Phase 2 Trial of Safety, Immunogenicity, and Efficacy Against Plasmodium Falciparum Malaria of PfSPZ Vaccine in Children in Mali
Phase: PHASE2
Conditions: Malaria, Malaria,Falciparum
Sponsor: Sanaria Inc.

 
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Abbreviated Title: PfSPZ Vaccine in Malian Children 
FMPOS EC #: N°2021/151/CE/USTTB  
 
Project Assurance: FWA #00001769 
Sponsor (Sanaria) Protocol #:  MLSPZV5 
 
Version Date: 4.0, 01 December 2022 
 
Title: Phase 2 Trial of Safety, Immunogenicity, and Efficacy against Plasmodium falciparum 
Malaria of PfSPZ Vaccine in Children in Mali 
 
University of Sciences, Techniques and Technology, Bamako (USTTB)  
 
 

 
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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 
 
Associate Investigators: 
 
Mahamadou S. Sissoko, MD, MSPH, PhD  
Mahamadoun Hamady Assadou, MD, MPH  
Sadio dite Koni Diarra, MD 
Kourane Sissoko, MD 
Seydou Sankare, MD  
 
 
Amagana Dolo, PharmD, PhD 
Amatigue Zeguimé, PharmD  
Kadidia Baba Cisse, BSc 
Kalifa Diarra, PharmD 
Balla Diarra, PharmD 
Karim Bengaly, PharmD 
Amadou Niangaly, PharmD 
 
Independent Safety 
Monitor: 
 
 
Prof. Abdoul Aziz Diakite, MD 
Head, Department of General Pediatrics 
Gabriel Touré University Hospital 
Bamako, Mali 
Phone : + 223 66 74 49 56 
Email: doc abdela@yahoo.fr 
 
National Institute of Allergy and Infectious Diseases (NIAID)/National Institutes of Health 
(NIH)  

 
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Senior Investigator: 
Patrick Duffy, MD 
Laboratory of Malaria Immunology and Vaccinology (LMIV) 
National Institute of Allergy and Infectious Diseases 
National Institutes of Health 
BG 29B RM 4NN06 
9000 Rockville Pike 
Bethesda, MD 20814 
Phone: 301-761-5089 
Email: patrick.duffy@nih.gov  
 
Associate Investigators: 
 
 
David Cook, MD  
Judith Epstein, MD  
John Woodford, MBBS 
Aye Sousa, PA-C 
Joel Goldberg, MD, PhD 
 
Biostatistician: 
Zonghui Hu, PhD  
NIAID/NIH/BRB 
BG 5601FL RM 4C21 
5601 Fishers Lane, Room 4C21 
Rockville, Maryland 20852 
______________________________________________________________________________ 
 
Investigational Agents (if applicable): 
Drug Name: 
PfSPZ Vaccine 
 
 
Sponsor: 
Sanaria Inc 
Manufacturer: 
Sanaria Inc 
 

 
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Clinical Trial Site 
 
Bancoumana and surrounding villages, Mali 
Malaria Vaccine Center  
Malaria Research and Training Center 
Faculty of Medicine, Pharmacy and Odonto-Stomatology 
 
Institutional Review Board (IRB)   
Faculty of Medicine, Pharmacy and Odonto-Stomatology Ethics 
Committee (FMPOS EC) 
 
Data Safety and Monitoring Board: 
The DSMB is chartered by the IND Sponsor 
Contact: 
 
Thomas L Richie MD PhD 
 
Chief Medical Officer, Sanaria Inc. 
 
trichie@sanaria.com 
 
 
 
Clinical Laboratory:  
Malaria Research and Training Center 
DEAP Clinical Laboratory 
Faculty of Medicine, of Odonto-Stomatology Faculty of Pharmacy, 
University of Sciences, Techniques & Technologies, Bamako 
 
 
 
 
BP 1805, Point G 
 
 
 
 
Bamako, Mali 
 
Rodolphe Merieux Laboratory 
Bamako, Mali 
 
 
Additional Laboratories 
Laboratory of Malaria Immunology and Vaccinology 
National Institute of Allergy and Infectious Diseases 
National Institutes of Health 
 
Malaria Research and Training Center/DEAP Immunology 
Laboratory 
Faculty of Medicine, Pharmacy and Odonto-Stomatology, 
University of Sciences, Techniques & Technologies, Bamako 
 
Protein Potential LLC 
Rockville, MD 20850, USA 
 
National Institute of Allergy and Infectious Diseases 
National Institutes of Health 
Vaccine Research Center (VRC) Laboratories 
Bethesda, MD 20892, USA 

 
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Sanaria Incorporated  
Rockville, MD 20850, USA 
 
