Phase 1, Dose-Escalating, Double-Blind, Randomized, Comparator-Controlled Trial of the Safety, Tolerability, and Immunogenicity of the Transmission-Blocking Vaccine Pfs230D1-EPA/Matrix-M™ Against Plasmodium Falciparum in Adults in Mali
Phase: PHASE1
Conditions: Malaria,Falciparum
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

Phase 1, Dose-Escalating, Double-Blind, Randomized, Comparator-Controlled Trial of the 
Safety, Tolerability, and Immunogenicity of the Transmission-Blocking Vaccine 
Pfs230D1-EPA/Matrix-M™ against Plasmodium falciparum in Adults in Mali 
 
 
 
FMPOS Protocol Number: 2021/210/CE/USTTB 
FMPOS Project Federalwide Assurance (FWA): #00001769 
 
NIH Protocol Number: 000576 
NIH Project FWA: #00005897 
 
 
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: 27670 
 
Version 3.0 
November 09, 2022 
 
 
CONFIDENTIAL 
 
 

 
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Team Roster 
NIH Collaborators 
 
NIAID Senior Investigator: 
Patrick Duffy, MD 
LMIV/NIAID 
BG 29B RM 4NN06 
9000 Rockville Pike 
Bethesda, MD 20814 USA 
Tel: 301-761-5089 
Email: patrick.duffy@nih.gov  
 
NIAID Associate Investigators: 
Judith Epstein, MD 
David Cook, MD 
Sara Healy, MD, MPH 
Jen Hume, DPhil 
Jennifer Kwan, PhD 
 
Biostatisticians: 
Jennifer Kwan, PhD  
Jonathan Fintzi, PhD 
Bruce Swihart, PhD 
 
 
MRTC/FMPOS/USTTB Investigators  
 
MRTC Principal Investigator:  
 
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 
 
MRTC Sub-Investigators: 
 
Mahamadou S. Sissoko, MD, MSPH, PhD   (mssissoko@icermali.org) 
Abdoulaye Katile, MD, MSPH (katile@icermali.org) 
Mahamadoun Hamady Assadou, MD, MPH (mmaiga@icermali.org) 
Mamady Kone, MD, MPH (mamady@icermali.org) 
Bourama Kamate, MD (boukamate@icermali.org) 
Kourane Sissoko, MD (kouranes@icermali.org) 
Bayaya Haidara, MD (bayayah@icermali.org) 

 
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Amatigue Zeguimé, PharmD (amatigue@icermali.org) 
Kadidia Baba Cisse, BSc (kadidia@icermali.org) 
Kalifa Diarra, PharmD (kalifad@icermali.org) 
Daman Sylla, MD (dsylla@icermali.org) 
Mamadou B. Coulibaly, PharmD, PhD (doudou@icermali.org) 
Amagana Dolo, PharmD, PhD (adolo@icermali.org) 
Amadou Niangaly, PharmD, PhD (niangaly@icermali.org) 
 
 
Safety Oversight 
 
Data and Safety Monitoring Board (DSMB): 
NIAID DSMB 
 
Sponsor Medical Monitor (SMM): 
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 
 
Independent Safety Monitor (ISM): 
Yacouba Cissoko, MD, MSc, MBA  
Infectious Diseases, Immunology, Health Program Management 
Assistant Professor, Faculty of Medicine and Dentistry/USTTBBP 1805, Point G & Point G 
Teaching Hospital 
Bamako, Mali 
Cell:+ 223 74 56 76 49 
Email: ycissoko@icermali.org 
 
Ethics Committee (EC) 
 
FMPOS USTTB EC 
Point G 
Bamako, Mali 
 
 
Clinical Trial Site 
 
Sotuba Mali, West Africa and surrounding villages 
MRTC, 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, 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 
 
NIH Intramural Sequencing Center 

 
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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 
 
 
 
 

 
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Table of Contents 
 
Team Roster .................................................................................................................................. 2 
Table of Contents .......................................................................................................................... 6 
List of Tables ................................................................................................................................. 9 
List of Figures ................................................................................................................................ 9 
List of Abbreviations .................................................................................................................. 11 
Protocol Synopsis ........................................................................................................................ 14 
1 
Background Information and Scientific Rationale .......................................................... 17 
 
Development of the Study Agent, Pfs230D1-EPA/ Matrix-M ............................. 18 
 
Study Plan ............................................................................................................. 22 
2 
Previous Preclinical Experience with Pfs230D1-EPA/Matrix-M ................................... 23 
 
Pfs230D1-EPA/Matrix-M Serology Assessments by ELISA............................... 25 
 
Pfs230D1-EPA/Matrix-M Serology Assessments by ELISA............................... 26 
 
Toxicology Study in Rabbits ................................................................................ 27 
3 
Previous Clinical Experience with Pfs230D1M-EPA ...................................................... 27 
 
Safety of Pfs230D1M-EPA/Alhydrogel in Healthy Adults .................................. 27 
 
Immunogenicity and Functional Activity of Pfs230D1M-EPA/Alhydrogel in 
Healthy Adults ...................................................................................................................... 28 
 
Safety of Pfs230D1M-EPA/AS01 in Healthy Adults ........................................... 32 
 
Immunogenicity and Functional Activity (SMFA) of Pfs230D1M-EPA/AS01 in 
Healthy Adults (Primary Series + 4th Dose; Year 1 + 2) ...................................................... 33 
 
Functional Activity by Direct Skin Feeds ............................................................. 37 
 
DSF Analysis as an endpoint ................................................................................ 38 
 
Safety .................................................................................................................... 39 
 
Immunogenicity in Healthy Malian Children ....................................................... 39 
 
Vaccine Efficacy in Healthy Malian Children ...................................................... 41 
 
Summary of Clinical Experience with Matrix-M ................................................. 42 
4 
Study Objectives .................................................................................................................. 45 
5 
Study Design ........................................................................................................................ 45 
6 
Study Population ................................................................................................................. 48 

 
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Justification of Exclusion of Pregnant Women .................................................... 50 
 
Justification for Exclusion of Children ................................................................. 51 
7 
Study Agents ........................................................................................................................ 51 
 
Manufacturing ....................................................................................................... 51 
 
Disposition and Dispensation ............................................................................... 51 
 
Formulation, Packaging, and Labeling ................................................................. 52 
 
Storage, Shipping, and Stability ........................................................................... 53 
 
Preparation and Dosage ........................................................................................ 53 
 
Manufacturing ....................................................................................................... 53 
 
Disposition and Dispensation ............................................................................... 53 
 
Formulation, Packaging, and Labeling ................................................................. 53 
 
Storage, Shipping, and Stability ........................................................................... 54 
 
Preparation and Dosage ........................................................................................ 54 
8 
Study Schedule .................................................................................................................... 56 
9 
Study Procedures/Evaluations ........................................................................................... 58 
 
Blood Smears ........................................................................................................ 59 
 
ELISA ................................................................................................................... 59 
 
Transmission Assays ............................................................................................. 60 
 
Standard Membrane Feeding Assays .................................................................... 60 
 
Antibody Assay ..................................................................................................... 61 
 
B-Cell and T-Cell Assays ..................................................................................... 61 
 
Transcriptional Profiling ....................................................................................... 62 
10 Research Use of Stored Human Samples, Specimens, or Data ....................................... 63 
11 Data Sharing Plan ............................................................................................................... 64 

 
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12 Assessment of Safety ........................................................................................................... 64 
 
Severity ................................................................................................................. 69 
 
Causality ............................................................................................................... 70 
 
Adverse Events ..................................................................................................... 72 
 
Serious Adverse Events ........................................................................................ 72 
 
Unanticipated Problems ........................................................................................ 73 
 
Pregnancy .............................................................................................................. 73 
 
Medically Attended Adverse Events (MAAEs) that are Potential Immune-
Mediated Medical Conditions (PIMMCs) ............................................................................ 74 
 
Definitions............................................................................................................. 75 
 
Expedited Reporting to FMPOS EC ..................................................................... 76 
 
Annual Reporting to FMPOS EC ......................................................................... 76 
 
Pausing Rules for an Individual Subject ............................................................... 77 
 
Reporting a Pause ................................................................................................. 77 
 
Resumption of a Paused Study ............................................................................. 77 
 
Reporting a Study Halt.......................................................................................... 78 
 
Resumption of a Halted Study .............................................................................. 78 
 
Replacement of Withdrawn Participants .............................................................. 80 
 
Safety Review and Communications Plan ............................................................ 80 
 
Sponsor Medical Monitor ..................................................................................... 80 
 
Independent Safety Monitor (ISM) ....................................................................... 80 
 
Data and Safety Monitoring Board (DSMB) ........................................................ 81 
13 Site Monitoring Plan ........................................................................................................... 81 
14 Statistical Considerations and Sample Size ...................................................................... 82 
 
ELISA Analysis .................................................................................................... 83 
 
SMFA Analysis ..................................................................................................... 84 
 
Analysis................................................................................................................. 84 
 
Randomization ...................................................................................................... 84 
 
Blinding................................................................................................................. 84 

 
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Unblinding ............................................................................................................ 85 
15 Ethics/Protection of Human Subjects ............................................................................... 85 
 
Mali Site Community Permission and Individual Informed Consent Process ..... 86 
 
Study Vaccines...................................................................................................... 88 
 
Treatment for Malaria ........................................................................................... 90 
 
Venipuncture ......................................................................................................... 90 
16 Data Handling and Record Keeping ................................................................................. 91 
17 Role of the NIH Collaborators/Investigators.................................................................... 93 
Appendix A. Schedule of Assessments and Day-to-Day Schedule .......................................... 94 
Appendix B. Toxicity Tables .................................................................................................... 101 
Appendix C. Mali Adult Institutional Normal Laboratory Values ...................................... 107 
Appendix D.  Potential Immune-Mediated Medical Conditions........................................... 108 
Appendix E. References............................................................................................................ 109 
 
 
List of Tables 
Table 1. Immunization Schedule (example) ................................................................................. 23 
Table 2.  Immunogenicity of Pfs230D1-EPA Formulated with Alhydrogel or Matrix-M in CD-1 
Mice .............................................................................................................................................. 24 
Table 3. NIAID Protocol #15-I-0044 Enrollment and Vaccinations. ........................................... 27 
Table 4. Summary of DSF Results. .............................................................................................. 38 
Table 5. Vaccine efficacy based on DSFs for Year 1 and Year 2, full dose arm. ........................ 38 
Table 6. Vaccine Efficacy Based on DSFs for Year 1 and Year 2. .............................................. 41 
Table 7. Key Clinical Experience of Matrix-M with Various Vaccine Antigens. ........................ 43 
Table 8. Transmission-Blocking Assay ........................................................................................ 60 
Table 9. Solicited Adverse Events ................................................................................................ 69 
Table 10. Definitions for Severity of AE Grading ........................................................................ 70 
Table 11. Estimated Compensation Schedule 1 ............................................................................. 91 
 
List of Figures 
Figure 1. Life Cycle of Malaria Parasite Plasmodium ssp. (Su, Hayton et al. 2007) ................... 17 

 
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Figure 2. Protein Alignments of Recombinant Pfs230D1M to its Respective Native Protein or 
Protein Fragment. .......................................................................................................................... 19 
Figure 3. Comparison of Rhesus Antibody Responses (IgG) by ELISA (A) and TRA by SMFA 
(B) Following Immunization with Conjugated Pfs230D1-EPA in AS01 (GlaxoSmithKline) or 
Matrix-M (Novavax). .................................................................................................................... 21 
Figure 4. Dose dependent Pfs230D1M specific IgG responses by ELISA in CD-1 mice on 
multiple bleed days (28-154) following IM immunization on days 0 and 28. ............................. 24 
Figure 5. Rabbit antibody responses following immunization with Control (Saline), 40 μg 
Pfs230D1-EPA/50 μg Matrix-M (Group 4). ................................................................................. 25 
Figure 6. Transmission blocking activity by SMFA in rabbits ..................................................... 26 
Figure 7. Pfs230-specific Antibody Responses in Subjects Receiving Pfs230D1M, US Cohort 
(#15-I-0044). ................................................................................................................................. 29 
Figure 8. Pfs25 and Pfs230 Functional Activity by Standard Membrane Feeding Assay. ........... 30 
Figure 9. Pfs230 Functional Activity by Standard Membrane Feeding Assay in the Presence or 
Absence of Complement. .............................................................................................................. 30 
Figure 10. Pfs230-specific Antibody Responses in Subjects Receiving Pfs230D1M, 40 µg, in 
Mali (#15-I-0044). ........................................................................................................................ 31 
Figure 11. Antibody Responses to Pfs230D1M-EPA/AS01 after Low Dose and High Dose 
Vaccinations of Adults in Sotuba, Mali during Pilot Phase trial. ................................................. 33 
Figure 12. 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.
....................................................................................................................................................... 34 
Figure 13. Antibodies Against Pfs230D1M Measured by ELISA 12 Weeks Post Second and 
Third Vaccination in Bancoumana/Doneguebougou, Mali During Main Phase Trial. ................ 35 
Figure 14. ELISA Against Pfs230D1 Antigen. ............................................................................ 36 
Figure 15. Transmission-Reducing Activity Measured by SMFA 12 Weeks Post Second and 
Third Vaccinations in Bancoumana/Doneguebougou, Mali during Main Phase trial. ................. 37 
Figure 16. Pfs230 ELISA Results During Year 1 in Pilot Subjects.............................................. 40 
Figure 17. Comparison of Subset of DSF Cohort (9- to 18-Year-Old Subjects) by Vaccine Arm 
and DSF Positivity. ....................................................................................................................... 40 
Figure 18. Formulation of Pfs230D1M-EPA and Matrix-M for each dose level of Pfs230D1-
EPA/Matrix-M vaccine. ................................................................................................................ 52 
 
 
 

 
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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’Odonto Stomatologie 
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 
MAAEs 
Medically Attended Adverse Events  
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 
PIMMCs  
Potentially Immune-Mediated Medical Conditions  
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 
thick blood smear  
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 

 
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WBC 
white blood cell 
WHO 
World Health Organization 
WMW 
Wilcoxon-Mann-Whitney test 
WSRT 
Wilcoxon signed rank test 
 
 
 

 
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Protocol Synopsis 
Full Title:  
 Phase 1, Dose-Escalating, Double-Blind, Randomized, 
Comparator-Controlled Trial of the Safety, Tolerability, and 
Immunogenicity of the Transmission-Blocking Vaccine 
Pfs230D1-EPA/ Matrix-M™ against Plasmodium falciparum in 
Adults in Mali 
Short Title: 
Pfs230 Antigen/Adjuvant Study in Mali 
Clinical Phase: 
Phase 1 
IND Sponsor: 
OCRPRO/NIAID/NIH 
Clinical Sponsor:  
OCRPRO/NIAID 
Conducted by: 
MRTC, in collaboration with LMIV 
Supported by:  
PfTBV European & Developing Countries Clinical Trials 
Partnership (EDCTP) Consortium (University of Bamako)  
Principal Investigators:  
MRTC: Issaka Sagara, MD, MSPH, PhD (MRTC/DEAP/FMPOS)  
 
LMIV: Patrick Duffy (LMIV/NIAID/NIH) 
Study Agent Description: 
Pfs230D1-EPA/Matrix-M: Recombinant Pfs230 domain 1 
(Pfs230D1; a subdomain of a surface antigen of gametocytes, 
gametes, and zygotes, in the mosquito stage of Plasmodium 
falciparum [Pf]) conjugated to a recombinant Pseudomonas 
aeruginosa ExoProtein A (EPA) and adjuvanted with Matrix-M. It 
is administered as an intramuscular (IM) injection at the following 
dose levels: 
• 
12.5 µg Pfs230D1-EPA/25 µg Matrix-M 
• 
20 µg Pfs230D1-EPA/50 µg Matrix-M 
• 
40 µg Pfs230D1-EPA/50 µg Matrix-M 
Verorab Rabies Vaccine: One dose consists of the administration 
of 0.5 mL of vaccine via the intramuscular route and contains 
rabies virus, WISTAR Rabies PM/WI38 1503-3M strain 
(inactivated) ≥ 2.5 IU. 
Subject Sample Size: 
N=80 (n=60 Pfs230D1-EPA/Matrix-M; n=20 rabies control)  
Accrual Ceiling: 
N=150 
Accrual Period: 
September 2021-October 2021  
Study Duration:  
 
Start Date: approximately September 2021 

 
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End Date: approximately June2023 (includes data analysis)  
 
 
Study participants will be enrolled for a total of approximately 14 
to 16 months depending upon timing of screening   
Study Population: 
 
Healthy male and female adults (18 to 50 years of age) who reside 
in Sotuba and surrounding villages, Mali 
Study Design: 
 
This is a Phase 1, dose-escalating, randomized, double-blind, 
comparator-controlled study to assess the safety, tolerability, 
immunogenicity and transmission-blocking activity (TBA) of a 
3-dose regimen of Pfs230D1-EPA/Matrix-M versus rabies vaccine 
in healthy adults. This will be a first-in-human assessment of 
Pfs230D1-EPA/Matrix-M. Participants will be randomized to one 
of the study arms. Participants will be followed for 12 months from 
the last dose of study vaccine for safety and tolerability, as well as 
immunogenicity and functional antibody responses. 
 
