – 10.3 
8.0 – 9.4 
6.5 – 7.9 
< 6.5 and/or requiring 
transfusion 
Hemoglobin (Female)  
gm/dL  
8.0 – 9.0 
7.0 – 7.9 
6.0 – 6.9 
< 6 and/or requiring 
transfusion 
WBC Increase - 103/µL 
11.5 – 15.0 
15.1 – 20.0 
20.1 – 25.0 
> 25.0 
WBC Decrease - 103/µL 
2.5 – 3.3 
1.5 – 2.4 
1.0 – 1.4 
< 1.0 with fever 
Neutrophil/Granulocyte 
Decrease3 - 103/µL 
0.80 – 1.00 
0.50 – 0.79 
< 0.50 
< 0.50 with fever 
Platelets Decreased - 
103/µL 
100 – 110 
70 – 99 
25 – 69 
< 25 
Creatinine (Male) - 
µmol/L 
124.00 – 150.99 
151.00 – 176.99 177.00 – 221.00 
> 221.00 and requires 
dialysis 
Creatinine (Female) - 
µmol/L 
107.00 – 132.99 
133.00 – 159.99 160.00 – 215.99 
> 216.00 and requires 
dialysis 
Liver Function 
Tests/ALT - U/L 
75.0 – 150.9 
151.0 – 300.9 
301.0 – 600.0 
> 600.0 
Abbreviations: ALT, alanine transaminase; WBC, white blood cell. 
1 The laboratory values provided in the tables serve as guidelines and are dependent upon institutional normal 
parameters. Institutional normal reference ranges should be provided to demonstrate that they are appropriate.  
2 The clinical signs or symptoms associated with laboratory abnormalities might result in characterization of the 
laboratory abnormalities as Potentially Life Threatening (Grade 4). For example, a low sodium value that falls 
within a grade 3 parameter should be recorded as a grade 4 hyponatremia event if the subject had a new seizure 
associated with the low sodium value.  
3 Note, neutropenias are graded and followed, but based on previous experience in African populations, should be 
interpreted with caution since lower values are more frequently observed in people of African descent. 88,89 
 
 
 
 
 
 
 

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Mali Laboratory AE Grading <15 years of age 
Hematology and 
Biochemistry Values1, 2 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially Life 
Threatening (Grade 4) 
Hemoglobin 
(Male/Female) - gm/dL  
 
7.5 – 8.4 
 
6.1 – 7.4 
 
5.0 – 6.0 
 
< 5.0 g/dL 
 
WBC Increase - 103/µL 
14.5 – 16.0 
16.1 – 20.0 
20.1 – 30.0 
>30.0 
WBC Decrease - 103/µL 
2.5 – 3.3 
1.5 – 2.4 
1.0 – 1.4 
< 1.0 with or without 
fever 
Neutrophil/Granulocyte 
Decrease3 - 103/µL 
0.75 – 0.99 
0.50 – 0.74 
< 0.50 
< 0.50 with fever 
Platelets Decreased - 
103/µL 
100 – 120 
70 – 99 
25 – 69 
< 25 
Creatinine 
(Male/Female) - µmol/L 
95.00 – 119.99 
120.00 – 149.99 150.00 – 200.00 
> 200.00 and requires 
dialysis 
Liver Function 
Tests/ALT - U/L 
75.0 – 150.9 
151.0 – 300.9 
301.0 – 600.0 
> 600.0 
Abbreviations: ALT, alanine transaminase; WBC, white blood cell. 
1 The laboratory values provided in the tables serve as guidelines and are dependent upon institutional normal 
parameters. Institutional normal reference ranges should be provided to demonstrate that they are appropriate.  
2 The clinical signs or symptoms associated with laboratory abnormalities might result in characterization of the 
laboratory abnormalities as Potentially Life Threatening (Grade 4). For example, a low sodium value that falls 
within a grade 3 parameter should be recorded as a grade 4 hyponatremia event if the subject had a new seizure 
associated with the low sodium value.  
3 Note, neutropenias are graded and followed, but based on previous experience in African populations, should be 
interpreted with caution since lower values are more frequently observed in people of African descent. 88,89 

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Appendix D: Institutional Normal Laboratory Values 
 
Mali Adult (≥15 years of age): Institutional Normal Parameters  
Chemistry 
Chemistry1 
Reference Range 
Creatinine (Female) - µmol/L 
< 72 
Creatinine (Male) -µmol/L 
48 – 98 
ALT - U/L 
< 41 
Abbreviations: ALT, alanine transaminase. 
1 The laboratory values provided in the table are based on Bancoumana, Malian adults (age 18-45 years old). 
 
Hematology 
Hematology1 
Reference Range 
Hemoglobin (Female) - gm/dL 
9.1 – 13.8 
Hemoglobin (Male) - gm/dL 
10.8 – 15.8 
WBC - 103/µL 
3.6 – 9.0 
Absolute Neutrophil/Granulocyte Count - 
103/µL 
1.3 – 4.4 
Absolute Lymphocyte Count - 103/µL 
1.3 – 4.4 
Platelet Count (Female) - 103/µL 
144 – 413 
Platelet Count (Male) - 103/µL 
114 – 335 
Abbreviations: ALT, alanine transaminase; WBC, white blood cell. 
1 The laboratory values provided in the table are based on Bancoumana, Malian adults (age 18-45 years old). 
 
Urine Dip/Urinalysis 
Urine1 
Reference Ranges 
Protein 
None or Trace 
Blood 
(microscopic) – 
RBC/HPF 
None or Trace 
 
< 5 
Abbreviations: HPF, high power field; RBC, red blood cell. 
1 The laboratory values provided in the table are based on Bancoumana, Malian adults (age 18-45 years old) 
 
 
 
 
 
 
 
 
 
 

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Children (5-15 years of age) 
 
Hematology Reference Intervals* 
Hematology 
Reference 
Interval 
Units 
WBC- Leukocytes 
4.5 – 10.5 
103/μL 
Hemoglobin 
9.6 – 13.5 
g/dL 
Platelet Count 
138– 455 
103/μL 
Absolute Lymphocyte Count 
1.8 – 5.4 
103/μL 
Absolute Granulocyte Count** 
1.12 – 6.86 
103/μL 
Based on children ranging in age from 6 to 14 years old (based on sampling done at Doneguebougou site, Mali) 
**Based on reference intervals for a Ugandan population, 
 
Biochemistry Reference Intervals* 
 
Chemistry 
Reference 
Interval 
Units 
ALT 
5.06 – 53.4 
U/L 
Creatinine 
< 48.9 
μM/L 
Parameters from Pediatric Tables in Mali; MRTC, Dept of Epidemiology and Parasitic Diseases, 
Faculty of Medicine, Pharmacy and Odontostomatology, University of Bamako, Mali 
. 
 

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