National Institutes of Health Research Technologies Branch 
 
 
 
 
Genomic Technologies Section 
 
 
 
 
Bethesda, MD 20892, USA 
 
 
 
 
 
 
 
 
 
Laboratory of Parasitic Diseases 
 
 
 
 
National Institute of Allergy and Infectious Diseases 
National Institutes of Health 
 
 
 
 
Bethesda, MD 20892, USA 
 
 
 
 
 
Center for Human Immunology, Autoimmunity, and Inflammation 
 
 
 
 
National Heart, Lung and Blood Institute (NHLBI) 
 
 
 
 
Bethesda, MD 20892-1655, USA 
 
 
 
 
 
University of Bamako 
 
 
 
 
Laboratory of Immunogenetics and Parasitology 
 
 
 
 
Malaria Research and Training Center 
Bamako, Mali, West Africa 
 
Clinical Microbiology Laboratory 
Laboratory Medicine & Pathology 
University of Washington Medical Center 
Seattle, Washington, 98109, USA 
 
 
 
 
 
 
 

 
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TABLE OF CONTENTS  
TABLE OF CONTENTS ................................................................................................................ 6 
TABLE OF FIGURES AND TABLES ........................................................................................ 10 
1. 
ABBREVIATIONS .............................................................................................................. 11 
STATEMENT OF COMPLIANCE .............................................................................................. 13 
2. 
PROTOCOL SUMMARY .................................................................................................... 14 
2.1 
Synopsis ......................................................................................................................... 14 
2.2 
Study Schema and Study Flow Diagram ....................................................................... 18 
2.3 
Schedule of Activities (SOA) ......................................................................................... 22 
3. 
INTRODUCTION ................................................................................................................ 25 
3.1 
Study Rationale .............................................................................................................. 25 
3.2 
Background .................................................................................................................... 26 
 
Nonclinical Investigations of PfSPZ Vaccine......................................................... 26 
 
Clinical Experience with PfSPZ Vaccine ............................................................... 27 
3.3 
Risk/Benefit Assessment ................................................................................................ 36 
 
Known Potential Risks ............................................................................................ 36 
 
Known Potential Benefits ....................................................................................... 45 
 
Assessment of Potential Risks and Benefits ........................................................... 45 
4. 
OBJECTIVES AND ENDPOINTS ...................................................................................... 47 
5. 
STUDY DESIGN.................................................................................................................. 50 
5.1 
Overall Design................................................................................................................ 50 
5.2 
Scientific Rationale for Study Design ............................................................................ 52 
 
Justification for Dose .............................................................................................. 52 
 
Rationale for Age Groups Selected......................................................................... 52 
 
Rationale for Antimalarial Treatment prior to V1 .................................................. 53 
 
Rationale for Parasitemia Threshold Selected ........................................................ 55 
6. 
STUDY POPULATION ....................................................................................................... 60 
6.1 
Inclusion Criteria ............................................................................................................ 60 
6.2 
Exclusion Criteria ........................................................................................................... 60 
6.3 
Inclusion of Vulnerable Participants .............................................................................. 62 

 
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Participation of family members of study team members ...................................... 62 
6.4 
Inclusion of Pregnant Women, fetuses or neonates ....................................................... 62 
6.5 
Lifestyle Considerations ................................................................................................. 62 
6.6 
Screen Failures ............................................................................................................... 62 
6.7 
Strategies for Recruitment and Retention ...................................................................... 62 
 
Costs ........................................................................................................................ 62 
 
Compensation ......................................................................................................... 62 
7. 
STUDY INTERVENTION ................................................................................................... 63 
7.1 
Study Interventions(s) Administration ........................................................................... 63 
 
Study Intervention Description ............................................................................... 63 
 
Dosing and Administration ..................................................................................... 64 
7.2 
Preparation/Handling/Storage/Accountability ............................................................... 65 
 
Acquisition and Accountability. ............................................................................. 65 
 
Formulation, Appearance, Packaging, and Labeling .............................................. 66 
 
Product Storage and Stability.................................................................................. 67 
7.3 
Measures to Minimize Bias: Randomization and Blinding ........................................... 68 
 
Randomization ........................................................................................................ 68 
 
Blinding................................................................................................................... 68 
 
Unblinding .............................................................................................................. 69 
7.4 
Concomitant Therapy ..................................................................................................... 69 
8. 
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL..................................................................................... 69 
8.1 
Suspension of Study Intervention .................................................................................. 69 
8.2 
Discontinuation of Study Intervention ........................................................................... 72 
8.3 
Participant Discontinuation/Withdrawal from the Study ............................................... 72 
8.4 
Lost to Follow-up ........................................................................................................... 73 
9. 
STUDY ASSESSMENTS AND PROCEDURES ................................................................ 73 
9.1 
Screening Procedures ..................................................................................................... 73 
 