 
The study groups and arms are as follows. For all participants, the 
assigned study vaccine will be administered at Days 1, 29, and 57.  
Group 1: Pilot Group 
• Arm 1a (n=5): 12.5 µg Pfs230D1-EPA/25 µg Matrix-M 
• Arm 1b (n=5): 20 µg Pfs230D1-EPA/50 µg Matrix-M 
• Arm 1c (n=5): 40 µg Pfs230D1-EPA/50 µg Matrix-M 
• Arm 1d (n=4): rabies vaccine (standard dose) 
Group 2: Main Group 
• Arm 2a (n=15): 12.5 µg Pfs230D1-EPA/25 µg Matrix-M 
• Arm 2b (n=15): 20 µg Pfs230D1-EPA/50 µg Matrix-M 
• Arm 2c (n=15): 40 µg Pfs230D1-EPA/50 µg Matrix-M 
• Arm 2d (n=16): rabies vaccine (standard dose) 
 
Study Objectives:  
 
Primary Objective:  
• To assess in African adults the safety and the reactogenicity of 
administration of Pfs230D1-EPA/Matrix-M (first-in-human) as 
compared to the rabies vaccine control 
 
Secondary Objectives: 
 

 
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 To assess level of and duration of humoral immune responses 
as measured by enzyme-linked immunosorbent assay (ELISA) 
titer response to Pfs230D1M after third immunization   
 To assess the functional antibody response by standard 
membrane feeding assay (SMFA) to Pfs230D1M 
 
Exploratory Objectives: 
• To explore cellular and humoral responses to Pfs230D1M 
• To analyze innate and adaptive states before and at early 
timepoints after vaccination by using RNA sequencing  
• Estimation of interaction between host factors including but not 
limited to hemoglobinopathies, immune genes signatures, co-
infections, and environmental, demographic, and 
socioeconomic characteristics and primary and secondary 
endpoints  
 
Study Endpoints:  
 
Primary Endpoint: 
• Incidence of local and systemic adverse events (AEs) and 
serious adverse events (SAEs) 
Secondary Endpoints: 
• Anti-Pfs230D1M immunoglobulin (Ig) G levels as measured 
by ELISA 
• Transmission-reducing activity (TRA)/TBA of induced 
antibody in SMFA 
Exploratory Endpoints: 
• Cellular immune responses and antibody repertoire of 
functional antibody responses to vaccination 
• RNA transcriptome quantification as detected by RNA 
sequencing comparing vaccinees to controls 
• Estimation of interaction between host factors including but not 
limited to hemoglobinopathies, immune signatures, co-
infections, and environmental, demographic, and 
socioeconomic characteristics and primary and secondary 
endpoints 

 
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1 
Background Information and Scientific Rationale 
 
Background Information 
According to the World Health Organization (WHO), progress in malaria control has recently 
stalled, with no reductions in the number of malaria cases worldwide in the past several years 
(WHO 2020). In 2019, there were 229 million cases and 409,000 deaths. 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  (Sachs and Malaney 2002). These factors—as well as 
growing drug resistance of the causative parasite P. falciparum, widespread resistance of 
mosquitoes to insecticides, 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 (Duffy and Gorres 2020)  
 
Malaria is transmitted by Anopheles mosquitoes. Figure 1 shows the life cycle of the malaria 
parasite. During a blood meal from an infected person, the female mosquito ingests parasites, 
including gametocytes, the sexual forms of the parasite. Inside the mosquito midgut, male and 
female gametes fertilize to form a zygote, which further develops into an elongated ookinete 
form. The ookinete migrates to the outer surface of the midgut and develops into an oocyst, 
which undergoes multiple rounds of nuclear division to produce thousands of sporozoites that 
then migrate to the salivary glands. When the mosquito takes another blood meal, it injects 
sporozoites with its saliva, transmitting the malaria parasite to another person. 
 
Figure 1. Life Cycle of Malaria Parasite Plasmodium ssp. (Su, Hayton et al. 2007) 
 
 

 
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Since transmission by mosquitoes is a biological bottleneck for malaria, measures to block 
transmission have been integral components of the malaria control strategy. 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 TBA 
in SMFAs (Kaushal, Carter et al. 1983, Williamson, Keister et al. 1995, MacDonald, Nguyen et 
al. 2016, Coelho, Tang et al. 2021).  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 
 (Williamson, Criscio et al. 1993). 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, Pfs230D1-EPA/ Matrix-M 
The recombinant protein Pfs230D1M was developed at 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 (MacDonald, Nguyen et al. 
2016). 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 2). These same mutations were also observed in an analysis of over 2,000 parasite 
isolates  (MacDonald, Nguyen et al. 2016). In West Africa, the minor allelic frequency was 
reported to be 0.111 and 0.339 for G605S and K661N, respectively, shown in Figure 2. Of note, 
rabbit antisera raised against Pfs230D1M blocked parasite transmission of a Thailand isolate 
with the G605S mutation, suggesting efficacy of Pfs230D1M-EPA against the variant. The 
biological impact of the K661N mutation remains to be determined. 
 

 
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Figure 2. 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 (Farrance, Rhee et 
al. 2011, Tachibana, Wu et al. 2011). 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  (Lin, Ho et al. 2001, Passwell, Ashkenzi et al. 2010, Thiem, Lin et al. 2011) 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 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). 
 

 
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Pfs230D1M-EPA formulated in the more potent adjuvant AS01 has completed recent evaluation 
over two years in NIAID protocol #17-I-N006, with initial results supporting higher potency 
(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).  
 
Rationale for Trial and Study Design 
Pfs230D1 has become one of the leading transmission-blocking antigens for consideration as a 
licensed TBV to be used either alone or in combination with other transmission-blocking 
antigens. Clinical trials with this antigen adjuvanted with either Alhydrogel or AS01 have 
provided very encouraging results (Section 3). With the execution of the recent age de-escalation 
trial with Pfs230D1M-EPA adjuvanted with AS01 (Section 3), it is clear that research efforts 
with this vaccine should proceed forward.  
 
Four factors have spurred the programmatic decision to proceed with a trial evaluating 
Pfs230D1M-EPA in combination with Matrix-M, rather than focusing the next trial on further 
assessment of Pfs230D1M-EPA adjuvanted with AS01, as follows: 
 
1. Inaccessibility of AS01 as an adjuvant – Although the combination of Pfs230D1M-EPA 
with AS01 has proven to be safe, well-tolerated, immunogenic and efficacious in terms of 
preventing transmission of P. falciparum in the field, the adjuvant is not available for 
combination with Pfs230D1M-EPA unless it is purchased on the open market. 
GlaxoSmithKline, the manufacturer of AS01, has declined any additional collaborations 
using AS01 in malaria vaccine trials pending the completion of trials that test new 
regimens of their RTSS/AS01B malaria vaccine. Because of this inaccessibility, LMIV 
has had to seek an alternative adjuvant to combine with Pfs230D1M-EPA.   
 
2. Data from non-human primates indicating that Matrix-M has a similar immunogenicity 
profile to AS01 as an adjuvant for Pfs230 – In recent pre-clinical studies conducted by 
Dr. David Narum and his laboratory team at LMIV in non-human primates, Matrix-M 
appeared to have a very similar immunogenicity profile to AS01. Figure 3 shows a 
comparison of rhesus antibody responses (IgG) by ELISA (A) and TRA by SMFA (B) 
following immunization with conjugated Pfs230D1-EPA in AS01 or Matrix-M. Groups 
of 6 non-human primates were immunized with 40 ug Pfs230D1-EPA/50 µg Matrix-M 
on days 0, 28, and 112. Multiple bleeds were performed through day 301 or 308 for AS01 
or Matrix-M, respectively. No marked differences were observed in the IgG responses 
between AS01 and Matrix-M. The conjugated Pfs230D1M-EPA antisera were evaluated 
by SMFA in presence of human complement two weeks following the primary and 
secondary boost and at the end of the study, approximately 6 months following the final 

 
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Page 21 of 112 
boost. AS01 and Matrix-M ELISA titers and % TRA were compared on days 42, 140 and 
301/308 using a Wilcoxon test.  No significant differences were observed (ELISAs days 
42, 140 and 301/308, p = 0.48 for all; % TRA days 42, 140 and 301/308, p = 0.17, 0.86 
and 0.81, respectively). Historical antibody responses by ELISA for a conjugated 
Pfs230D1-EPA formulated with Alhydrogel are also included (in panel A) for reference. 
The study with Alhydrogel as an adjuvant was performed independently but with the 
same procedures, a slight variation in immunization schedule (days 0, 28, and 168), and 
the final bleed at day 308. 
 
Figure 3. Comparison of Rhesus Antibody Responses (IgG) by ELISA (A) and TRA 
by SMFA (B) Following Immunization with Conjugated Pfs230D1-EPA in AS01 
(GlaxoSmithKline) or Matrix-M (Novavax). 
 
 
3. Potential for antigen dose-sparing – Immunization with Matrix-M has been shown to 
lead to increased numbers of CD169+ macrophages and activated dendritic cells 
(Magnusson, Altenburg et al. 2018) in draining lymph nodes, resulting in increased 
antigen presentation. CD169+ macrophages have been shown to have a role in 
transporting antigens to B lymphocytes and facilitating cross-presentation of antigen to 
CD8+ T lymphocytes (Carrasco and Batista 2007, Gray and Cyster 2012). Subsequent 
generation of cross-reactive antibodies and multi-functional CD4+ T lymphocytes 
(Bengtsson, Song et al. 2016, Shinde, Cai et al. 2020) may lead to increased antibody and 
cellular responses with the potential for antigen dose-sparing. For this reason, we have 
included an arm with only 20 µg of Pfs230D1M in addition to the arm with 40 µg of 
Pfs230D1M.  
 
4. Collaborative research -– LMIV is part of a PfTBV Consortium, funded by the European 
and Developing Countries Clinical Trials Partnership (EDCTP). This Consortium, led by 
Dr. Issaka Sagara of MRTC, is evaluating a portfolio of three innovative candidate 
malaria vaccines that aim to block P. falciparum transmission. An “Antigen Combination 
Trial” in Mali is planned for the first quarter of 2022. If Pfs230D1-EPA/Matrix-M is 

 
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Page 22 of 112 
shown to be safe, well-tolerated and immunogenic in the trial proposed here, this vaccine 
will be combined with another malaria candidate vaccine, R0.6C/Matrix-M in the 
Antigen Combination Trial. R0.6C, a fusion protein manufactured by SSI (Denmark) is 
about to undergo “first in humans” testing in Denmark and Burkina Faso. A combination 
of Pfs230D1 and Pfs48/45 (6C) specific mAbs demonstrated enhanced transmission 
blocking by SMFA (Singh et al. 2020). We will review the ELISA results from samples 
collected 2 weeks after the final dose of Pfs230D1-EPA/50 µg Matrix-M prior to starting 
the Combination Antigen Trial. 
 
As background, the R0.6C fusion protein is a chimera consisting of the 6-cysteine C-
terminal fragment of PfS48/45 (6C) coupled to the N-terminal region of asexual stage 
Glutamate Rich Protein GLURP (R0) produced in Lactococcus lactis. The sexual stage 
Pfs48/45 antigen is a well-established lead candidate for a P. falciparum TBV because of 
its critical role in parasite fertilization. Male gametes lacking Pfs48/45 are unable to bind 
female gametes in the mosquito midgut, thus preventing ookinete, oocyst, and ultimately 
sporozoite development. Immunization with R0.6C in rodents induces functional 
antibodies against the 6C subunit (Singh, Roeffen et al. 2017). Anti-6C antibodies are 
ingested during the blood meal and can bind male sexual forms in the mosquito gut, 
preventing their fertilization of female gametes and thus ookinete and oocyst 
development. Sera of vaccinated animals were able to reduce transmission in the SMFA 
with cultured gametocytes. Anti-6C antibody titres were further increased by immunizing 
with R0.6C adjuvanted with Alhydrogel or Alhydrogel and Matrix-M. 
 
The other vaccine which will be tested alongside these vaccines will be a “chimera” 
vaccine known as PRO.C.6C, a recombinant Pfs48/45-Pfs230 fusion protein expressed in 
L. lactis, also manufactured by SSI and being tested in a “first-in-humans” trial during the 
second half of 2021. This chimera contains the pro-domain of Pfs230 (at the n-terminus), 
a 23 native amino acid linker potion of the circumsporozoite protein and the 48/45 
antigen. 
 Study Plan 
This “first-in-human” trial with the Pfs230D1-EPA/Matrix-M vaccine will be conducted as a 
dose-escalation trial. Because of the limitations related to formulation, the lowest dose of 
antigen, 12.5 µg, can only be combined with 25 µg of Matrix-M (and not 50 µg of Matrix-M). Of 
note, a recent publication with the pre-erythrocytic stage candidate malaria vaccine R21 
adjuvanted with Matrix-M demonstrated that R21 adjuvanted with 25-µg Matrix-M 
demonstrated only marginally lower efficacy as compared to R21 adjuvanted with 50-µg Matrix-
M (Datoo, Natama et al. 2021). For the 20-µg and 40-µg antigen doses, we have elected to move 
forward with the full-dose Matrix-M as recommended by our collaborating partner, Novavax. 
Although, at LMIV we have generally dose-escalated directly from our low dose to the 40-µg 

 
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Page 23 of 112 
dose, since we have included the 20-µg dose to determine whether we can achieve dose-sparing, 
we will take advantage of the opportunity to look for any safety signals, staggering the 20-µg and 
40-µg doses. For all three Pfs230D1-EPA/Matrix-M antigen dosages, we will start with a Pilot 
Group of 5 subjects in the Pfs230D1-EPA/Matrix-M arm and a sub-group of rabies vaccine 
controls. For the Pilot Group, the different dosages are separated by approximately 2 weeks 
(Table 1). After the first injections of the 40-µg dose to 5 participants, a report will be prepared 
for the Data Safety and Monitoring Board (DSMB) which will include trial safety data through 
72 hours after the first injections of the  40-µg dose.  If the DSMB concurs, immunization of the 
Main Group can proceed with 15 subjects from each arm plus the rabies control subjects as 
shown in Table 1. (Table 1 is provided as an example of what may occur.) In order to lessen the 
chance for errors when handling different dosages of Pfs230D1-EPA/Matrix-M and to ease the 
workload on the staff on any given day, we will attempt to divide the immunizations for the 
Main Group over 3 days. We will do the same for the second and final immunizations for the 
Main Group.  
 
Table 1. Immunization Schedule (example) 
 
* For simplicity, the table shows only the first of 3 doses of the Rabies Vaccine in the Control Group.  
2 
Previous Preclinical Experience with Pfs230D1-EPA/Matrix-M  
 
Immunogenicity of Pfs230D1-EPA with Alhydrogel and Matrix-M in CD-1 Mice 
Nine groups of CD-1 mice, 3 groups of 15 animals and 6 groups with 10 animals, were 
immunized on Days 0 and 28 by IM injection with 50 µL (Groups 1-6) or 80 µL (2 sites x 40 µL 
for Groups 7-9), of formulation per vaccination day. The formulations and dosing 
concentrations/volumes are shown in Table 2 below. Immune sera were collected on Days 0, 28, 
42, 70, 98, 126 and 154.  
 
Day 1 
Day 15 
Day 29
Start after 
DSMB 
Approval  
Day 43 
Day 57 
28 days 
after Dose 1 
for Main 
Group 
Day 71
Day 85
56 days 
after Dose 1 
for Main 
Group 
Dose 1      
n=5 pilot  
Dose 2      
n=5 pilot
Dose 3      
n=5 pilot
Dose 1   
n=15 Main
Dose 2   
n=15 Main
Dose 3 n=15 
Main 
Dose 1    
n=5 pilot 
Dose 2      
n=5 pilot 
Dose 3     
n=5 pilot 
Dose 1      
n=15 Main 
Dose 1       
n=15 Main 
Dose 1       
n=15 Main 
Dose 1    
n=5 pilot
Dose 2    
n=5 pilot
Dose 3    
n=5 pilot
Dose 1      
n=15  Main 
Dose 1       
n=15  Main 
Dose 1       
n=15  Main 
Rabies Vaccine*
Dose 1         
n=1   
Dose 1      
n=1   
Dose 1      
n=2   
Dose 1      
n=16   
12.5 ug Pfs230/  
25 ug Matrix-M 
20ug Pfs230/    
50 ug Matrix-M 
40ug Pfs230/    
50 ug Matrix-M 
DSMB 
Meeting  - 
Review 
data 
collected 
through 72 
hours after 
all Pilot 
Subjects 
receive at 
least one 
vaccination 

 
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Page 24 of 112 
Table 2.  Immunogenicity of Pfs230D1-EPA Formulated with Alhydrogel or Matrix-M in 
CD-1 Mice 
Group 
No. 
Formulation 
Antigen Dose 
(µg 
Pfs230D1M) 
Group 
Size 
Dose 
Volume 
(mL) 
Vaccinations 
(Days) 
Bleed 
Days 
(ELISA) 
Termination 
Day 
42 
Day 
154 
1 
Pfs230D1-
EPA/Alhydrogel 
1 
10 
0.05 
0, 28 
0, 28, 42, 
70, 98, 
126, 154 
5 
5 
2 
0.3 
15 
10 
5 
3 
0.03 
10 
5 
5 
4 
Pfs230D1-
EPA/Matrix-M 
1 
10 
5 
5 
5 
0.3 
15 
10 
5 
6 
0.03 
10 
5 
5 
7 
*Pfs230D1-
EPA/Alhydrogel + 
Matrix-M 
1 
10 
** 
2 sites x 
0.04 
5 
5 
8 
0.3 
15 
10 
5 
9 
0.03 
10 
5 
5 
* Pfs230D1-EPA/Alhydrogel (1600 µg/mL Alhydrogel) was formulated on Day -1 and mixed at bedside with 
Matrix-M adjuvant on Day 0 and Day 28.  
** Formulations were prepared for dosing 80 µL per animal and delivered in two sites with 40 µL per site. 
 