Screening activities performed prior to obtaining informed consent ...................... 73 
 
Screening activities performed after a consent for screening has been signed ....... 73 
9.2 
Enrollment and Artemether Lumefantrine Visit ............................................................ 74 
9.3 
Vaccination Visits .......................................................................................................... 74 

 
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9.4 
Safety Follow-up Visits .................................................................................................. 74 
9.5 
Efficacy Assessments ..................................................................................................... 74 
9.6 
Unscheduled Visits ......................................................................................................... 75 
9.7 
Clinical Evaluations ....................................................................................................... 75 
9.8 
Unscheduled Blood Smear Positive Visits ..................................................................... 76 
9.9 
Malaria qPCR ................................................................................................................. 76 
 
Samples for Genetic/Genomic Analysis ................................................................. 78 
9.10 
Safety and Other Assessments .................................................................................... 78 
9.11 
Adverse Events and Serious Adverse Events ............................................................. 78 
 Definition of Adverse Event ................................................................................... 78 
 Definition of Serious Adverse Events (SAE) ......................................................... 78 
 Classification of an Adverse Event ......................................................................... 79 
 Time Period and Frequency for Event Assessment and Follow-Up ....................... 80 
 Adverse Event Reporting ........................................................................................ 82 
 Serious Adverse Event Reporting ........................................................................... 82 
 Events of Special Interest........................................................................................ 84 
 Reporting of Pregnancy .......................................................................................... 84 
9.12 
Unanticipated Problems .............................................................................................. 84 
 Definition of Unanticipated Problems (UP) ........................................................... 84 
 Reporting Procedures to the FMPOS EC................................................................ 85 
10. STATISTICAL CONSIDERATIONS.................................................................................. 85 
10.1 
Statistical Hypothesis ................................................................................................. 85 
10.2 
Sample Size Determination ........................................................................................ 87 
10.3 
Populations for Analyses ............................................................................................ 89 
10.4 
Statistical Analyses ..................................................................................................... 90 
 General Approach ................................................................................................... 90 
 Safety Analyses ....................................................................................................... 91 
 Efficacy Analysis .................................................................................................... 91 
 Exploratory Analyses .............................................................................................. 92 
 Planned Unblinded Analyses .................................................................................. 92 
11. REGULATORY AND OPERATIONAL CONSIDERATIONS ......................................... 93 

 
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11.1 
Informed Consent Process .......................................................................................... 93 
 Consent Procedures and Documentation ................................................................ 93 
 Consent for minors when they reach the age of majority ....................................... 94 
 Considerations for Consent of family members of study team members ............... 95 
 Consent of Subjects who are, or become, decisionally impaired ........................... 95 
11.2 
Study Discontinuation and Closure ............................................................................ 95 
11.3 
Confidentiality and Privacy ........................................................................................ 95 
11.4 
Future use of Stored Specimens and Data .................................................................. 96 
11.5 
Safety Oversight ......................................................................................................... 97 
11.6 
Clinical Monitoring .................................................................................................... 97 
11.7 
Quality Assurance and Quality Control ...................................................................... 98 
11.8 
Data Handling and Record Keeping ........................................................................... 98 
 Data Collection and Management Responsibilities ................................................ 98 
 Study Records Retention......................................................................................... 99 
11.9 
Protocol Deviations and Non-Compliance ............................................................... 100 
 Definition of Protocol Deviation .......................................................................... 100 
11.10 
Publication and Data Sharing Policy ........................................................................ 100 
 Human Data Sharing Plan ..................................................................................... 100 
 Genomic Data Sharing Plan .................................................................................. 100 
12. REFERENCES ................................................................................................................... 106 
 
 
 

 
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TABLE OF FIGURES AND TABLES 
Figure 1. Study Schema ................................................................................................................ 19 
Figure 2. Study Schema (For details of study visits please see Section 2.3, Schedule of 
Activities). ..................................................................................................................................... 20 
Figure 3. Difference between post-immunization and pre-immunization antibody results in 
BSPZV2 trial in Tanzania by age group. ...................................................................................... 30 
Figure 4. PfSPZ-specific memory CD4 T-cell responses pre- and post-vaccination. .................. 31 
Figure 5. Vaccine efficacy at 3, 6, 9, and 12 months for the three vaccine groups (Oneko and 
Steinhardt, unpublished data)........................................................................................................ 32 
Figure 6. Anti-CSP responses in vaccinees in the KSPZV1 clinical trial (Oneko, unpublished). 34 
Figure 7. Frequency of Vδ2 γδ T cells prior to immunization in the BSPZV2 trial in Tanzania 
(Oneko, submitted). ...................................................................................................................... 35 
Figure 8. Effect of vaccine on incidence of any solicited systemic adverse events per study 
subject in randomized, double-blind, placebo-controlled trials of PfSPZ Vaccine in infants and 
children aged 5 months to <18 years in sub-Saharan Africa. ....................................................... 41 
Figure 9: Pattern of Fever and Parasites ....................................................................................... 57 
Figure 10: Acute Malaria vs Parasite Density .............................................................................. 58 
Figure 11: Attributable Fraction using Fever as a Symptom of Diseases..................................... 59 
Table 1. PfSPZ Vaccination Period ........................................................................................... 22 
Table 2. PfSPZ Post-vaccination malaria assessment period ....................................................... 24 
Table 3. Chronological listing of trials of PfSPZ Vaccine ........................................................... 27 
Table 4. Solicited adverse events recorded days 0-7 post-all immunizations. ............................. 37 
Table 5. Total number (percent) of Grade 2 laboratory abnormalities by pediatric age group in 
BSPZV2 trial in Bagamoyo, Tanzania. ......................................................................................... 38 
Table 6.  Total number (percent) of adverse events by pediatric age group in KSPZV1 trial in 
Kenya ............................................................................................................................................ 38 
Table 7. Day 8 post-immunization laboratory abnormalities of any grade by dose group, number 
of subjects (percent). ..................................................................................................................... 39 
Table 8. Solicited adverse events recorded days 0-7 post-all immunizations. ............................. 39 
Table 9. Laboratory abnormalities Grade 2 or higher by age group, days 1 to 28 post-
immunization. ............................................................................................................................... 40 
Table 10. Incidence of symptomatic malaria infection per person per year in Bancoumana, Mali 
by age and season.......................................................................................................................... 53 
Table 11. Estimated compensation for the study. ......................................................................... 63 
Table 12. Solicited Adverse Events .............................................................................................. 81 

 
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Table 13. Probability of observing 0, 1 and 2 or more events, within an arm of size 120 for 
different true event rates. .............................................................................................................. 87 
Table 14. Two-sided 95% confidence intervals based on observing a particular rate of safety 
endpoint arms for an arm of size 120............................................................................................ 88 
Table 15. Power calculation for plausible Pf malaria with symptom rates for two-sided log-rank 
test under type 1 error rate of alpha=0.05. .................................................................................... 89 
 
 
1. ABBREVIATIONS 
AE 
Adverse Event 
AL 
Artemether/lumefantrine  
ANC 
absolute neutrophil count 
ANCOVA 
Analysis of Covariance 
ATP 
According to Protocol  
CFR 
Code of Federal Regulations 
CLIA 
Clinical Laboratory Improvement Amendments 
CMP 
Clinical Monitoring Plan 
COC 
Certificate of Confidentiality 
CONSORT Consolidated Standards of Reporting Trials 
Cr 
creatinine  
CRF 
Case Report Form 
DCC 
Data Coordinating Center 
DHHS 
Department of Health and Human Services 
DSMB 
Data Safety Monitoring Board 
DRE 
Disease-Related Event 
ECG 
electrocardiogram 
EC 
Ethics Committee 
eCRF 
Electronic Case Report Forms 
FDA 
Food and Drug Administration 
FDAAA 
Food and Drug Administration Amendments Act of 2007 
FFR 
Federal Financial Report 
GCP 
Good Clinical Practice 
GLP 
Good Laboratory Practices 
GMP 
Good Manufacturing Practices 
GWAS 
Genome-Wide Association Studies 
Hgb 
Hemoglobin  
HIPAA 
Health Insurance Portability and Accountability Act  
HR 
hazard ratio  
HSA 
human serum albumin  
IATA 
International Air Transport Association 
IB 
Investigator’s Brochure 

 
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ICH 
International Conference on Harmonisation  
ICMJE 
International Committee of Medical Journal Editors 
IDE 
Investigational Device Exemption 
IFA 
immunofluorescence assay  
IgE 
immunoglobulin E  
IgG 
immunoglobulin G  
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
ISI 
inhibition of sporozoite invasion assay 
ISM 
Independent Safety Monitor 
ISO 
International Organization for Standardization 
ITT 
Intention-To-Treat 
LSMEANS Least-squares Means 
LMIV 
Laboratory of Malaria Immunology and Vaccinology 
MedDRA 
Medical Dictionary for Regulatory Activities 
MOP 
Manual of Procedures 
MSDS 
Material Safety Data Sheet 
NASBA 
nucleic acid sequence-based amplification 
NCT 
National Clinical Trial 
NIH  
National Institutes of Health 
NIH IC 
NIH Institute or Center 
NS 
normal saline  
OCICB 
Office of Cyber Infrastructure and Computational Biology  
OHRP 
Office for Human Research Protections 
PBS 
phosphate buffered saline 
PCR 
polymerase chain reaction  
PI 
Principal Investigator 
QA 
Quality Assurance 
QC 
Quality Control 
RNA 
ribonucleic acid 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SMC 
Seasonal Malaria Chemoprophylaxis 
SNP 
single nucleotide polymorphisms  
SOA 
Schedule of Activities 
SOC 
System Organ Class 
SOP 
Standard Operating Procedure 
UP 
Unanticipated Problem 
US 
United States 
USTTB 
University of Sciences, Techniques and Technology, Bamako  
 