 
Figure 4 shows the dose dependent Pfs230D1M specific IgG responses by ELISA.  
 
                Error bars and line represent geometric mean (GM) and 95% confidence intervals. 
Figure 4. Dose dependent Pfs230D1M specific IgG responses by ELISA in CD-1 mice on 
multiple bleed days (28-154) following IM immunization on days 0 and 28. 

 
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Page 25 of 112 
 
Conclusion: CD-1 mice immunized with Pfs230D1-EPA/Matrix-M or Pfs230D1-EPA/ 
Alhydrogel+Matrix-M had greater Pfs230D1M specific IgG titers than Pfs230D1-EPA/ 
Alhydrogel groups at 0.3 and 1 µg dose levels for up to 154 study days. The Pfs230D1-EPA/ 
Alhydrogel+Matrix-M group at 0.03 µg maintained higher titers than either Alhydrogel or 
Matrix-M alone groups.  
 
Repeated Dose Toxicity Study by Intramuscular Administration in Rabbits 
 Pfs230D1-EPA/Matrix-M Serology Assessments by ELISA 
A repeat dose toxicity study was performed on rabbits with administration of 40 μg Pfs230D1-
EPA/50 μg of Matrix-M, or saline controls. Immunizations occurred on Days 0, 15, 29 and 43. 
Antibody responses were assessed as shown in Figure 5 below. Bleed days included the pre-
bleed (Day 0), Day 15, Day 29, Day 43 and final bleeds (Day 46 or 71) for Pfs230D1M, and pre-
bleed pools, Day 15, Day 29, Day 43 and final bleeds (Day 46 or 71) for EPA. Arrows represent 
days of vaccination (Day 0, 15, 29, and 43). 
 
Figure 5. Rabbit antibody responses following immunization with Control (Saline), 40 μg 
Pfs230D1-EPA/50 μg Matrix-M (Group 4). 
0
10
20
30
40
50
60
70
80
-500
4500
9500
14500
19500
Anti-Pfs230D1 EU
0
10
20
30
40
50
60
70
80
-500
4500
9500
14500
19500
Anti-EPA EU
Group 1: Control, Saline
Group 4: Pfs230D1-EPA/Matrix-M(TM)
Study Day
  
Graphs show the geometric mean of individual animal antibody titers against Pfs230D1M or EPA in vaccine 
groups as a function of time; error bars represent the Geomean with 95% CI.  
 
Conclusion:  
All immunized animals in Group 4 responded to the Pfs230D1-EPA vaccine, as evaluated by 
ELISA against Pfs230D1M and EPA for specific antibody (IgG) responses. No animals in the 
Control group (Group 1) had detectable antibodies against Pfs230D1M or EPA. The vaccine was 
immunogenic in Group 4. 

 
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Page 26 of 112 
 Pfs230D1-EPA/Matrix-M Serology Assessments by ELISA 
The terminal sera collected from individual rabbits on D71 (28 days after the 4th vaccination) 
were tested for transmission blocking activities by a SMFA. Each assay sample was prepared by 
mixing 60 µL of heat-inactivated rabbit sera with 100 µL of non-heat-inactivated human serum 
pool and added to 100 µL gametocyte-infected RBCs. A pool of D71 sera from 10 Group 1 
(control) rabbits was used as control for calculation of transmission reducing activity (TRA, 
defined as reduction of oocyst count in individual midgut in comparison to that in controls) and 
transmission blocking activity (TBA, defined as reduction of infected mosquito in comparison to 
the controls). The results are summarized in Figure 6.   
 
Figure 6. Transmission blocking activity by SMFA in rabbits 
4003
4004
4005
4009
4010
4503
4504
4505
4509
4510
Control
0
50
100
150
200
No. oocysts per mosquito
Evaluation of the transmission reducing activity of rabbit antisera from 
Group 4 of the rabbit toxicology study 48599. The average transmission 
reducing activity is shown (red line) and the individual number of oocysts 
per mosquito, using in general 20 mosquitoes. Rabbits 4004 and 4509 had a 
reduced number of total mosquitoes evaluated (18/20 and 8/20, 
respectively) so those antisera were used in a repeat study using the same 
negative control. The results for % TRA were similar to those shown here 
(% TRA > 99.5%).   
 
Conclusion: Pfs230D1-EPA/Matrix-M is immunogenic in rabbits. The antisera induced by the 
vaccine induced strong activity with an average level of transmission reducing activity of 99.6% 
and the reduction in prevalence of 75.2%.  

 
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Page 27 of 112 
 Toxicology Study in Rabbits  
The toxicology study was done to evaluate the local tolerance and the potential toxicity in rabbits 
induced by four intramuscular injections of Pfs230D1M-EPA formulated with Matrix-M. On 
completion of the treatment period, designated animals were euthanized after the last injection 
(early euthanasia) or after a 4-week treatment-free period (late euthanasia) in order to evaluate 
the reversibility of any findings and potential delayed adverse effects. The toxicology study has 
been completed and an initial draft report has been received with no marked indications noted.   
3 
Previous Clinical Experience with Pfs230D1M-EPA 
 
Pfs230D1M-EPA/Alhydrogel in Healthy Adults (#15-I-0044) 
Summary: 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 US and Bancoumana, Mali (NIAID protocol #15-I-0044; clinicaltrials.gov: 
NCT02334462). Another TBV candidate, Pfs25M-EPA/Alhydrogel, was also assessed as a 
stand-alone vaccine and also co-administered with Pfs230D1M-EPA/Alhydrogel (Table 3). In 
summary, the studies in the US and Mali established that Pfs230 vaccine was superior to the 
Pfs25 vaccine for inducing functional activity, and that the combination of Pfs230 and Pfs25 was 
not superior to Pfs230 vaccine alone (Healy, Anderson et al. 2021). 
 
Table 3. 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. 
 Safety of Pfs230D1M-EPA/Alhydrogel in Healthy Adults 
In both the US and Mali, 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). Safety analysis of the high dose (40 µg) of 

 
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Pfs230D1M-EPA/Alhydrogel showed that many reported AEs were mild (Grade 1; 708/1431; 
49%), with the most commonly reported AEs being injection site pain, headache, malaria, 
neutropenia, nasopharyngitis, and rhinitis. The majority of laboratory abnormalities were 
Grade 1. The most commonly reported related AEs were injection site reactogenicity (pain, 
induration, pruritus, and edema), leukopenia, neutropenia, and headache, which were all Grade 1 
or 2. The related AE reported with the highest frequency was injection site pain, which did not 
increase in frequency with subsequent vaccination. However, overall, reported local 
reactogenicity, but not solicited systemic symptoms, appeared to increase (in frequency and 
duration of symptoms) with increasing antigen dose of Pfs230. In comparison to the comparator 
arms, more related AEs were reported for the Pfs230D1M vaccinees, alone or in combination, 
and the majority of these were Grade 1 or 2 local reactogenicity. 
 
In a single Pfs230-vaccinated subject in Mali, a Grade 3 gastroenteritis was reported with 
associated Grade 4 laboratory abnormalities (leukocytosis, increased blood creatinine), all 
deemed unlikely related to vaccination and all of which resolved shortly after resolution of the 
gastroenteritis symptoms. Other than this case, there were no Grade 3 or 4 AEs. There were 
3 unrelated SAEs, one of which was a cerebrovascular accident that led to death. No SAEs were 
reported in the Pfs230-vaccinated arms. No participants were removed from study participation 
due to a related AE of any severity. 
 Immunogenicity and Functional Activity of Pfs230D1M-EPA/Alhydrogel in Healthy 
Adults 
Pfs230D1M induced antibody responses in most US vaccinees (Figure 7C-E) and resulted in 
high TRA (100%, 98% TRA) in 2 out of 5 individuals and significant TRA (73%, 62% TRA) in 
2 others (Figure 8A) after just 2 vaccine doses. In the Pfs25M + Pfs230D1M combination group, 
antibody responses were similar to the individual antigen arms (Figure 7F-I), and 2 individuals 
had appreciable functional activity after 2 vaccinations (one had 90% and the other had 68% 
(Figure 8A). The activity correlated well with anti-Pfs230D1M titers, demonstrating that the 
functional activity was due to the vaccine (Figure 8C). 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 9). 
 

 
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Figure 7. Pfs230-specific Antibody Responses in Subjects Receiving Pfs230D1M, US 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 8. Pfs25 and Pfs230 Functional Activity by Standard Membrane Feeding Assay. 
 
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 9. 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 
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. 
 

 
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Evaluation of immunogenicity by ELISA in healthy Malian 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 10). 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 10) and 
percentage of responders (detectable antibody responses).  
 
Figure 10. 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. 
 
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.  
 
In summary, preliminary ELISA and SMFA results suggested that the two doses induced similar 
activity. In the main phase of the study, the 40-µg dosage was used for vaccination. Unblinding 
of a larger cohort of this trial, with 60 subjects in the full-dose arm (3 doses of 40 µg at 0, 1, and 

 
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6 months) and 60 subjects in a fractional-dosing arm (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), occurred in March 
of 2018. The results of the trial established the full 3-dose regimen using 40-ug dosage as the 
benchmark for future trials, and demonstrated safety and tolerability of the regimen. A booster 
dose of Pfs230D1M (40 µg) was administered approximately 12 months after the third 
immunization. 
 Safety of Pfs230D1M-EPA/AS01 in Healthy Adults 
Pilot Phase  
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 were mild (Grade 1; 13 µg: 23/29, 79%; 40 µg: 42/51, 82%), with the 
majority being injection site pain. Laboratory abnormalities were also observed, with the 
majority being transient, asymptomatic Grade 1 (mild) and Grade 2 (moderate) neutropenias. 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.  
 
Main Phase  
In the main phase of the trial, there was a full-dose arm (n=56) and a fractional-dose arm (n=61). 
The full-dose arm received Pfs230D1M-EPA/AS01 at 40 µg at 0, 1, 6, and 18 months. The 
fractional-dose arm received Pfs230D1M-EPA/AS01 at 40 µg at 0, 1, and 18 months and 
received a fractional dose of 8 µg of Pfs230D1M-EPA/AS01 at dose #3 at 6 months. Because 
there is no fractional dosing in the proposed trial, full details of the fractional dosing arm are not 
provided here; overall, the AE profile was similar to that of the full-dose arm, and further details 
can be found in the Investigator Brochure.  
 
First 3 vaccinations: In the full-dose arm (n=56), the first three vaccinations were well tolerated 
with 66% (252/383) of the total AEs being mild. The most common Grade 1 AEs were injection 
site pain (95/252; 38%) followed by headache and malaria. 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. 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. Grade 2 injection site pain was also significantly greater in the full-dose and 
fractional-dose arms compared to the control. Grade 1 headaches were significantly greater in the 
full-dose arm than the control. Pyrexia was reported more frequently in the full-dose arm than 
the control arm.  
 
Booster dose: The fourth dose of Pfs230D1M-EPA/AS01 was well tolerated; most AEs were 
mild (Grade 1 AEs; 160/363, 44%). The most commonly reported Grade 1 AE was injection site 

 
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pain, which was observed in about half of Pfs230D1M vaccinees. Of the related Grade 2 AEs, 
injection site pain was most common (7/11, 63%) followed by headache, fatigue, and injection 
site movement impairment in one subject. There were no related Grade 3 AEs. Participants in the 
Pfs230D1M booster arm experienced significantly more Grade 1 injection site pain and headache 
than the comparator arm. Grade 2 injection site pain was also reported more frequently in the 
Pfs230D1M arm but was not significantly different from the comparator.  
 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 11). 
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 12). Anti-Pfs230 was sufficient to 
induce 100% TRA 2 weeks post dose 2, which was still >90% 12 weeks post dose 2. In contrast, 
anti-Pfs25 did not exceed 80% TRA post dose 2. The combination of Pfs25 and Pfs230 was not 
superior to Pfs230 alone for inducing functional serum activity.  
 
Figure 11. 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 12. 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 US 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-dose 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 13). Antibody titers were also assessed after the third vaccination, and as expected, the 
full-dose 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 third vaccination compared 
to second vaccination (p=0.20 for full dose group; p=>0.99 for fractional dose group; Figure 13). 

 
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Notably, Pfs230D1 titers were significantly higher in the full versus fractional dosing regimen at 
12 weeks post dose 3.  
 
Figure 13. Antibodies Against Pfs230D1M Measured by ELISA 12 Weeks 
Post Second and Third 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, 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 14). 
 

 
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Figure 14. ELISA Against Pfs230D1 Antigen. 
 
Titer increase was greater with full versus fractional regimen after the third 
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 second and third 
vaccinations. Both fractional and full dose arms were found to have significantly higher TRA 
(Figure 15) (the decrease in the number of oocysts per infected mosquito) after both second and 
third 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 the third vaccination 
compared to the second vaccination; this difference was not observed in the fractional-dose arms 
(Figure 15). Together with the ELISA data (Figure 13), 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 15. Transmission-Reducing Activity Measured by SMFA 12 Weeks 
Post Second and Third Vaccinations in Bancoumana/Doneguebougou, Mali 
during Main Phase trial. 
 
Note: Error bars are median and 95% CI. 
 
Functional activity, measured by SMFA by TRA, was maintained after the fourth dose at a high 
level of activity. TBA (reduction in infected mosquitoes) by SMFA was significantly higher in 
both Pfs230D1M vaccine arms compared to the comparator at 3 months post dose 4 but did not 
significantly differ between full and fractional dosing regimens. 
 Functional Activity by Direct Skin Feeds 
Direct skin feeds (DSFs) 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 5065 DSFs were completed in 2018. There were 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 4). A trend of lower infections in the full-dose regimen was 
observed in 2017 but was not significant. Positive DSFs were significantly less frequent in the 
full-dose regimen in 2018, a first and extremely important achievement in the field of malaria 
TBVs – in vivo functional activity. 
 
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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Table 4. Summary of 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. 
 DSF Analysis as an endpoint 
Results of vaccine efficacy have been calculated to be similar to the Age De-escalation study 
below for biostatistical purposes only. Table 5 provides the preliminary results of DSFs for Year 
1 and Year 2 of the study, and both years combined for the full dose arm of the study, showing 
strong vaccine efficacy in the adult trial.  
 
Table 5. Vaccine efficacy based on DSFs for Year 1 and Year 2, full dose arm. 
 
Vaccine Efficacy 
95% CI 
p value 
Year 1 
0.4972 
-1.0672      0.8777     
0.341 
Year 2 
0.8119        
0.3875      0.9423      
0.006 
Year 1 and 2  
0.7250 
0.3041      0.8913      
0.006 
 
 
Age De-Escalation/Family Compound Trial of Pfs230D1M-EPA/AS01 Vaccine (#19-I-
N086) 
This phase 2 study of the safety, immunogenicity, vaccine activity, and vaccine efficacy of 
Pfs230D1M-EPA/AS01 against P. falciparum malaria in vaccine units (VU) in Doneguebougou, 
Mali and surrounding villages began in April 2019. The trial is currently undergoing full 
unblinding and analysis so only partial results are available, described below.  
 
The trial has been conducted to assess Pfs230D1M-EPA/AS01 versus comparators (Havrix, 
Typhim Vi, and Menactra) at a community level. The sample was drawn using a census of 
compounds in Doneguebougou, Mali and an adjacent village. Family compounds were 
aggregated by proximity and mosquito habitat into VUs (n=137), which were randomly assigned 
to receive Pfs230D1M-EPA/AS01, 40 µg, or comparators at 0, 1, and 2 months in all eligible 
subjects 5 years of age or older. Unvaccinated children 1-4 years of age received AL treatment 
prior to their VU receipt of dose #3, for parasitemia endpoints. All vaccinated subjects were 

 
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treated with AL prior to dose #1, and children 5-8 years of age were treated with AL prior to 
dose #3. The trial began with age de-escalation pilot phases (to ensure safety) with smaller 
numbers of subjects prior to moving to main phases of the trial. In June 2020, available subjects 
were re-enrolled to receive a fourth vaccine dose approximately 1 year post dose #3. Subjects 
aged 1-4 years were also re-enrolled in the corresponding VU.  
 