 

 
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STATEMENT OF COMPLIANCE 
The trial will be carried out in accordance with International Conference on Harmonisation Good 
Clinical Practice (ICH GCP) and the United States (US) Code of Federal Regulations (CFR) 
applicable to clinical studies (45 CFR Part 46, 21 CFR Part 50, 21 CFR Part 56, 21 CFR Part 
312, and/or 21 CFR Part 812). 
National Institutes of Health (NIH)-funded investigators and clinical trial site staff who are 
responsible for the conduct, management, or oversight of NIH-funded clinical trials have 
completed Human Subjects Protection and ICH GCP Training. 
The protocol, informed consent form(s), recruitment materials, and all participant materials will 
be submitted to the Institutional Review Board (IRB) for review and approval.  Approval of both 
the protocol and the consent form must be obtained before any participant is enrolled.  Any 
amendment to the protocol will require review and approval by the IRB before the changes are 
implemented to the study.  In addition, all changes to the consent form will be IRB-approved; an 
IRB determination will be made regarding whether a new consent needs to be obtained from 
participants who provided consent, using a previously approved consent form. 
 
 

 
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2. PROTOCOL SUMMARY 
2.1 
SYNOPSIS 
Title: 
Phase 2 Trial of Safety, Immunogenicity, and Efficacy against 
Plasmodium falciparum Malaria of PfSPZ Vaccine in Children in Mali 
 
Study 
Description: 
This is a clinical trial of Sanaria® PfSPZ Vaccine, a non-adjuvanted, live 
(metabolically active), radiation-attenuated, non-replicating, whole 
sporozoite (SPZ) vaccine designed to prevent malaria infection caused by 
Plasmodium falciparum (Pf).  
 
In this randomized, double-blind, placebo-controlled trial, approximately 
268 (up to 290) healthy children aged 6-10 years will be administered 
three doses of 9.0x105 Pf sporozoites (PfSPZ) of PfSPZ Vaccine (or 
placebo) at 1, 8, and 29-days using direct venous inoculation (DVI).  
 
The study is composed of a single cohort with two arms enrolled 
concurrently: 
 
 
 
Arm 1 (PfSPZ Vaccine): (n = ~134) children ages 6 – 10 will receive 
three doses of PfSPZ Vaccine (9.0x105 PfSPZ) via direct venous 
inoculation (DVI) at 1, 8, and 29 days  
 
Arm 2 (normal saline): (n = ~134) children ages 6 – 10 will receive 
normal saline via DVI at 1, 8, and 29 days  
 
All subjects will receive artemether-lumefantrine (AL) approximately 1- 2 
weeks before the first and, if there is evidence of significant malaria 
transmission in the community or the rainy season begins, a third dose of 
PfSPZ Vaccine or normal saline for clearance of Pf parasitema.  
 
Vaccinated participants and non-immunized placebo controls will be 
monitored using passive and active case detection for development of Pf 
malaria with symptoms for 26 weeks after V3 through the natural malaria 
transmission season to estimate vaccine efficacy (VE).  Case 
ascertainment will be prompted by clinical signs and symptoms of acute 
malaria, with subsequent confirmation of parasitemia by TBS.  
Participants will be monitored retrospectively for Pf malaria (parasitemia) 
as a secondary endpoint. 
 