To assess vaccine efficacy, DSFs (for 9- to 18-year-old subjects only) were performed by 
feeding 60 mosquitoes directly on the subject’s skin for approximately 15 minutes. 
 Safety  
Thus far, AEs and laboratory abnormalities have been reported in an unblinded manner only. 
Analysis of the data is in progress. Overall, no safety signals have been observed in this trial. The 
majority of AEs have been mild injection site pain, headache, and pyrexia. A trend previously 
noted with the adjuvant AS01, increased frequency and severity of fever episodes post dose #2, 
has been seen, particularly in the youngest participants. The majority of laboratory abnormalities 
have been Grade 1. There have been no related SAEs noted. A more detailed description can be 
found in the Investigator’s Brochure accompanying this submission. 
 Immunogenicity in Healthy Malian Children  
Initial ELISA results have been generated for Year 1 as shown in Figure 16. 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; represented by group 
vaccine assignment) appear to have similar antibody responses to Pfs230D1M-EPA/AS01 
vaccine as healthy adults receiving a similar vaccine regimen and same Pfs230D1M-EPA/AS01 
vaccine dose. Also similar to what was seen in protocol #17-I-N006 (adult AS01-only study), 
children had significantly higher antibody titer measured by ELISA against Pfs230 compared to 
the control arm after only 2 doses given at 0 and 28 days. Antibody titers were also assessed after 
the third vaccination, and as expected, the Pfs230 vaccine arms had significantly higher titers 
compared to control arms, but again similar to the adult-only study (protocol #17-I-N006), the 
antibody titer was not significantly higher after the third vaccination compared to the second 
vaccination. 
 

 
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Figure 16. Pfs230 ELISA Results During Year 1 in Pilot Subjects. 
 
Vaccinations administered on day 0, 28, 126 (black arrows). ELISA timepoints = day 0 
(pre-vaccination), day 14 (14 days post dose 1), day 42 (14 days post dose 2), and day 
140 (14 days post dose 3).  
 
In an initial comparison of Pfs230 antibody responses in Pfs230D1M-EPA/AS01-vaccinated 
children at 12 weeks post dose #3, children who were found to have a DSF-positive feed (at least 
1 mosquito with ≥1 oocyst) had a statistically significant (p<0.0001) lower Pfs230 antibody level 
than children with a negative DSF feed (Figure 17). No differences between DSF+ and DSF- 
were seen in antibody responses to Pfs230 in the comparator arm (p=0.9998). 
 
Figure 17. Comparison of Subset of DSF Cohort (9- to 18-Year-Old Subjects) by Vaccine 
Arm and DSF Positivity. 
 
Vaccinations administered on day 0, 28, 56 (black arrows). 
 

 
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 Vaccine Efficacy in Healthy Malian Children 
Results of vaccine efficacy have been unblinded for biostatistical purposes only. Table 6 
provides the preliminary results of DSFs for Year 1 and Year 2 of the study. It is important to 
emphasize that these are preliminary results only.  
 
Table 6. Vaccine Efficacy Based on DSFs for Year 1 and Year 2. 
 
Vaccine Efficacy 
95% CI 
p value 
Year 1 
72% 
42; 86 
< 0.001 
Year 2 
75% 
42; 89 
< 0.001 
 
These preliminary efficacy results are extremely promising and support the idea that 
Pfs230D1M-EPA is an excellent candidate antigen for a TBV. Future plans for additional 
analyses of these positive DSFs include oocyst speciation, sieving analysis of Pfs230, and 
parasite genotyping. 
 
EPA 
Recombinant EPA is not a component of any licensed vaccines, but has been extensively studied 
as a carrier for polysaccharide conjugate vaccines. These include a typhoid vaccine containing 
22-µg recombinant EPA tested in children as young as 2 months old (Lin, Ho et al. 2001, Thiem, 
Lin et al. 2011), and a shigellosis vaccine containing 75-µg recombinant EPA tested in children 
1-7 years old (Ashkenazi, Passwell et al. 1999, Passwell, Ashkenazi et al. 2003, Passwell, 
Ashkenzi et al. 2010). No safety issues have been identified to date with the use of the 
recombinant EPA. 
 
MATRIX M 
Matrix-M is a saponin-based adjuvant manufactured by Novavax AB (Uppsala, Sweden). 
Although it is not yet fully understood how the Matrix-M adjuvant achieves its stimulatory 
effects, this adjuvant is known to transiently enhance the number of activated immune cells in 
the draining lymph nodes which may in turn lead to increased uptake and presentation of vaccine 
antigens to elicit a competent immune response (Reimer, Karlsson et al. 2012). Specifically, it 
has been shown that there is an increase of CD169+ macrophages, as well as activated dendritic 
cells, to the draining lymph nodes after immunization with Matrix-M adjuvanted vaccines, which 
may help to increase antigen presentation (Magnusson, Altenburg et al. 2018). Hence, CD169+ 
macrophages have previously been shown to have a role in transporting antigens to B 
lymphocytes by trapping them in the draining lymph node and to facilitate cross-presentation of 
the antigen to CD8+ T lymphocytes (Carrasco and Batista 2007, Gray and Cyster 2012). This 
may lead to increased humoral and cellular immune responses, manifested by cross-reactive 
antibodies and multi-functional CD4+ T lymphocytes (Bengtsson, Song et al. 2016, Shinde, Cai 

 
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et al. 2020). Consequently, Matrix-M has been shown to contribute to antigen dose-sparing and 
increased duration of humoral and cellular vaccine responses. 
 Summary of Clinical Experience with Matrix-M 
The Matrix-M adjuvant technology (Bengtsson, Karlsson et al. 2013) is a promising technology 
which has been explored for various infectious diseases and has a good safety profile in humans 
(Shinde, Fries et al. 2018). To date, Matrix-M has been utilized in more than 25 clinical trials, 
including multiple Phase III trials (Table 7). In these studies, Matrix-M has been combined with 
multiple malaria vaccine candidates, influenza, COVID-19, and other disease indications. The 
most common dosage of Matrix-M has been 50 µg in adults, with a maximum dosage of 75 µg.  
 
Datoo et al reported on a double-blind, randomized, controlled trial (NCT02925403) of a low-
dose circumsporozoite protein-based 37 vaccine, R21, with two different doses of adjuvant, 
Matrix-M (25 µg and 50 µg), in 450 children aged 5-17 months in Nanoro, 38 Burkina Faso, a 
highly seasonal malaria transmission setting. Three vaccinations were administered at 4-week 
39 intervals prior to the malaria season with a fourth dose one year later. R21/MM had a 
favorable safety profile and was well-tolerated. Vaccine efficacy (VE) was 74% (95% CI, 63-82) 
and 46 77% (95% CI, 67-84) in the low- and high-dose adjuvant groups, respectively. At 1 year, 
VE remained high at 47 77% (95% CI, 67-84) in the high-dose adjuvant group (Datoo, Natama 
et al. 2021).  
 
Keech et al (Keech, Albert et al. 2020) published the results of a randomized, placebo-controlled, 
phase 1–2 trial to evaluate the safety and immunogenicity of the rSARS-CoV-2 vaccine (in 5-μg 
and 25-μg doses, with or without Matrix-M1 adjuvant in 131 healthy adults (NCT04368988). 
The vaccine, NVX-CoV2373 appeared to be safe and was shown to elicit immune responses that 
exceeded levels in Covid-19 convalescent serum. The addition of Matrix-M resulted in enhanced 
immune responses, was antigen dose–sparing, and induced a T helper 1 (Th1) response.  
 
Shinde et al (Shinde, Bhikha et al. 2021) reported on rSARS-CoV-2 vaccine (containing 50 µg of 
Matrix-M) administered intramuscularly to adult subjects in South Africa (NCT04533399). 
Among 2,684 baseline seronegative participants (94% HIV-negative and 6% HIV-positive), 
predominantly mild-to-moderate COVID-19 developed in 15 participants in the vaccine group 
and in 29 in the placebo group (vaccine efficacy, 49.4%; 95% confidence interval [CI], 6.1 to 
72.8).  Among the vaccine recipients, the most common solicited systemic adverse events after 
the first dose and second dose were headache, muscle pain, and fatigue. The mean duration of 
such events was slightly longer after the second dose but generally less than 3 days.  
 
In yet another recent example (NCT01444482), an influenza vaccine containing Matrix-M was 
tested in a randomized, observer-blinded, active comparator-controlled trial during the 2019-
2020 influenza season (Shinde, Cho et al. 2020). In brief, 2,654 clinically stable, community-

 
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dwelling adults ≥65 years of age were randomized to receive a single IM dose of either Matrix-
M-adjuvanted quadrivalent nanoparticle influenza vaccine (qNIV) or a licensed inactivated 
influenza vaccine (IIV4). Local reactogenicity, primarily mild to moderate and transient pain, 
was higher in the qNIV group. qNIV was generally well tolerated and produced a qualitatively 
and quantitatively enhanced humoral and cellular immune response in older adults.  
 
Table 7. Key Clinical Experience of Matrix-M with Various Vaccine Antigens.  
NCT Number 
Title 
Conditions 
Interventions 
Characteristics 
Population 
NCT04201431 
Safety, Immunogenicity and Efficacy of 
the Blood-stage Plasmodium Vivax 
Malaria Vaccine Candidate PvDBPII in 
Matrix M1 
Malaria, Vivax 
• Biological: PvDBPII/Matrix M1 
Phase 1 
Phase 2 
18 Years to 45 
Years (Adult) 
NCT01669512 
Adjuvanting Viral Vectored Malaria 
Vaccines With Matrix M 
Malaria 
• Biological: Low Dose Matrix M 
Regimen 
• Biological: Standard Dose 
Matrix M Regimen 
Phase 1 
18 Years to 50 
Years (Adult) 
NCT04130282 
VAC077: Safety and Immunogenicity of 
the Pfs25-IMX313/Matrix-M Vaccine 
Malaria 
• Biological: Pfs25-IMX313/ 
Matrix-M1 
Phase 1 
18 Years to 45 
Years (Adult) 
NCT04318002 
Safety and Immunogenicity of RH5.1/ 
Matrix-M in Adults and Infants Living in 
Tanzania 
Malaria 
• Biological: RH5.1/Matrix-M 
Phase 1 
6 Months to 45 
Years (Child, 
Adult) 
NCT03896724 
Safety, Immunogenicity and Efficacy of 
R21 Matrix-M in 5-17 Month Old Children 
in Nanoro, Burkina Faso 
Malaria 
• Biological: R21 adjuvanted with 
25mcg Matrix-M 
• Biological: R21 adjuvanted with 
50mcg Matrix-M 
Phase 1 
Phase 2 
5 Months to 17 
Months (Child) 
NCT04271306 
Safety, Immunogenicity and ex Vivo 
Efficacy of Pfs25-IMX313/Matrix-M in 
Healthy Volunteers in Bagamoyo, 
Tanzania. 
Malaria 
• Biological: Pfs25-IMX313 
(10ug)/Matrix-M (50ug) 
• Biological: Pfs25-IMX313 
(50ug)/Matrix-M (50ug) 
• Biological: Pfs25-IMX313 
(50ug)/Matrix-M (50ug) 
& Pfs25-IMX313 (10ug)/ 
Matrix-M (50ug) 
Phase 1 
5 Years to 45 Years 
(Child, Adult) 
NCT02572388 
A Study to Assess the Safety and 
Immunogenicity of the Malaria Vaccine, 
R21, Administered With and Without 
Matrix-M1 
Malaria 
• Biological: R21 
• Biological: Matrix-M1 
Phase 1 
18 Years to 50 
Years (Adult) 
NCT02925403 
A Study to Assess the Safety and 
Immunogenicity of the Malaria Vaccine, 
R21, With Matrix-M1 Adjuvant 
Malaria 
• Biological: R21/Matrix-M1 
• Other: Saline 
Phase 1 
Phase 2 
18 Years to 45 
Years (Adult) 
NCT01444482 
Study of Parenterally Administrated 
Adjuvanted Seasonal Influenza Vaccine in 
Healthy Elderly Volunteers 
Influenza 
• Biological: Matrix M 
• Biological: Seasonal influenza 
vaccine 
Phase 1 
65 Years to 75 
Years (Older Adult) 
NCT04611802 
A Study Looking at the Efficacy, Immune 
Response, and Safety of a COVID-19 
Vaccine in Adults at Risk for SARS-CoV-2 
COVID-19 
• Biological: SARS-CoV-2 rS/ 
Matrix-M1 Adjuvant 
• Other: Placebo 
Phase 3 
18 Years and older 
(Adult, Older 
Adult) 
NCT04368988 
Evaluation of the Safety and 
Immunogenicity of a SARS-CoV-2 rS 
Nanoparticle Vaccine With/Without 
Matrix-M Adjuvant 
COVID-19 
• Biological: SARS-CoV-2 rS - 
Phase 1 
• Biological: SARS-CoV-2 rS/ 
Matrix-M Adjuvant, Days 0 and 
21 - Phase 2 
• And more 
Phase 1 
Phase 2 
18 Years to 84 
Years (Adult) 
NCT04533399 
A Study Looking at the Effectiveness and 
Safety of a COVID-19 Vaccine in South 
African Adults 
COVID-19 
• Biological: SARS-CoV-2 rS/ 
Matrix-M1 Adjuvant 
• Other: Placebo 
Phase 2 
18 Years to 84 
Years (Adult) 
NCT03580824 
A Study to Determine if a New Malaria 
Vaccine is Safe and Induces Immunity 
Among Kenyan Adults, Young Children 
and Infants 
Malaria 
• Biological: R21 in Matrix- M 
adjuvant vaccine 
Phase 1 
Phase 2 
5 Months to 45 
Years (Child, 
Adult) 

 
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NCT Number 
Title 
Conditions 
Interventions 
Characteristics 
Population 
NCT04583995 
A Study Looking at the Effectiveness, 
Immune Response, and Safety of a 
COVID-19 Vaccine in Adults in the United 
Kingdom 
COVID-19 
• Biological: SARS-CoV-2 rS/ 
Matrix M1-Adjuvant 
• Other: Placebo 
• Biological: Licensed seasonal 
influenza vaccine 
Phase 3 
18 Years to 84 
Years (Adult, Older 
Adult) 
NCT02078674 
A(H7N9) VLP Antigen Dose-Ranging 
Study With Matrix-M1™ Adjuvant 
Influenza 
(Pandemic) 
• Biological: Monovalent Avian 
Influenza VLP (H7N9) 
• Biological: Matrix-M1™ 
adjuvant 
Phase 1 
Phase 2 
18 Years to 64 
Years (Adult) 
NCT02300142 
Rollover Trial for Placebo Subjects 
Previously Enrolled Into GEN-003-002 
Study 
Genital Herpes 
• Biological: GEN-003 Vaccine 
(30-60µg of each antigen) 
• Biological: Matrix-M2 Adjuvant 
(25-75µg) 
Phase 2 
18 Years to 50 
Years (Adult) 
NCT01667341 
Safety and Immunogenicity Study of 
Therapeutic HSV-2 Vaccine 
Genital Herpes 
• Biological: GEN-003 with 
Matrix M-2 
• Biological: GEN-003 
Phase 1 
Phase 2 
18 Years to 50 
Years (Adult) 
NCT03026348 
Safety and Immunogenicity Study to 
Evaluate Single- or Two-Dose Regimens 
Of RSV F Vaccine With and Without 
Aluminum Phosphate or Matrix-M1™ 
Adjuvants In Clinically-Stable Older 
Adults 
Respiratory 
Syncytial 
Viruses 
• Biological: RSV F Vaccine with
Aluminum Phosphate Adjuvant 
• Biological: RSV F Vaccine 
• Biological: Matrix-M1 Adjuvant 
Phase 2 
60 Years to 80 
Years (Adult, Older 
Adult) 
NCT02114060 
Dose Ranging Safety and Efficacy of 
Therapeutic HSV-2 Vaccine 
Genital Herpes 
• Biological: GEN-003 Vaccine 
(30µg of each antigen) 
• Biological: Matrix-M2 Adjuvant 
(75µg) 
• And more 
Phase 2 
18 Years to 50 
Years (Adult) 
NCT03947190 
A Study to Determine if New Types of 
Malaria Vaccines Are Safe, Effective and 
Lead to Immunity in Kenyan Adults 
Malaria 
• Biological: R21/Matrix-M 
• Biological: ChAd63/MVA ME-
TRAP 
• Biological: intradermal injection 
(ID) or direct venous injection 
(DVI) of PfSPZ Challenge 
Phase 2 
18 Years to 45 
Years (Adult) 
NCT03293498 
Evaluation of the Safety and 
Immunogenicity of a Recombinant 
Trivalent Nanoparticle Influenza Vaccine 
With Matrix M-1 Adjuvant (NanoFlu) 
Influenza 
• Biological: NanoFlu 
• Biological: Fluzone HD - Day 0 
• Biological: Fluzone HD - Day 21 
• Other: Saline - Day 21 
Phase 1 
Phase 2 
60 Years and older 
(Adult, Older 
Adult) 
NCT03658629 
Phase 2 Dose and Formulation 
Confirmation of Quad-NIV in Older Adults 
Influenza 
• Biological: NanoFlu (Quad- 
NIV) 
• Other: Matrix-M Adjuvant 
• Biological: Fluzone HD 
• Biological:Flublok  
Phase 2 
65 Years and older 
(Older Adult) 
NCT03970993 
VAC 072-An Efficacy Study of R21/MM 
in Different Dose Schedules 
Malaria 
• Biological: R21 Matrix-M 
vaccination 
• Biological: R21 Matrix-M 
vaccination and CHMI 
Phase 1 
Phase 2 
18 Years to 45 
Years (Adult) 
NCT02370589 
Study to Evaluate the Immunogenicity and 
Safety of an Ebola Virus (EBOV) 
Glycoprotein (GP) Vaccine in Healthy 
Subjects 
Ebola 
• Biological: Base Dose EBOV 
GP Vaccine 
• Biological: 2-8x Base Dose 
EBOV GP Vaccine 
• Biological: Matrix-M Adjuvant 
Phase 1 
18 Years to 50 
Years (Adult) 
NCT02515175 
Evaluating New Formulation of 
Therapeutic HSV-2 Vaccine 
Genital Herpes 
• Biological: Matrix-M2 
• Biological: GEN-003 
Phase 2 
18 Years to 50 
Years (Adult) 
NCT04120194 
Phase 3 Pivotal Trial of NanoFlu™ in 
Older Adults 
Influenza 
• Biological: NanoFlu 
• Biological: Fluzone 
Quadrivalent 
Phase 3 
65 Years and older 
(Older Adult) 
NCT04645147 
Safety and Immunogenicity of an Epstein-
Barr Virus (EBV) gp350-Ferritin 
Nanoparticle Vaccine in Healthy Adults 
With or Without EBV Infection 
EBV 
• Biological: EBV gp350- Ferritin 
Vaccine 
• Other: Matrix-M1 
Phase 1 
18 Years to 29 
Years (Adult) 
NCT03146403 
Maintenance Dose Study of GEN-003 in 
Subjects With Genital Herpes Infection 
Genital Herpes 
• Biological: GEN-003 
• Biological: Matrix-M 
Phase 2 
Child, Adult, Older 
Adult 