Note: for the purposes of this protocol the following definitions apply: 
Primary case definition: 

 
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Pf malaria with symptoms is defined as a positive thick blood smear at a 
density of >1000 parasites/uL (P/uL) plus: 
 Measured axillary temperature ≥ 37.5 degrees Celsius or 
history of fever (subjective or objective) in the last 24 
hours, or,  
 Symptoms of malaria: 
o Verbal individual (individual able and willing to 
answer questions): A verbal individual is 
considered symptomatic if reporting at the time of 
evaluation at least two of the following 
symptoms/symptom groups: headache, chills and/or 
rigors, malaise and/or fatigue, dizziness and/or 
light-headedness, myalgias and/or arthralgias; or 
o Non-verbal individual (small child or any 
individual unable or unwilling to answer 
questions): A non-verbal individual is considered 
symptomatic if manifesting at the time of 
evaluation at least two of the following signs/sign 
groups: drowsiness, irritability and/or fussiness, 
inability and/or refusal to eat or drink, prostration; 
or 
 Any individual: Signs of severe malaria (e.g. impairment 
of consciousness, severe anemia, hemoglobinuria, acute 
kidney injury, etc.) 
Secondary case definition: 
Pf malaria with symptoms is defined as a positive thick blood smear at a 
density of > 0 P/uL plus: 
 Measured axillary temperature ≥ 37.5 degrees Celsius or 
history of fever (subjective or objective) in the last 24 
hours, or,  
 Symptoms of malaria as defined in the primary case 
definition; or 
 Meeting criteria for severe malaria 
 
Pf malaria is defined as: 
 At least one unambiguous asexual parasite on thick blood 
smear identified by two independent microscopists after 
each examining 0.50 µL of blood in a study participant 

 
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Objectives: 
 
Primary Objectives:  
1. To describe the safety of PfSPZ Vaccine in children with respect 
to the occurrence of possibly, probably, or definitely related 
serious adverse events (SAEs). 
2. To measure VE against first episode of Pf malaria with symptoms 
by time-to-event analysis. 
 
 
Secondary Objectives: 
1. To assess the safety of PfSPZ Vaccine with respect to the 
occurrence of unsolicited AEs, laboratory abnormalities, solicited 
Aes. 
2. To measure VE against first episode of Pf malaria (parasitemia) 
with or without associated symptoms by time-to-event analysis. 
3. To measure antibody responses to Pf circumsporozoite protein 
(CSP) and their association with protection. 
 
Exploratory Objectives:  
1. To assess the immune response to PfSPZ Vaccine in children. 
2. To assess genetic relatedness of the PfSPZ Vaccine parasite strain 
to malaria infection parasites.  
3. To measure VE using other outcome measures such as against all 
episodes of Pf malaria with symptoms and Pf malaria rather than 
against the first episode, using proportional analysis, or using ITT 
or ATP populations, or severity of malaria infections or 
hemoglobin concentration. 
4. To explore the relationship between detectable parasitemia prior to 
vaccination and VE.  
 
Endpoints: 
Primary Endpoints: 
1. Proportion of vaccinees compared to controls experiencing related 
SAEs from V1 to 26 weeks after V3 
2. VE computed as one minus the estimated hazard ratio (HR) (time-
to-event analysis) for first episode of Pf malaria with symptoms 
from 2 weeks after V3 to 26 weeks after V3 in the modified ITT 
(mITT) population. 
 
Secondary Endpoints: 
1. Safety and tolerability in children 
- 
The differences in proportions of vaccinees compared to 
controls experiencing unsolicited Aes from the time of V1 
to 14 days after V3 (or last immunization).  

 
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- 
The differences in proportions of vaccinees compared to 
controls experiencing laboratory abnormalities from time 
of V1 to 14 days after V3. 
- 
The differences in proportions of vaccinees compared to 
controls experiencing solicited Aes during the 7 days 
following each immunization. 
2. VE computed as one minus the estimated hazard ratio (HR) for 
first episode of Pf malaria detected by thick blood smear (TBS), 
from 2 weeks after V3 to 26 weeks after V3 in the mITT 
population. 
3. Antibody levels to PfCSP by standardized ELISA comparing 
protected and unprotected vaccinees and controls. 
 
Exploratory Endpoints: 
1. Humoral and cellular immune responses to PfSPZ Vaccine  
2. Genotyping of peripheral blood Pf parasites 
3. Additional measures of VE 
- 
VE against all episodes (rather than first episode) of Pf 
malaria with symptoms and Pf malaria by Cox regression 
analysis 
- 
VE computed by proportional (binary) analysis (1 – risk ratio) 
for first episode of Pf malaria detected by TBS, from 2 weeks 
after V3 to 26 weeks after V3 in the intention-to-treat (ITT), 
mITT and according to protocol (ATP) populations (whole 
group and subgroups) 
- 
VE computed as one minus the estimated HR for first episode 
of Pf malaria detected by TBS, from 2 weeks after V3 to 26 
weeks after V3 in the ITT and ATP populations (whole group 
and subgroups) 
- 
VE for Pf malaria measured by and nucleic acid detection in 
children from 2 weeks after V3 to 26 weeks after V3 using 
both (time to event, proportional/binary) 
- 
VE by both time-to-event and  proportional/binary analyses for 
first episodes of Pf malaria with symptoms with  
o parasite density > 500 parasites/µL, or  
o parasite density > 5000 parasites/µL 
- 
or Pf malaria with symptoms characterized by: 
o grade 3 signs or symptoms,  
o associated with fever and convulsions, or  
o hospitalization, or 
o other clinical measures in vaccinees vs. controls 
- 
Rates of anemia (grade 1, grade 2, grade 3) in vaccinees vs. 
controls. 
4. Retrospective malaria qPCR values taken prior to presumptive AL 
treatment and vaccination 

 
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2.2 
STUDY SCHEMA AND STUDY FLOW DIAGRAM 
This study will enroll healthy Malian children between 6 and 10 years of age to participate in a 
randomized, double blind, placebo-controlled study to assess the safety, immunogenicity and 
protective efficacy of PfSPZ Vaccine. Participants will be immunized with a 3-dose series of 9.0 
x105 PfSPZ of PfSPZ Vaccine or normal saline (placebo) at 1, 8, and 29 days  Randomization 
into the two arms will be in a 1:1 ratio (Erreur ! Source du renvoi introuvable.). The primary 
follow-up will be for six months. The study flow is shown in Figure 1. 
 
Study 
Population: 
Approximately 268 (maximum 290) healthy children (6 to10 years of 
age), resident in Bancoumana and surrounding villages, Mali. Given the 
potential for dropouts between AL dosing and first vaccinations, up to 290 
participants will be dosed with AL prior to first vaccination in order to 
ensure the target vaccination numbers are met.  All participants dosed 
with AL will also be offered vaccination (or placebo). 
 
Phase: 
2 
Description of 
Sites/Facilities 
Enrolling 
Participants: 
 
Malaria Research and Training Center (MRTC) research clinic in 
Bancoumana, Mali 
 
   
Description of 
Study 
Intervention: 
Radiation attenuated, aseptic, purified, vialed, cryopreserved, NF54 
Plasmodium falciparum sporozoites (referred to as PfSPZ Vaccine) 
produced by Sanaria Inc. will be administered via DVI at dose 9.0x105 
PfSPZ at 1, 8, and 29 days 
 
Normal saline administered via DVI at 1, 8, and 29 days  
 
Study Duration: 
22 months 
March 2022-July 2022 (screening + enrollment + vaccinations) 
January 2023 (completion of follow-up) 
January 2024 (completion of data analyses) 
 
Participant 
Duration: 
Approximately 9 months 
 
 
 
 

 
PfSPZ Vaccine in Children 
 
 
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Figure 2. Study Schema (For details of study visits please see Section 2.3, Schedule of Activities). 
 
Prior to  
Enrollment  
 
 
  
Day -14   
 
 
  
Day 1  
 
 
 
Day 8  
 
 
 
 
Day 12      
 
 
 
 
Day 29  
 
 
 
Day 29-211 
Malaria  
Transmission  
Season 
 
 
 
 
 
 
 
 
 
 
Final Assessment:  
Clinic visit, malaria surveillance, and research 
labs approximately 6 months after V3 
Enroll (n=~268): artemether-lumefantrine (AL) dosing #1 
Screen (n = up to 1500): Obtain informed consent. Screen potential participants by 
inclusion and exclusion criteria; obtain history, document. 
Randomize (n=~268): 1:1 to PfSPZ Vaccine or Placebo: Dose #1 
Follow-up assessments for safety 
Follow-up assessments for safety 
PfSPZ Vaccine or Placebo: Dose #3 
PfSPZ Vaccine or Placebo: Dose #2 
Surveillance for Incident Pf malaria with symptoms x 24 weeks (TBS every 4 weeks): 
In-clinic visits every 2 weeks (if indicated by signs/symptoms of malaria) starting 2 
weeks after V3  
Passive surveillance by self-reporting symptoms or signs at any time 
Artemether-lumefantrine (AL) dosing #2 (an option, per Section 5.1) 
Follow-up assessments for safety 

 
PfSPZ Vaccine in Children 
 
 
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PfSPZ Vaccine in Children 
 
 
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2.3 
 
SCHEDULE OF ACTIVITIES (SOA) 
The schedule of assessment activities is detailed in Erreur ! Référence non valide pour un 
signet. and  
 
 
 
 
 
Table 2).   
Table 1. PfSPZ Vaccination Period 
 

 
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PfSPZ Vaccine in Children 
 
 
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Table 2. PfSPZ Post-vaccination malaria assessment period 