 
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NCT Number 
Title 
Conditions 
Interventions 
Characteristics 
Population 
NCT02905019 
A Safety and Efficacy Study of R21 +/- 
ChAd63/MVA ME-TRAP 
Malaria 
• Biological: R21 with Matrix- M1 
• Biological: ChAd63 ME-TRAP 
• Biological: MVA ME-TRAP 
Phase 1 
Phase 2 
18 Years to 45 
Years (Adult) 
4 
Study Objectives  
Primary Objective:  
• To assess in African adults the safety and the reactogenicity of administration of      
Pfs230D1-EPA/Matrix-M (first-in-human) as compared to the rabies vaccine control 
 
Secondary Objectives: 
 
 To assess level of and duration of humoral immune responses as measured by ELISA titer 
response to Pfs230D1M after third immunization 
 To assess the functional antibody response by SMFA to Pfs230D1M  
 
Exploratory Objectives: 
 
• To explore cellular and humoral responses to Pfs230D1M 
• To analyze innate and adaptive states before and at early timepoints after vaccination by 
using RNA sequencing  
• Estimation of interaction between host factors including but not limited to 
hemoglobinopathies, immune genes signatures, co-infections, and environmental, 
demographic, and socioeconomic characteristics and primary and secondary endpoints  
5 
Study Design  
This is a Phase 1, dose-escalating, randomized, double-blind, comparator-controlled study 
designed to evaluate the safety, tolerability immunogenicity, and transmission-blocking activity 
of Pfs230D1M conjugate vaccine formulated on Matrix-M. Eighty adult subjects will be drawn 
from Sotuba, Mali and the surrounding areas. Participants will be randomized to one of the study 
arms (see below) to receive 1 of 3 dose levels of Pfs230D1-EPA/Matrix-M or a standard dose of 
comparator rabies vaccine administered as an IM injection at 3 timepoints (study days 1, 29, and 
57).  
Group 1: Pilot Group 
• Arm 1a (n=5): 12.5 µg Pfs230D1-EPA/25 µg Matrix-M 
• Arm 1b (n=5): 20 µg Pfs230D1-EPA/50 µg Matrix-M 
• Arm 1c (n= 5): 40 µg Pfs230D1-EPA/50 µg Matrix-M 
• Arm 1d (n=4): rabies vaccine (2.5 IU rabies virus antigen) 
 

 
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Group 2: Main Group 
• 
Arm 2a (n=15): 12.5 µg Pfs230D1-EPA/25 µg Matrix-M 
• 
Arm 2b (n=15): 20 µg Pfs230D1-EPA/50 µg Matrix-M 
• 
Arm 2c (n=15): 40 µg Pfs230D1-EPA/50 µg Matrix-M 
• 
Arm 2d (n=16): rabies vaccine (2.5 IU rabies virus antigen) 
 
All vaccinated subjects will be followed for 12 months from the last study vaccination. Follow 
up will include safety and tolerability, as well as immunogenicity via ELISA and functional 
antibody responses via SMFA. Subjects will be monitored for patent parasitemia through 6 
months after the final vaccination.  See Appendix A for a detailed schedule of assessments.  
 
Study Endpoints 
Primary Endpoint: 
• Incidence of local and systemic AEs and SAEs  
 
Secondary Endpoints: 
• Anti-Pfs230D1 IgG levels as measured by ELISA 
• TRA/TBA of induced antibody in SMFA  
 
Exploratory Endpoints: 
 
• Cellular immune responses and antibody repertoire of functional antibody responses 
to vaccination 
• RNA transcriptome quantification as detected by RNA sequencing comparing 
vaccinees to controls 
• Host factors including but not limited to hemoglobinopathies, immune signatures, co-
infections, and environmental, demographic, and socioeconomic characteristics  
 
Sample Size and Estimated Duration of Study 
A total of 80 subjects will be vaccinated with either Pfs230D1-EPA/Matrix-M (n=60) or the 
rabies vaccine comparator (n=20). The last study visit will occur at Month 14 (12 months after 
the final immunization). Up to 150 subjects will be screened to accommodate possible screening 
failures. 
 
The sample size was derived to be able to demonstrate safety, tolerability, and immunogenicity 
of the study vaccine as described in Section 14.1.  

 
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Study Definitions 
Screened: Subjects will receive a screening identification number when the informed consent is 
signed, and will either be determined as “enrolled” or “screen failures” as noted below. 
• Screening may be completed over the course of multiple visits. 
• Screening will occur within 56 days prior to enrollment into the study. 
• If screening laboratories are obtained >56 days prior to planned enrollment (Day -7), then 
the subject will need to have a repeat physical exam, medical history review and all 
laboratories outside of the window will need to be collected again (inclusive of safety 
labs, human immunodeficiency virus [HIV],) to confirm whether the subject may proceed 
to enrollment  
 
Enrolled: Subjects will be considered enrolled beginning on Day -7, and the final study number 
will be assigned at this point.  
 
Randomized: Participants will be randomized to one of the study groups. See Section 14.5 for 
details.  
 
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 8.1 
• Subject withdraws consent before Day -7 (i.e., enrollment). 
 
Withdrawn: Participants will be considered to be withdrawn from the study if they meet any of 
the withdrawal criteria in Section 12.11 prior to completing the final study visit. 
 
Completed: Subjects are considered completed when they complete the final study visit for their 
arm. 
 
Lost to follow-up: A participant will be considered lost to follow-up if he or she fails to attend a 
required study visit and cannot be located. Study site staff will make at least 3 attempts to contact 
participants to complete the remaining study visits. These contact attempts should be 
documented in the participant’s study file. Should the participant continue to be unreachable, 
they will be considered to have withdrawn from the study with a primary reason of lost to 
follow-up. 

 
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6 
Study Population 
 
Study Site 
This study will be conducted in the area of Sotuba, Mali, a village near the Malian capital city of 
Bamako. Bamako is located on the Niger river and has a population of about 2.7 million people. 
The district of Bamako is divided into urban (hypoendemic) and periurban (mesoendemic) areas 
and peripheral villages (hyperendemic).  
  
Sotuba is a village located on the outskirts of Bamako on the bank of the Niger River, consisting 
of ∼7,500 inhabitants. Malaria transmission follows the same seasonality as in Bancoumana and 
Donéguébougou with the transmission season taking place from June until December. However, 
entomological inoculation rates are historically much lower than in Bancoumana and 
Donéguébougou. From February 2017 to March 2019, for 268 pediatric cases of symptomatic 
malaria in Sotuba, the mean parasitemia was ~256.7 Pf trophozoïtes (min=1; max=5840; 
STD=630.593), and the mean parasitemia at the time of presentation was 10,268 p/uL (range 
40 to 233,600) (Sissoko, MS, unpublished data). The annual rainfall varies from  800 mm to 
1000 mm and occurs from June to October. Many clinical trials of malaria vaccines and other 
drugs, as well as epidemiological and entomologic malaria studies, have been conducted 
in  Sotuba. The MRTC maintains a medical clinic and laboratory in Sotuba and has been working 
at this site since 1993. 
 
Recruitment Plan 
Community permission will be obtained from village elders and other community members in 
Sotuba after explanation and discussion of the study at a community meeting. A general 
announcement about the study will be made at the time of community permission, using local 
radio or any traditional channel of communication. The announcement will include general 
information about the study as well as contact information for the study site/staff for those 
interested in participating. 
 
Inclusion Criteria  
All of the following criteria must be fulfilled for a volunteer to participate in this trial: 
1. Age: > 18 years old and < 50 years old.  
2. Available for the duration of the trial. 
3. Known resident or long-term resident (more than 1 year) of Sotuba, 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. 

 
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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:  
o Confirmed pharmacologic contraceptives (parenteral) delivery. 
o Intrauterine or implantable device.  
• EXCEPTIONS to required pregnancy prevention includes the following:  
o Postmenopausal state: defined as no menses for 12 months without an 
alternative medical cause.   
o Surgical sterilization. 
7. Willing to have blood samples stored for future research. 
 
Exclusion Criteria  
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. 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. 
3. 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.) 
4. 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.) 
5. Infected with HIV.   
6. Evidence of clinically significant neurologic, cardiac, pulmonary, hepatic, endocrine, 
rheumatologic, autoimmune, hematological, oncologic, or renal disease by history, 
physical examination, and/or laboratory studies. 
7. History of receiving any investigational product within the past 30 days. 
8. Current or planned participation in an investigational vaccine study until the time period 
of the last required study visit under this protocol. 
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. 

 
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11. Known: 
• 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. 
• Autoimmune or antibody-mediated disease including but not limited to: systemic 
lupus erythematosus, rheumatoid arthritis, multiple sclerosis, Sjögren’s syndrome, 
or autoimmune thrombocytopenia. 
• Immunodeficiency syndrome. 
• Seizure disorder (exception: history of simple febrile seizures). 
• Asplenia or functional asplenia. 
• 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. 
• Allergy to latex or neomycin. 
12. Receipt of: 
• Live vaccine within 4 weeks prior to enrollment or a killed vaccine within 
2 weeks prior to enrollment. 
• Immunoglobulins and/or blood products within the past 6 months. 
• Investigational malaria vaccine in the last 2 years. 
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. 
 
Co-enrollment guidelines: Co-enrollment in other trials is limited. 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. Enrolled subjects will be informed that they may be invited to 
participate in other subsequent studies.  
 
Justification for Exclusion of Special Populations 
 Justification of Exclusion of Pregnant Women 
Pregnant women are excluded from participation in this study. The effects of Pfs230D1-EPA/ 
Matrix-M on the developing human fetus are unknown with the potential for teratogenic or 
abortifacient effects. 

 
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 Justification for Exclusion of Children  
This is a “first-in-human” trial with the Pfs230D1-EPA/Matrix-M vaccine. As such, safety and 
tolerability should be established in adults prior to testing in children.  
7 
Study Agents 
 
Pfs230D1-EPA/Matrix-M  
 Manufacturing 
Pfs230D1M-EPA:  PpPfs230D1M and EcEPA lots, both manufactured at the Pilot 
Bioproduction Facility, Walter Reed Army Institute of Research (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 maleimide-activated EcEPA, followed by 
purification using size-exclusion chromatography. The Pfs230D1M-EPA conjugate was 
manufactured at the Walter Reed Army Institute of Research Pilot Bioproduction Facility in 
compliance with cGMP standards. 
 
Matrix-M: The Matrix-M adjuvant is manufactured by Novavax AB (Uppsala, Sweden), a 
subsidiary of Novavax, Inc. The adjuvant contains purified saponin components derived from an 
extract of the bark of the Quillaja saponaria tree; a phospholipid, egg-derived phosphatidyl-
choline (PC); and semi-synthetic cholesterol of non-animal origin. The manufacturing process 
starts with extraction of the Quillaja bark to provide the Quillaja extract which is fractionated by 
chromatography into two distinct fractions (Matrix-A and Matrix-C). 
 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 the Pilot Bioproduction Facility, 
Walter Reed Army Institute of Research (Silver Spring, Maryland) where the materials are 
formulated and packaged, or from ThermoFisher BioServices (Gaithersburg, Maryland) where 
additional product manufactured at the Biopharmaceutical Development Program at the 
Frederick National Laboratory of Research is stored.  
 
The adjuvant will be supplied under refrigerated conditions (2°C to 8°C) for mix with 
Pfs230D1M-EPA at the site. Vials and cartons containing the adjuvant will be labelled as 
appropriate. See Section 7.8 for information about study agent accountability. 

 
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 Formulation, Packaging, and Labeling 
Each single-use vial of Pfs230D1M-EPA contains 160 µg/mL of conjugated Pfs230D1M and 
124 µg/mL or 143 µg/mL of conjugated EPA in 4-mM phosphate-buffered saline (PBS), in a 
volume of 0.5 mL. The vial label reads “160 ug/mL Conjugated Pfs230D1M in 4-mM PBS.” 
Vaccines will be labeled “Caution: New Drug – Limited by Federal (or United States) law to 
investigational use.”  
 
The active ingredient in Matrix-M, specifically Matrix-M1, are saponin-based fractions. Matrix-
M1 has a ratio of Matrix-A and Matrix-C of 85:15 (by weight). Both Matrix-A and Matrix-C are 
individual fractions (separated by chromatography) derived from extracts from the Quillaja 
saponaria tree. Each vial of Matrix-M1 contains saponin content of 0.375 mg/mL in PBS, at a 
pH of 7.2, in a final volume of 0.75 mL. The Matrix-M1 adjuvant is analyzed according to set 
specifications for saponin content (Matrix-A and Matrix-C) by reversed-phase high-performance 
liquid chromatography (RP-HPLC), appearance, turbidity, endotoxins, and sterility. The adjuvant 
is supplied for on-site reconstitution under refrigerated conditions (+2-8°C).  
 
Formulation for each dose level  
Figure 18 provides an illustration of the formulation of Pfs230D1M-EPA and Matrix-M for each 
of the three dose levels of vaccine.  
 
Figure 18. Formulation of Pfs230D1M-EPA and Matrix-M for each dose level of Pfs230D1-
EPA/Matrix-M vaccine. 
 

 
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 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 
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. However, the long-term storage in a 
freezer cannot exceed -65°C. Vaccines are always shipped on dry ice, and generally remain 
below -70°C. The container with Matrix M1 is stored at +2-8°C until use. 
 
Vials will be transported and stored at temperature-controlled conditions, according to 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 indicated temperature conditions 
until usage approval has been obtained from the Sponsor. 
 Preparation and Dosage 
The Pfs230D1M-EPA conjugates are stored at -65°C to -90°C until just before transport to the 
field for use, then stored at 2-8°C as described above. The final vaccine for administration is 
obtained by admixing Pfs230D1M-EPA with Matrix-M as appropriate for each dose level (see 
Figure 18) and must be administered within 4 hours of reconstitution. See Section 7.3 for 
administration information. 
 
Comparator vaccine 
 Manufacturing 
Verorab Rabies Vaccine is manufactured by Sanofi Pasteur. 
 Disposition and Dispensation 
Verorab Rabies Vaccine will be supplied to the study site according to the manufacturer’s 
recommendations. See Section 7.8 for information about study agent accountability. 
 Formulation, Packaging, and Labeling 
Verorab Rabies Vaccine is a purified inactivated rabies vaccine (Wistar rabies PM/WI 38 1503-
3M strain) prepared on Vero cells. It is supplied as a powder and solvent for suspension for 
injection in a prefilled syringe. Before reconstitution, the powder is a white and homogeneous 
pellet. The solvent is a limpid solution. 

 
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 Storage, Shipping, and Stability 
The product should be stored in the original outer package, refrigerated (2°C - 8°C), and 
protected from light. It should not be frozen. After reconstitution, the vaccine may be used up to 
8 hours after reconstitution provided it is maintained at 2°C to 8°C and protected from light. 
Unused vaccine must be discarded after 8 hours. 
 Preparation and Dosage 
Verorab Rabies Vaccine should be reconstituted immediately prior to use, according to 
instructions provided in the package insert. After reconstitution, 1 dose (0.5 mL) contains rabies 
virus, WISTAR Rabies PM/WI38 1503-3M strain (inactivated) ≥ 2.5 IU. 
 
Administration 
Each dose of the Pfs230D1-EPA/Matrix-M vaccine or Verorab (or other rabies vaccine approved 
by Malian health regulatory) is administered as an IM injection into the deltoid muscle. 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 subjects for any immediate allergic 
reactions or other injury resulting from participation in this research study.  
 
Contraindications to Vaccination 
The following criteria should be checked prior to each study vaccination and are 
contraindications to further vaccination with either the experimental vaccine or the rabies 
vaccine:  
• Hypersensitivity reaction following administration of the study vaccine or comparator. 
• Positive urine or serum β-hCG test prior to vaccination. 
 
Subjects who receive at least 1 dose of study vaccine under this protocol prior to developing a 
contraindication 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. Subjects who have a positive β-hCG test prior to their first study 
vaccination may be withdrawn from the study and replaced. 
 
Indications for Deferral of Vaccination 
If any of the following criteria are met at the time of the scheduled study vaccination, the 
vaccination will be deferred pending resolution of the issue: 
• Oral temperature >38.0ºC at the time of vaccination. 
• Receipt of a prohibited medication/procedure as described in Section 7.7. 

 
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• 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. 
 
Symptomatic individuals may 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.  
 
If the subject meets any of the above criteria for deferral on the day of first immunization, the 
investigator may elect to withdraw 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, they 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. 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. 
• 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. 
 

 
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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. 
8 
Study Schedule 
The study schedule and approximate amounts of blood drawn are detailed in Appendix A.  
 
At all visits during the trial, appropriate measures will be taken to prevent transmission of SARS-
CoV-2, the organism which causes COVID-19. This will include proper use of Personal 
Protective Equipment (PPE) such as face masks and gloves, social distancing, good hand 
hygiene and other control and prevention measures as outlined in procedures. 
 
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. 
 
If a subject is found to be HIV positive at screening, local counseling will be provided first and 
then the subject will be referred to the national management system for further follow-up. 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.  
 
The following screening evaluations must be completed for all subjects within the 56 days prior 
to enrollment (Day -7 visit):  

 
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• Explain the study and informed consent/assent documents to the subject  
• Ensure the subject has correctly answered ≥80% of the questions on the Malaria 
Comprehension Exam.  
• 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, and 
medication use.  
• Females of childbearing potential must be willing to use reliable contraception from at 
least 21 days prior to first vaccination through 1 month after the last vaccination.  
• EXCEPTIONS to required pregnancy prevention includes the following:  
• Postmenopausal state: defined as no menses for 12 months without 
an alternative medical cause   
• Surgical sterilization 
• Administer a complete physical examination, including vital signs (blood pressure, 
temperature, and heart rate), height/length, and weight.  
• 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.  
• Obtain blood for complete blood count with differential (CBC w/diff) and platelet count, 
ALT, Cr, Hepatitis B, Hepatitis C, HIV antibody and hemoglobin typing.  
• For females of childbearing potential, obtain urine (or serum) for pregnancy testing.  
If screening laboratories are completed within ≤2 days prior to Study Day -7, 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 -7, the following screening 
procedures will need to be repeated before enrollment: updated medical history, repeat physical 
exam and all laboratories outside of the window (inclusive of safety labs and human 
immunodeficiency virus [HIV]). Subjects will then be reassessed for eligibility based on the re-
screening information. 
 
 
Enrollment and On-Study Visits 
Individuals who are deemed eligible for study participation will be enrolled and begin study 
participation with Day -7 procedures. See Appendix A for a detailed study schedule.  
 
The study visits scheduled for Study Days 281, 337, and 393 (8, 10 and 12 months after the final 
vaccination) may be conducted as phone calls if the participant is not able to come to the study 
site in person.  

 
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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 end-of-study visit procedures as 
possible. 
9 
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. 
 
Blood Draw 
The total amount of blood collected is well within the American Association of Blood Banks 
recommendations and the current NIH guidelines, and will not compromise these otherwise 
healthy subjects (Howie 2011). 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. Laboratory 
reference ranges are provided in Appendix C: Mali Adult Institutional Normal Laboratory 
Values.  
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 is collected for research purposes 
2. 
Serum Creatinine 
3. 
ALT 
4. 
Hepatitis B and C  
5. 
HIV antibody test (can include rapid diagnostics, ELISA, western blot if 
indicated) 
6. 
Urine and/or serum pregnancy testing (β-hCG) in females of childbearing 
potential 

 
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Malaria Diagnostics  
 Blood Smears 
Blood Smears: 
Blood smears (BS) will also be collected periodically and will be read in real time.  
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.  
 
Blood BS Reading: Giemsa-stained thick and thin films will be examined for asexual and sexual 
parasites in the MRTC clinical laboratory. BS are prepared in duplicate according to standard 
procedures and evaluated by trained study microscopists. 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. 
9.4.1.1 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.  
 
For clinical diagnostics, RDTs will be utilized for determination of an acute malarial illness. The 
RDT will be paired with collection and reading of a thick BS.  
 
Participants diagnosed with malaria will be treated with either AL or another approved/licensed 
anti-malarial medication per Malian Government treatment guidelines. 
 
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 

 
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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 8. 
 
Table 8. 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  
 
Feeding assays demonstrate biologic activity of transmission-blocking antibodies and are critical 
to selection of TBV candidates. Subjects will be screened periodically by BS (see Appendix A) 
for the presence of asexual parasites and gametocytes (slides read retrospectively).  
 Standard Membrane Feeding Assays 
Membrane-feeding assays demonstrate biologic activity of TBA 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  (Cheru, Wu et al. 2010). The SMFAs will be conducted at LMIV and LMVR 
in Rockville, Maryland using laboratory-strain mosquitoes and parasites. Assays will compare 
feedings with the following: 
• Plasma/sera. 

 
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• 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. 
 
Immunology Assays  
 Antibody Assay 
Anti-Pfs230 ELISAs will be performed on sera or plasma obtained from immunized subjects at 
LMIV in Bethesda, Maryland, and may also be performed at collaborating laboratories. 
 
For Pfs230, briefly, microwell plates are coated with antigen solution. Plates are washed with 
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 #3. 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. 
 B-Cell and T-Cell Assays 
Specimens collected for B-cell and T-cell studies will undergo initial processing and cell 
separation at the clinical site and will be transported to LMIV according to standard procedures. 
 
B-cell studies will be done at LMIV and associated immunology laboratory partners. The 
analysis of the generation and maintenance of antigen-specific memory B cells will be carried 
out to determine if these cells can be elicited and maintained by vaccination. Peripheral blood 
lymphocytes will be obtained and assayed for the presence of antigen-specific memory B cells, 

 
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total number of memory B cells, and plasmablast responses using flow cytometry and ELISPOT 
assays. 
 
T-cell studies will be performed at LMIV. Antigen-specific T-cell responses to vaccination will 
be determined by ELISPOT and/or intracellular cytokine staining flow cytometry. 
 
Ex vivo studies will be performed at MRTC using whole blood to enumerate various immune 
subsets (T, B, NK cells) prior to and after each vaccination. 
 Transcriptional Profiling 
Whole genome transcriptional profiling will be performed to explore possible gene expression 
profiles or pathways that predict optimal responses to vaccination and to determine if innate 
immune responses are sensitively reflected in the PBMC transcriptome shortly after vaccination. 
Gene expression profiling following vaccination will allow the predictive capacity of eventual 
high and low responders, and thus will assist in defining the correlates of protection induced by 
vaccination. 
 
Transcriptional analyses will be performed on whole blood collected as outlined in Appendix A. 
Blood will be collected via venous puncture and placed in PAXGene tubes to preserve RNA 
integrity until the RNA is extracted. Specimens will be analyzed at the Research Technologies 
Branch, NIAID, the NIH Intramural Sequencing Center, and/or by other LMIV laboratory 
collaborators. The molecular profiling encompasses the identification of RNA transcripts present 
in all humans, which are induced or repressed after each vaccination. This does not represent 
genetic testing of individuals or their DNA. 
 
Other Laboratory Assays 
If there is adequate remaining blood sample available to fulfill the laboratory objectives, other 
laboratory assays may be performed as follows: 
• qPCR may be used to detect gametocytes using whole blood collected on the day of 
positive BS.  
• 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  (Fanello, 
Santolamazza et al. 2002). 
• Cytokine levels may be evaluated during and following vaccination. 
 

 
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Study physicians may ask for additional laboratory exams related to subject care. Results of 
clinically indicated laboratory evaluations may be collected for research use. 
 
Collection of Malaria Prevention Measures During the Transmission Season 
Enrolled subjects may be asked at one or more of their study visits about other malaria 
prevention measures being utilized by the individual, including use of bed nets, indoor residual 
spraying, seasonal malaria chemoprophylaxis, personal use of insecticide, and/or intermittent 
preventive treatment.  
10 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.  
 
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 (with no personally identifiable information) will be 
shipped to LMIV during and after the study as appropriate. These samples will 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. The code key will be maintained by the Malian investigators. 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 16.1. 
 
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. In that case, the principal investigators 
will review the request. If the planned research falls within the category of “human subjects 
research” on the part of the investigators, FMPOS EC review and approval will be obtained. This 
includes the investigators sending out coded and linked specimens or data and getting results that 
they can link back to their subjects. 
 
Data will be archived by the study team in compliance with requirements for retention of 
research records; alternatively, after FMPOS and study sponsor approval, the data may be either 
destroyed or transferred to another repository. 
 

 
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Reporting the Loss or Destruction of Samples/Specimens/Data to the EC: 
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 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 FMPOS EC. This decision will not affect the individual’s participation in any other protocols 
at MRTC. 
11 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, and 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. 
12 Assessment of Safety 
 Definitions 
AE: 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. 
 

 
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Adverse Reaction (AR): An AE that is caused by a study agent. ARs are a subset of all 
suspected adverse reactions (defined below) where there is reason to conclude that the study 
agent caused the event. 
 
Suspected Adverse Reaction (SAR): An AE for which there is a reasonable possibility that the 
study agent caused the AE. ‘Reasonable possibility’ means that there is evidence to suggest a 
causal relationship between the study agent and the AE. An SAR implies a lesser degree of 
certainty about causality than AR, which implies a high degree of certainty. 
 
SARs are the subset of all AEs for which there is a reasonable possibility that the study agent 
caused (see “Causality” below) the event. Inherent in this definition, and in the requirement to 
report SARs, is the need for the sponsor to evaluate the available evidence and make a judgment 
about the likelihood that the study agent actually caused the AE.  
 
The sponsor is responsible for making the causality judgment. 
 
SAE: An SAE: 
• is an AE that results in death 
• is an AE that is life-threatening (places the participant at immediate risk of death from the 
event as it occurred) 
• is an AE that requires inpatient hospitalization or prolongs an existing hospitalization 
NOTE: 
o Hospitalization is considered required if outpatient treatment would generally be 
considered inappropriate.  
o Same-day surgical procedures that are required to address an AE are considered 
hospitalizations, even if they do not involve an overnight admission. 
o Hospitalization due to a condition that has not worsened and that pre-dates study 
participation (e.g., elective correction of an unchanged baseline skin lesion), or 
due to social circumstance (e.g., prolonged stay to arrange aftercare), or that is 
planned/required “per protocol” and that proceeds without prolongation or 
complication, is not considered an SAE by this criterion. 
• is, or results in, a persistent or significant incapacity or substantial disruption of the 
ability to conduct normal life functions 
• is a congenital anomaly/birth defect/miscarriage/stillbirth 
NOTE: This definition is more inclusive than some commonly published definitions. It 
includes an affected conceptus/neonate whose: 
• biological mother was exposed to a study agent at any point from conception through the 
end of the pregnancy, and/or, if breastfeeding, the 30-day neonatal period; or  

 
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• biological father was exposed to a study agent at any point during the 90 days prior to 
conception. 
• This is separate from, and in addition to, general reporting of pregnancy in a study 
participant or female partner of a male participant (see Section 12.5.4 below). 
• a medically important event 
NOTE: Medical and scientific judgment should be exercised. Events that significantly 
jeopardize the subject and/or require intervention to prevent one of the SAE outcomes 
listed above are generally considered medically important, and are thus SAEs. 
 
Unexpected AE: An AE is unexpected if it is not listed in the investigator’s brochure or package 
insert (for marketed products) at the frequency, specificity, and severity that has been observed.  
NOTE: 
o Such events should also be evaluated for possible reporting as unanticipated problems 
(UPs) (see Section 12.5.3 below).  
o Unexpected, as used in this definition, also refers to AEs or SARs that are mentioned in 
the investigator’s brochure as occurring with a class of drugs/biologics, or as anticipated 
from the pharmacological properties of the study agent but are not specifically mentioned 
as occurring with the particular study agent under investigation. 
 
Serious and Unexpected Suspected Adverse Reaction (SUSAR): A SUSAR is an SAR 
(defined above) that is both serious and unexpected. 
 
UP: 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 (including but not limited to risks) as described in the EC-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 is 
• possibly, probably, or definitely related to participation in the research; and 
• suggests the research 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 documents currently approved by the EC. 
NOTE: 
o Per the sponsor, an SAE always meets this “greater risk” criterion. 
o An incident, experience, or outcome that meets the definition of a UP 
generally will warrant consideration of changes to the protocol or informed 
consent form, or to study procedures (e.g., the manual of procedures [MOP] 
for the study), in order to protect the safety, welfare, or rights of participants 

 
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or others. Some UPs may warrant a corrective and preventive action plan 
(CAPA) at the discretion of the sponsor or other oversight entities. 
 
Serious UP: A UP that meets the definition of a Serious Adverse Event or compromises the 
safety, welfare or rights of subjects or others. 
 
UP that is not an AE (UPnonAE): A UPnonAE belongs to a subset of UPs that 
• meets the definition of a UP, and 
• does not meet the definition of an AE or SAE 
NOTE: Examples of UPnonAEs include, but are not limited to: 
o a breach of confidentiality 
o prolonged shedding of a vaccine virus beyond the anticipated timeline 
o unexpectedly large number of pregnancies on a study 
o subject departure from an isolation unit prior to meeting all discharge criteria 
o accidental destruction of study records 
o unaccounted-for study agent 
o overdosage, underdosage, or other significant error in administration or use of 
study agent or intervention, even if there is no AE/SAE  
o development of an actual or possible concern for study agent purity, sterility, 
potency, dosage, etc. 
NOTE: A decision to temporarily quarantine, or to permanently not use all or part 
of study agent supply due to an unexpected finding or event (e.g., particulate, 
cloudiness, temperature excursion), even if there is no known or proven issue 
(i.e., out of an “abundance of caution”), is considered a UPnonAE. 
 
New Onset of Chronic Illness (NOCI): A NOCI is 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 AE Case Report Form (CRF) or electronic database, and 
• reported as outlined below (e.g., IND sponsor, FMPOS EC, US Food and Drug 
Administration [FDA]). 
 

 
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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. 
 
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 below. 
 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 9 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 (i.e., asymptomatic parasitemia) will not 
be reported as an AE. Clinical or symptomatic malaria will be reported as an AE. 
 

 
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Table 9. Solicited Adverse Events 
Systemic adverse events 
Fever (temperature ≥38.0 °C) 
Headache  
Nausea/Vomiting  
Diarrhea 
Abdominal pain 
Fatigue 
Malaise  
Myalgia  
Arthralgia  
Urticaria  
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, , all 
laboratory AEs will be collected and graded through 14 days after the first and second 
vaccination and 28 days after the third 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. 
 Severity 
Severity of AEs will be assessed by the investigator according to the toxicity tables provided in 
Appendix B. AEs not included in the Appendices will be graded for severity using the definitions 
provided in Table 10. 

 
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Table 10. 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 
 
Unlikely Related 
• does not have a reasonable temporal relationship 
OR 

 
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• 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 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.  
 
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. 
 
Causality assessment will be provided by the Principal Investigator or designee and reviewed by 
the sponsor. The sponsor may make a separate and final determination on the “reasonable 
possibility” that the event was “related” or “unrelated” to the study agent, in keeping with 
applicable (US FDA) guidance on sponsor IND safety reporting.  
 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 

 
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lost to follow-up), the last known status and 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 by REDCap or 
on the Safety Expedited Report Form (SERF).  
 Investigator Reporting Responsibilities to the Sponsor 
 Adverse Events 
AE data will be entered into the research database no less than every other week and will include 
all data through one week prior to database entry. 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 by 
REDCap or 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  
https://crimsonredcap.cc.nih.gov/redcap/index.php 
 
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 ISM  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 

 
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 Unanticipated Problems 
Unless otherwise specified above, UPs that are also AEs or SAEs, must be reported to the CSO 
(by REDCap, or by email and SERF if REDCap is not available) no later than when they are due 
to be reported to the FMPOS EC. 
 
UPnonAEs are not reported to the CSO but must be reported to the clinical trials management 
(CTM) group and the EC according to their requirements and preferred methods. If the 
UPnonAE raises a significant potential subject safety concern, the SMM should be consulted by 
email or phone no later than when reports are made to the EC and/or CTM. 
 Pregnancy 
Pregnancy itself is not an AE. However, complications of pregnancies are AEs and may be 
SAEs. Events that meet AE or SAE criteria in relation to pregnancy, delivery, or the 
conceptus/neonate (see Section 12.1) are reportable (by REDCap, or by email and SERF if 
REDCap is not available). All pregnancies occurring up until 1 month after last vaccination will 
be reported (by REDCap, or by email and SERF if REDCap is not available) to the CSO within 
1 business day from site awareness. Pregnancies after that timepoint will be reported in a non-
expedited manner. 
 
Pregnancy outcome data (e.g., delivery outcome, spontaneous or elective termination of the 
pregnancy) will be reported to the CSO (by REDCap, or by email and SERF if REDCap is not 
available) and LMIV within 3 business days of the site’s awareness. 
 
In the event of pregnancy in a study subject who has possibly been exposed to study agent, the 
following steps will be taken: 
• Unblind the study subject  
• 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) and clinical visits.  
• Report to FMPOS EC as an informational item. 
• Report to the DSMB, SMM, and Malian ISM. 
• Advise research participant to notify the obstetrician of study participation and potential 
study agent exposure. 
 

 
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 Medically Attended Adverse Events (MAAEs) that are Potential Immune-Mediated 
Medical Conditions (PIMMCs)  
This trial includes plans for collection and analysis of data relating to medically attended adverse 
events (MAAEs) among subjects in all treatment groups through 12 months following the last 
study vaccination, due to the theoretical potential for induction of autoimmune or auto-
inflammatory diseases due to Matrix-M.  
 
Collection of MAAE data beyond 6 months after the last study vaccination will not delay 
submission of clinical trial reports and initiation of subsequent clinical trials. Final analyses of 
MAAEs will be submitted in clinical trial report addenda.   
 
As part of our analyses of MAAEs in all annual reports, clinical trial reports, and clinical trial 
report addenda, we will include tabulated summaries of available adverse event data for 
potentially immune-mediated medical conditions reported during the trial, categorized by 
MedDRA term. These summaries will include any MedDRA preferred term included in the 
Immune-mediated/Autoimmune disorders Standard MedDRA Query (SMQ).  Conditions 
reported during the trial that match the terms included in the SMQ (or list of terms provided by 
FDA) as well as any other potentially immune-mediated medical conditions reported during the 
trial that do not appear in the SMQ will be included. For each adverse event included in the 
summaries, we will provide a case narrative, along with an assessment of the seriousness of the 
event and causal relationship to study vaccine, as required by 21 CFR 312.32.  
 
Because the request for this information by the FDA is based on a theoretical potential, the 
occurrence of a potentially vaccine-related immune-mediated medical condition will be 
considered unexpected. We will adhere to expedited reporting requirements as provided in 21 
CFR 312.32 for any adverse event assessed as a serious and unexpected suspected adverse 
reaction (SUSAR). 
 
Based on the theoretical concern for the development of autoimmune diseases after vaccination 
with new vaccines containing novel adjuvants, a list of Adverse Events of Special Interest 
(AESI) specific to potential immune-mediated medical conditions (PIMMCs) is provided in 
Appendix D (Integrated Summary of Safety of Other Novavax Recombinant Nanoparticle 
Vaccine Antigens with Matrix-M1TM Adjuvant, Version 1.0 dated 20 April 2021).    
 
Reporting of MAAEs (which are PIMMCs) to the CSO will be included in monthly safety 
reports (in separate table[s]) unless they are considered to be SAEs, in which case they will be 
reported to the CSO as per Section 12.5.2 (SAE Reporting).  
 
In order to be certain that the study team is alerted to any PIMMCs as quickly as possible, study 
participants will be asked to report to the study team for any medical issues which arise from the 

 
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start of the study through 12 months after the final dose. They will also be asked to provide 
information about any Medically Attended Adverse Events (MAAEs) where the participant was 
seen by a Health Care Provider outside of the study (including visits to doctors, nurse 
practitioners, traditional healers and any other local practitioners). In this way, the study team 
can determine if any of these complaints may be considered PIMMCs.  
 Investigator Reporting Procedures to FMPOS  
 Definitions 
Protocol Deviation: Any change, divergence, or departure from the EC-approved research 
protocol.  
• Major Deviations: Deviations from the EC-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 EC, 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 affects 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 EC).  

 
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 Expedited Reporting to FMPOS EC 
Non-compliance: Any actual or suspected non-compliance by any investigator or entity 
associated with the protocol must be reported by the Malian PI/designee within 7 calendar days 
of any investigator or individual associated with the protocol first becoming aware, unless 
otherwise indicated in this policy. Non-compliance will be reported to the NIH Office of Human 
Subjects Research Protections as required per institutional policy. 
 
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.  
 
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: 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.  
 
Suspension or termination of activities: Any suspension or termination of research activities, 
including holds on new enrollment, placed upon the research by the study sponsor, NIH or 
ethical review committee leadership, or any regulatory agency must be reported within 
7 calendar days of an investigator becoming aware.  
 Annual Reporting to FMPOS EC 
Investigators must provide the following information to the EC in summary format at the time of 
continuing review: minor protocol deviations; AEs and SAEs that do not meet the definition of 
an UP.  
 Sponsor’s Reporting Responsibilities 
Events reported to the sponsor will be promptly evaluated and will be reported as required 
according to FDA IND safety reporting guidance and regulations. IND safety reports will be 
shared with other stakeholders according to applicable agreements (e.g., CRADAs and CTAs). 
 
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. 

 
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 Pausing Rules for the Protocol 
Pausing is the suspension of administration of study agent to a single subject 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: 
• A subject experiences one SAE that is determined as possibly, probably, or definitely 
related to a study agent 
• A subject experiences a hypersensitivity reaction (e.g. anaphylaxis, diffuse urticaria) that 
is determined to be possibly, probably, or definitely related to the vaccine 
• A subject experiences ≥1 Grade 3 or greater AEs or ≥1 Grade 3 laboratory abnormalities 
that are deemed not to be a SAE (solicited local/systemic or unsolicited; lasting 72 hours 
or more) that are possibly, probably, or definitely related to a study agent, 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 for any safety 
issue. The study safety oversight bodies (i.e., DSMB and/or ISM) may recommend a pause to the 
PI. 
 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 1 business day to the CSO and to the FMPOS EC according to their requirements. The PI 
will also notify the DSMB and ISM through the specified pathway. 
 Resumption of a Paused Study 
The CSO, in collaboration with the PI, DSMB, and ISM, will determine whether or not it is safe 
to resume administration of the study agent to the subject. The PI will notify the FMPOS EC of 
the decision on resumption of the study agent. A subject who does not resume study agent 
administration will continue to be followed for safety and may provide samples for immunology 
assays.  
  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. 
 

 
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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): 
• 1 or more subject(s) experience an SAE as defined in Section 12.1 of this protocol that is 
determined to be possibly, probably, or definitely related to the vaccine, or 
• 1 or more subjects experience a hypersensitivity reaction (e.g. anaphylaxis, diffuse 
urticaria) that is determined to be possibly, probably, or definitely related to the vaccine, 
or 
• 
Pilot Group: > 20% (2 or more of n=5) of vaccinees assigned to an experimental vaccine 
dosage (either low, middle or high dose) experience any Grade 3 AE (solicited or 
unsolicited) lasting 48 hours or more or any Grade 3 laboratory abnormality with either 
the AE or lab abnormality determined to be possibly, probably, or definitely related to the 
vaccine as defined in this protocol, within the 7 days post vaccination 
• 
Main Group*: >20% (4 or more of n=20) of vaccinees assigned to an experimental 
vaccine dosage (either low, middle or high dose) experience any Grade 3 AE (solicited or 
unsolicited) with signs or symptoms lasting for 48 hours or more or any Grade 3 
laboratory abnormality with either the AE or lab abnormality determined to be possibly, 
probably, or definitely related to the vaccine as defined in this protocol, within the 7 days 
post vaccination 
or 
• Any safety issue that the study PI or CSO determines should halt the study. 
*Once the Main Group starts, Halting Rules will be based on review of data from all 20 participants 
receiving a specific experimental vaccine dosage (n=20)  
 
If halting criteria are met based on blinded data, the list of research participants with the Grade 3 
AEs or laboratories will be sent to the DSMB Executive Secretary. A DSMB Meeting could be 
convened on short notice at the occurrence of such an event.   
 
In addition, the FDA, FMPOS EC and/or CSO may halt the study at any time.  
 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 and to the FMPOS EC according to their requirements. The PI 
will also notify the DSMB and ISM through the specified pathway. 
  Resumption of a Halted Study 
The CSO, in collaboration with the PI, DSMB, and ISM, 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 EC of the decision on resumption of the study. Subjects who do not resume study 

 
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agent administration will continue to be followed for safety. They may also provide samples for 
immunology assays if the PI agrees that this is appropriate.  
 Early Termination of Study 
The NIAID/OCRPRO as the study sponsor, FMPOS EC, or 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 be withdrawn from the study for any of the following reasons: 
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 after being enrolled (see Section 5.3). 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. At the PI’s discretion - for any event that may pose a safety risk to the subject or 
jeopardize data collected for the study 
5. Other – is used when previous categories do not apply and a written explanation is 
required. 
 
A withdrawn subject will not have any further study visits, safety evaluations, or research 
procedures for this protocol; however, any data collected from that subject prior to the 
withdrawal will be included in the safety and immunogenicity analysis if the subject completed 
at least 1 study vaccination. 
 
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. If the reason is not immediately 
clear, the investigator will make a reasonable effort to determine it. 

 
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 Replacement of Withdrawn Participants 
Subjects who withdraw or are withdrawn from the study after receiving at least 1 study 
vaccination 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. The ISM will 
remain blinded throughout the study.  
 
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 
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 the EC 
on a biannual basis or more frequently if a safety concern is raised.  

 
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 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. 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 EC 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 FMPOS EC. 
13 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 (or conduct virtual site visits) 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 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, representatives of the PfTBV EDCTP Consortium 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. 

 
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14 Statistical Considerations and Sample Size  
 Sample Size  
Safety: 
The arms are sufficiently sized for safety. For each dose level (n=20) in Arms 1a combined with 
2a, and 1b combined with 2b, 1c combined with 2c, and 1d combined with 2d, vaccination of 20 
subjects gives a power of at least 0.80 for detecting 1 or more serious or severe AEs that occur 
with a probability of 0.077 or more per subject (or 0.90 for an event with a probability of 0.109).  
 
If we combine all treated groups (arms 1a/2a, 1b/2b, 1c/2c), with a total of 60 subjects, we have 
80% power to detect 1 or more serious or severe AEs that occur with a probability of 0.026 or 
more per subject (or 0.90 for an event with a probability of .037).  
 
The aforementioned calculations do not assume any historical data; they are statistical 
consequences of the binomial distribution. 
 
We will compare all AE event proportions between the control arm and treated arm by Fisher’s 
exact test. 
 
Immunogenicity:  
We plan to do a test for an overall signal with a global analysis. That is, we will combine arms 
1a/2a, 1b/2b, and 1c/2c into an “any vaccination” group of n=60 and combine arms 1d and 2d 
into a “control” group of n=20. Using historical data from the AS01 trial, we are well powered to 
detect a log difference (>99.9% power) and ½ log difference (99.7% power). 
 
Following this global test, testing specific arms is of interest. To adjust for multiplicity, we use 
Bonferroni corrected alpha of 0.05/3. To investigate the specific arms we conduct three tests: 
arms 1a/2a vs controls, arms 1b/2b vs controls, and arms 1c/2c vs controls. The controls use 
arms 1d and 2d. This makes each analysis n=20 vs n=20.  As such, we are well powered to detect 
a log difference (>99.9% power) and ½ log difference (89.2% power). 
 
These calculations were made assuming normal distributions with mean and standard deviations 
from the AS01 trial. 1000 simulations were run to see how often a significant result came from a  
Wilcoxon-Mann-Whitney test (WMW). 
 Primary Objective 
To assess safety and reactogenicity of administration of Pfs230D1-EPA/Matrix-M (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. 

 
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• 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 Objectives 
 ELISA Analysis  
There are several questions of interest related to antibody response, which include the change in 
ELISA values from baseline after a given number of doses of vaccine and the change in ELISA 
values between doses. To address these questions, we will use an arm-specific WSRT within the 
20 subjects receiving a given vaccine dose.  
 
The data from subjects who had Pfs230 ELISA measurements after receiving two doses on 
protocol #17-I-N006 (Pfs230/AS01 trial) allow us to estimate the 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 protocol #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 protocol #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.   
 
ELISA results will be analyzed by Wilcoxon-Mann-Whitney test, as the simulated power 
calculations were conducted. 

 
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 SMFA Analysis 
For a group of 20 participants for each vaccine dosage, we anticipate about 90% power to detect 
a difference in TRAs of 32% (the observed difference in protocol #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 
previously established methods (Swihart, Fay et al. 2018). A nice feature of the standardized 
TBA is that its power is identical to those of the TRA.  
 
SMFA results will be analyzed by Wilcoxon-Mann-Whitney test, as the simulated power 
calculations were conducted. 
 Exploratory Objectives 
 Analysis 
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, 
as appropriate.  
 Measures to Minimize Bias: Randomization and Blinding 
  Randomization 
The randomization list will be prepared and the code maintained by the study biostatistician.  
Randomization will be assigned at the time of first vaccination with the next available subject. 
Once a subject has received their first vaccination, they cannot be replaced.  
 
During the study, the list linking randomization numbers to study product (Pfs230D1-EPA/ 
Matrix-M or Verorab or other Malian health regulatory approved rabies vaccine) will be made 
available only to the study statistician and associated team members, pharmacy team/syringe 
preparers (at the start of the study), ISM (if needed to review), and DSMB chair (if needed for 
closed session unblinded review). On vaccination days, the vaccines associated with each 
randomization number will be obtained from the pharmacist. 
 Blinding 
The study is double-blind (clinical staff and participants). Blinding extends to laboratory staff 
conducting clinical chemistry, hematology, and parasitology tests, and to laboratory staff 

 
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conducting research assays such as antibody and cellular immunity assays. Blinding will 
continue until completion of the last study visit and the cleaning and locking of the data set. The 
principal investigator will be responsible for strict maintenance of the blinding on site.  
 Unblinding 
Details of unblinding procedures are provided in the unblinding SOP.  
 
After unblinding, subjects who received the Pfs230D1-EPA/Matrix-M vaccine will be offered 
the opportunity to receive the Rabies Vaccine on the same dosing schedule as the Controls 
(3 doses given at 1-month intervals).  
 
Unblinding of individual participants: If knowledge of the treatment assignment is needed to 
provide appropriate medical care, and unblinding is recommended by the principal investigator, 
the ISM, or the DSMB, the treatment assignment of that research subject may be unblinded and 
provided to the treating clinician and other clinical staff on a need-to-know basis by the head of 
Pharmaceutical Operations at the study site or other designated unblinded staff who have access 
to the unblinded randomization list and pharmaceutical team records. The principal investigator 
must contact the Sponsor and provide documentation of the event and the reasons for unblinding.   
 
See Section 12.9 regarding unblinding if pausing or halting criteria are met based on blinded 
data.   
 
Unintentional unblinding:  If unintentional unblinding of study agent assignment occurs, the 
principal investigator will create a plan for ongoing management of the participant(s) involved 
and for preventing the recurrence of a similar incident, as appropriate. If the protocol team 
determines that the unintentional unblinding may have a significant impact on the study plan 
(e.g., if the randomization codes for multiple participants or an entire cohort were accidentally 
broken), the need for a protocol amendment will be addressed as soon as possible. 
 
Reporting: The PI will report all cases of intentional and unintentional unscheduled unblinding to 
the DSMB in writing within 1 business day after site awareness via email to the DSMB mailbox 
(niaiddsmbia@niaid.nih.gov) outlining the reason for the unblinding and the date it occurred. 
The report will also be submitted to the SMM. 
 
Subjects who are unblinded will be encouraged to remain in the study to be followed for safety.  
15 Ethics/Protection of Human Subjects 
This research will be conducted in compliance with the protocol, ICH GCP, and all applicable 
regulatory requirements. 

 
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 FMPOS USTTB EC 
A copy of the protocol, informed consent forms, and other study related information to be 
completed by subjects, such as questionnaires, medical history forms, and any proposed 
advertising/recruitment materials or letters to the subjects will be submitted to the FMPOS EC 
for written approval. The investigator must submit and obtain approval from the FMPOS EC for 
all subsequent amendments to the protocol, informed consent documents, and other study 
documentation referenced above. The investigator will be responsible for obtaining EC approval 
of the annual continuing review throughout the duration of the study. The investigators will 
notify the FMPOS EC of protocol violations and other reportable events 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 forms will be approved by the FMPOS EC. The subject will sign the informed consent 
document prior to undergoing any research procedures. The subjects may withdraw 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 and the person who conducted the 
informed consent discussion. The original signed informed consent form will be retained in the 
subject’s chart and a signed and dated copy will be provided to the subject. 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. 
 Mali Site Community Permission and Individual Informed Consent Process 
15.2.1.1 Community Permission 
Community permission will be obtained from village elders, family heads, and other community 
members after explanation and discussion of the study  (Diallo, Doumbo et al. 2005). 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’s 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. 

 
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• 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. 
15.2.1.2 Individual Informed Consent 
Potential subjects will be invited to come to the study clinic for review of the informed consent.  
 
At the consenting visit, the potential subject will read the consent form, or have it explained in 
cases of illiteracy. Subjects 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 
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.  
 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 

 
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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 FMPOS EC, the 
FDA, OHRP, or the sponsor’s designee.  
 Potential Risks 
Risks to the subjects are associated with vaccination and other study procedures. These risks are 
outlined below. 
 Study Vaccines 
15.4.1.1 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. 
15.4.1.2 General Study Vaccine Risks 
The study vaccine can cause non-specific inflammation and may be harmful while pregnant 
(Section 6.5). Therefore, pregnant women are excluded from study participation, and women of 
reproductive potential will be required to agree to use birth control as outlined in Section 6.3 and 
will be tested for pregnancy prior to each study vaccine administration.  
 
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. 
15.4.1.3 Pfs230D1-EPA/Matrix-M 
15.4.1.4 Pfs230D1M 
Common solicited AEs during past administrations of Pfs230D1M-EPA/Alhydrogel and 
Pfs230D1M-EPA/AS01 included injection site pain, redness, induration, and swelling. Other 
commonly reported solicited AEs included headache, malaise, diarrhea, nausea, and fever. 
 

 
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While several subjects in studies conducted in the US and Mali experienced changes in 
hematologic parameters (such as anemia, leukopenia, and neutropenia), some of which were 
moderate (Grade 2) in severity, an examination of trends in the US study and the unblinded 
portion of the Mali study 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, SMM, 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.  
15.4.1.5 Matrix-M 
Common solicited AEs during past administrations of other vaccines containing the adjuvant  
Matrix-M (including the COVID Vaccine candidate, NVX-CoV2373) are: injection site pain, 
redness, induration, and swelling and the following systemic AEs: fever, nausea/vomiting, 
headache, fatigue, malaise, myalgia and arthralgia.  
15.4.1.6 EPA 
EPA has been studied in both malaria transmission vaccine studies (as noted with Pfs230D1M) 
and other vaccination studies  (Ashkenazi, Passwell et al. 1999, Lin, Ho et al. 2001, Passwell, 
Ashkenazi et al. 2003, Passwell, Ashkenzi et al. 2010, Thiem, Lin et al. 2011). The use of EPA 
has identified no safety issues to date. 
15.4.1.7 Verorab Rabies Vaccine  
The following adverse reactions have been associated with Verorab Rabies Vaccine: 
 
Very common (≥ 1/10): adenopathy/lymphadenopathy; myalgia; injection-site pain; fever; 
malaise. 
 
Common (≥ 1/100): cutaneous allergic reactions such as rash, pruritus, and oedema; headache; 
dizziness; abdominal pain; nausea; somnolence; arthralgia; shivering; injection-site erythema, 
pruritus, hematoma, and induration; asthenia; influenza-like syndrome. 
 

 
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Uncommon (≥ 1/1000): injection-site swelling; urticaria; angioedema; dyspnea; diarrhea. 
 
Verorab Rabies Vaccine is considered a Category B vaccine. One animal toxicity study on 
reproduction and development led with another inactivated rabies vaccine produced in VERO 
cells, did not evidence any deleterious effect on female fertility and on pre- and post-natal 
development. Clinical use of rabies vaccines (inactivated "WISTAR Rabies PM/WI38 1503-3M 
strain") during a limited number of pregnancies did not show any malformative or fetotoxic 
effects to date.  
 
For complete safety details, please refer to the package insert provided for Verorab Rabies 
Vaccine. 
 Treatment for Malaria  
Participants who are diagnosed with clinical malaria will be treated with the standard of care as 
per the Mali Ministry of Health.  
 Venipuncture 
Risks occasionally associated with venipuncture include pain, bruising, bleeding, and infection at 
the site of venipuncture, lightheadedness, and rarely, syncope. 
 Potential Benefits 
Subjects will not receive any direct benefit from participation in this study. It is hoped that 
information gained in this study will contribute to the development of a safe and effective 
malaria vaccine.  
 Photography 
Taking pictures of the body or face may be embarrassing to some people. These photographs 
may be published in medical journals, without identifying the participant. We will attempt to 
preserve the anonymity of the participant as much as possible, while providing the information 
needed to support the research being published. Participants may decline photographs or place 
any restrictions on their use. Participants will be given the opportunity to discuss this with the 
principal or associate investigators. 
 Compensation 
Subjects will be given in kind (such as rice and/or millet) or cash equivalent payments, in 
multiple installments as outlined in Table 11, 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.  
 

 
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The FMPOS EC recommends compensating the study subject for their time lost for study 
procedures. The amount of compensation is equivalent to $6 in United States dollars (USD) for 
each scheduled visit with laboratory procedures and is equivalent to USD $3 for each scheduled 
visit without laboratory procedures. $3 is equivalent to 1500 CFA. $6 is equivalent to 3000 CFA.  
 
Table 11. Estimated Compensation Schedule 1 
Study Activity 
Number of Visits 
US Dollar 
Local Currency 
(CFA)2 
 
Screening (with blood draw) 
1 
$6 
3000 
Day -7 (with blood draw)  
1 
$6 
3000 
Vaccination #1 and follow-up visits 
with blood draw  
4 
$24 
12,000 
Vaccination #1- related visits  without 
blood draw 
1 
$3 
1,500 
Vaccination #2 and follow-up visits 
with blood draw 
4 
$24 
12,000 
Vaccination #2 - related visits  
without blood draw 
1 
$3 
1,500 
Vaccination #3 and follow-up visits 
with blood draw 
4 
$24 
12,000 
Vaccination #3 - related visits  
without blood draw 
1 
$3 
1,500 
Visits at 2, 3 and 6 months after last 
vaccination (with blood draws)  
4 
$24 
12,000 
Visits at 8, 10 and 12 months after 
last vaccination (without blood 
draws) 
3 
$9 
4,500 
Total 
24 
 
 
Unscheduled visits (all ages) 
Unscheduled visits 
TBD 
$3-6 
1500-3000 
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 
 
16 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 and 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 access coded data only, and will not have access to personally identifiable 
information.  
 
Collection and Storage of Biometric Data: In this study we are planning on using biometric data 
to identify study participants.  Biometrics is a science that measures certain physical 
characteristics in order to uniquely identify a person.  Common biometric measurements are 
fingerprints, photographs, DNA and face recognition.  In this study we will take fingerprint 
measurements and a photograph of each enrolled participant; these will be stored in a secure 
biometric database and kept separate from all other study data. Only study personnel will have 
access to this database.  
 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 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 FMPOS 
EC that granted the original approval for the study. Any change to the protocol will be submitted 
to the Sponsor and to the FMPOS 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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17 Role of the NIH Collaborators/Investigators 
The MRTC and the LMIV/NIAID/NIH clinical research teams will work collaboratively on this 
research. However, the NIH research staff will not be directly engaged in any clinical research 
activities (such as recruiting, obtaining informed consent of individuals to be study participants,  
making decisions about subject eligibility, administering the study product or assessing adverse 
events). The NIH study team will not have access to any identifiable private information from 
the study participants. The NIH collaborators will primarily be studying, interpreting, and 
analyzing unlinked data and specimens for purposes of this research. 
 
 
 
 

 
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Appendix A. Schedule of Assessments and Day-to-Day Schedule 

 
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Day to Day Schedule  
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 56 mL of blood for CBC with differential and platelet count, ALT, creatinine, malaria blood smear and 
PCR, hemoglobin typing, anti-Pfs230 antibody ELISA, standard membrane feeding assay (SMFA), cellular assays, 
transcriptional analysis.  
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. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is negative before proceeding. 
5. For females, ensure agreement and compliance with pregnancy prevention before AL dosing. 
 
Study Day 1 (±0 days; 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 46.5 mL of blood for CBC with differential and platelet count, ALT, creatinine, malaria blood smear and 
PCR, anti-Pfs230 antibody ELISA, cellular and ex vivo assays, SMFA, and transcriptional analysis.  
11. Administer the vaccine. 
The following evaluations/procedures will be completed following vaccination: 

 
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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 2 (±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 4 (±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 8 (±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 15 (±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 37 mL of blood for CBC with differential and platelet count, ALT, creatinine, malaria blood smear, anti-
Pfs230 antibody ELISA, cellular and ex vivo assays, SMFA and transcriptional analysis. 
 
 

 
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Study Day 29 (±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. 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 16.5 mL of blood for CBC with differential and platelet count, ALT, creatinine, malaria blood smear, 
anti-Pfs230 antibody ELISA, SMFA and transcriptional analysis. 
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 30 (±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 32 (±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. 
 
 

 
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Study Day 36 (±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 43 (±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 36.5 mL of blood for CBC with differential and platelet count, ALT, creatinine, malaria blood smear, 
anti-Pfs230 antibody ELISA, cellular and ex vivo assays, SMFA and transcriptional analysis. 
 
Study Day 57 (±7 days; day of 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, ensure agreement and compliance with pregnancy prevention before vaccination 
 
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 
5. Obtain approximately 16.5 mL of blood for CBC with differential and platelet count, ALT, creatinine, malaria blood smear, 
anti-Pfs230 anti body ELISA and transcriptional analysis. 
 
Study Day 58 (±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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Study Day 60 (±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, malaria smear, and 
transcriptional analysis.  
 
Study Day 64 (±2 day; 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 71 (±3 days; 14 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 16.5 mL of blood for CBC with differential and platelet count, ALT, creatinine, malaria blood smear, 
anti-Pfs230 antibody ELISA, SMFA and transcriptional analysis. 
 
Study Day 85 (±14 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 5.5 mL of blood for CBC with differential and platelet count, ALT, creatinine, and blood smear.  
 
Study Day 113  (±14 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 30.5 mL of blood for malaria blood smear and for cellular and ex-vivo assays.  

 
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Study Day 141 (±28 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. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is negative before proceeding.  
5. Obtain approximately 11.5 mL of blood for malaria blood smear, anti-Pfs230 antibody ELISA, standard membrane feeding 
assay (SMFA) and transcriptional analysis. 
 
Study Day 225 (±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 21.5 mL of blood for malaria blood smear, anti-Pfs230 antibody ELISA, standard membrane feeding 
assay (SMFA) and transcriptional analysis. 
 
Study Day 281 (±28 days; 224 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 337 (±28 days; 280 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 20.5 mL of blood for malaria blood smear, anti-Pfs230 antibody ELISA, and standard membrane 
feeding assay (SMFA). 
 
Study Day 393 (±28 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. 

 
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4. Obtain approximately 20.5 mL of blood for malaria blood smear, anti-Pfs230 antibody ELISA, and standard membrane 
feeding assay (SMFA). 
 
 
Appendix B. 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 
2.5 – 5 cm 
5.1 – 10 cm 
> 10 cm 
Necrosis or exfoliative 
dermatitis 
Induration/Swelling 
at site3   
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 
Bruising at site2     
2.5 – 5 cm 
5.1 – 10 cm 
> 10 cm 
Necrosis or exfoliative 
dermatitis 
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. 

 
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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 
101 – 115 
116 – 130 
> 130 
ER visit or hospitalization 
for arrhythmia 
Bradycardia - beats per 
minute4; at rest + calm 
50 – 54 
45 – 49 
< 45 
ER visit or hospitalization 
for arrhythmia 
Hypertension (systolic) -
mm Hg; at rest + calm 
141 – 150 
151 – 155 
> 155 
ER visit or hospitalization 
for malignant hypertension 
Hypertension (diastolic) 
-mm Hg; at rest + calm 
91 – 95 
96 – 100 
> 100 
ER visit or hospitalization 
for malignant hypertension 
Hypotension (systolic) -
mm Hg; at rest + calm 
85 – 89 
80 – 84 
< 80 
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.  
3 Oral temperature; no recent hot or cold beverages or smoking.  
4 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) 
Fever (°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 
Headache 
No interference with 
activity  
Repeated use of non-narcoti c 
pain reliever > 24 hours or some 
interference with activity 
Significant; any use of narcotic 
pain reliever or 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 
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 
Abdominal 
Pain  
No interference with 
activity 
Some interference with activity 
Significant; prevents daily 
activity 
ER visit or 
hospitalization 
Fatigue 
No interference with 
activity 
Some interference with activity 
Significant; 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 
Urticaria  
No interference with 
activity 
Requiring PO or topical treatment 
> 24 hours or IV medications or 
steroids for ≤24 hours  
Requiring IV medication or 
steroids for >24 hours  
ER visit or 
hospitalization 
Abbreviations: ER, emergency room; IV, intravenous; PO, “per os” or oral administration. 

 
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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: Adults 
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 – 9.3 
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 – 0.90 
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 
130.00 – 150.99 
151.00 – 176.99 
177.00 – 221.00 
> 221.00 and requires dialysis 
Creatinine (Female) - µmol/L 
110.00 – 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  (Bain 1996, Haddy, Rana et al. 1999). 
 

 
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Appendix C. Mali Adult Institutional Normal Laboratory Values 
 
Chemistry 
Chemistry1 
Reference Range 
Creatinine (Female) - µmol/L 
57.4 – 96.2  
Creatinine (Male) -µmol/L 
59.4 – 119.4 
ALT - U/L 
< 45.5 
Abbreviations: ALT, alanine transaminase. 
1 The laboratory values provided in the table are based on Bancoumana, Malian adults (age 18-45 years old). 
[Doucoure 2021, submitted] 
 
Hematology 
Hematology1 
Reference Range 
Hemoglobin (Female) - gm/dL 
9.6 – 14.6 
Hemoglobin (Male) - gm/dL 
10.8 – 16.6 
WBC - 103/µL 
3.7 – 9.4 
Absolute Neutrophil/Granulocyte Count - 103/µL 
1.0 – 4.8 
Absolute Lymphocyte Count - 103/µL 
1.5 – 4.5 
Platelet Count (Female) - 103/µL 
163.9 – 429.1 
Platelet Count (Male) - 103/µL 
119.7 – 368.5 
Abbreviations: WBC, white blood cell. 
1 The laboratory values provided in the table are based on Bancoumana, Malian adults (age 18-45 years old). 
[Doucoure 2021, submitted] 
 
 
 
 
 
 

 
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Appendix D.  Potential Immune-Mediated Medical Conditions  
 
Categories 
Diagnoses (as MedDRA Preferred Terms) 
Neuroinflammatory disorders: 
Acute disseminated encephalomyelitis (including site-specific variants: 
eg, non-infectious encephalitis, encephalomyelitis, myelitis, 
myeloradiculomyelitis), cranial nerve disorders including 
paralyses/paresis (eg, Bell’s palsy), generalized convulsion, Guillain- 
Barre syndrome (including Miller Fisher syndrome and other variants), 
immune-mediated peripheral neuropathies and plexopathies (including 
chronic inflammatory demyelinating polyneuropathy, multifocal motor 
neuropathy and polyneuropathies associated with monoclonal 
gammopathy), myasthenia gravis, multiple sclerosis, narcolepsy, optic 
neuritis, transverse myelitis, uveitis. 
Musculoskeletal and connective 
tissue disorders: 
Antisynthetase syndrome, dermatomyositis, juvenile chronic arthritis 
(including Still’s disease), mixed connective tissue disorder, 
polymyalgia rheumatic, polymyositis, psoriatic arthropathy, relapsing 
polychondritis, rheumatoid arthritis, scleroderma (including diffuse 
systemic form and CREST syndrome), spondyloarthritis (including 
ankylosing spondylitis, reactive arthritis [Reiter's Syndrome] and 
undifferentiated spondyloarthritis), systemic lupus erythematosus, 
systemic sclerosis, Sjogren’s syndrome. 
Vasculitides: 
Large vessels vasculitis (including giant cell arteritis such as Takayasu's 
arteritis and temporal arteritis), medium sized and/or small vessels 
vasculitis (including polyarteritis nodosa, Kawasaki's disease, 
microscopic polyangiitis, Wegener's granulomatosis, Churg–Strauss 
syndrome [allergic granulomatous angiitis], Buerger’s disease 
[thromboangiitis obliterans], necrotizing vasculitis and ANCA-positive 
vasculitis [type unspecified], Henoch-Schonlein purpura, Behcet's 
syndrome, leukocytoclastic vasculitis). 
Gastrointestinal disorders: 
Crohn’s disease, celiac disease, ulcerative colitis, ulcerative proctitis. 
Hepatic disorders: 
Autoimmune hepatitis, autoimmune cholangitis, primary sclerosing 
cholangitis, primary biliary cirrhosis. 
Renal disorders: 
Autoimmune glomerulonephritis (including IgA nephropathy, 
glomerulonephritis rapidly progressive, membranous glomerulonephritis, 
membranoproliferative glomerulonephritis, and mesangioproliferative 
glomerulonephritis). 
Cardiac disorders: 
Autoimmune myocarditis/cardiomyopathy. 
Skin disorders: 
Alopecia areata, psoriasis, vitiligo, Raynaud’s phenomenon, erythema 
nodosum, autoimmune bullous skin diseases (including pemphigus, 
pemphigoid and dermatitis herpetiformis), cutaneous lupus 
erythematosus, morphoea, lichen planus, Stevens-Johnson syndrome, 
Sweet’s syndrome. 
Hematologic disorders: 
Autoimmune hemolytic anemia, autoimmune thrombocytopenia, 
antiphospholipid syndrome, thrombocytopenia. 
Metabolic disorders: 
Autoimmune thyroiditis, Grave’s or Basedow’s disease, new onset 
Hashimoto thyroiditis, diabetes mellitus type 1, Addison’s disease. 
Other disorders: 
Goodpasture syndrome, idiopathic pulmonary fibrosis, pernicious 
anemia, sarcoidosis. 
 
 

 
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Pfs230 Antigen/Adjuvant Study Version 3.0                                                                                                          November 09, 2022  
 
 
 
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