PfSPZ Vaccine in Children 
 
 
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3.  INTRODUCTION 
3.1 
STUDY RATIONALE 
Malaria is a major global health problem, especially in sub-Saharan Africa. Progress in reducing 
worldwide morbidity and mortality has stalled, with the World Health Organization reporting an 
upward trend in the number of clinical cases from 2014 (217,000,000) to 2019 (229,000,000) (1) and 
no significant progress in reducing mortality during the past year (411,000 deaths in 2018, 409,000 
deaths in 2019). The burden of malaria is most severe in sub-Saharan Africa, where 17 countries 
reported an increase in the number of cases in 2018 compared to 2015-2017 (2). Mali is one of the 
eleven highest burden countries in the world, with the estimated rate of cases per 1000 population 
exceeded in 2018 only by the rate in Burkina Faso (1). Clearly, a vaccine that could prevent infection 
and thereby block Pf malaria with symptoms and transmission would strongly support the goal of 
malaria elimination.  
One of the most promising vaccine candidates is Sanaria® PfSPZ Vaccine, comprised of whole, 
radiation-attenuated P. falciparum (Pf) sporozoites (SPZ). Clinical trials of PfSPZ Vaccine in the US, 
Europe, and six African countries have shown PfSPZ Vaccine to be safe and well-tolerated in malaria-
naive adults and in African adults, teenagers, children, infants and HIV+ individuals, with no 
difference in adverse event profiles between vaccine recipients and normal saline controls in 
randomized, double blind, placebo-controlled trials (3-10). In these studies, and in additional non-
randomized studies, PfSPZ Vaccine has induced high levels of protection against homologous and 
heterologous controlled human malaria infection (CHMI) in malaria-naive and malaria exposed adults 
(4-6, 11-15). It has also shown modest protection against naturally transmitted Pf malaria infection 
over a six-month transmission season in malaria-exposed adults in four separate trials, including three 
conducted in Mali (Sissoko unpublished, Sirima unpublished)(9). Demonstration of vaccine efficacy 
(VE) in pediatric populations is limited, however. Three Phase 1 trials in Tanzania, Kenya and 
Equatorial Guinea and one Phase 2 trial in Kenya have been completed in infants and children ranging 
in age from 5 months to 17 years, and all four trials demonstrated excellent safety and tolerability, 
with no statistically significant difference in adverse event rates between vaccine recipients and 
normal saline placebo controls (Oneko submittted, Jongo unpublished)(3, 10). Data on VE in pediatric 
populations are now needed. 
The Phase 2 trial in Kenyan infants was the first pediatric study with PfSPZ Vaccine to measure VE. 
The trial, called KSPZV1, showed efficacy against both parasitemia and Pf malaria with symptoms at 
3 months in the highest dose group (1.8x106 PfSPZ Vaccine), but not at the primary 6-month endpoint, 
a result believed to be related to the absence of measurable cellular responses to the vaccine in these 
young children, all of whom were less than 1 year of age at the time of vaccination; it was 
hypothesized that the infant immune system is not mature enough to develop the CD8 T cell responses 
that underlie the durable protection induced by PfSPZ Vaccine in other age groups.  More recently, a 
second pediatric protection study was initiated in 1-to-12-year-olds in Gabon, also a randomized, 
double blind, placebo-controlled trial measuring VE against naturally transmitted Pf malaria. This 
trial, called LaSPZV1, is still underway in Lambaréné, a region of Central Africa characterized by 
year-round Pf transmission and thus different from the Sahel, where transmission is highly seasonal. 
Results from the LaSPZV1 study are expected in the next few months. To date, no studies have been 
performed in pediatric populations in the Sahel.  
This protocol describes the first such pediatric trial. It will be unique as the first pediatric trial in a 
region characterized by intense seasonal Pf transmission. It will also be the second trial looking at 
efficacy in pre-school and school-age children after LaSPZV1. The study is powered to show 
protection against first episodes of Pf malaria with symptoms over the six-month transmission season 
(primary objective), an important pediatric health need, and against first episodes of parasitemia 

PfSPZ Vaccine in Children 
 
 
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V4.0, 01 December 2022 
 
 
 
(secondary objective), a more stringent endpoint representing the prevention of both clinical disease 
and transmission.  Positive results from this trial will set the stage for future large-scale Phase 3 trials. 
In correspondence with the US FDA, the agency has emphasized the importance of field efficacy 
studies in both adults and children and requires promising Phase 2 results prior to approving the Phase 
3 testing plan. For this reason, the trial described herein is on the critical pathway to collecting the 
protection data needed for PfSPZ Vaccine licensure by the FDA.   
3.2 
BACKGROUND  
Sanaria® PfSPZ Vaccine is comprised of aseptic, purified, cryopreserved, Pf sporozoites (PfSPZ) that 
are manufactured according to current Good Manufacturing Practices (cGMPs) for parenteral 
injection. They are diluted in phosphate buffered saline (PBS) and human serum albumin (HSA) to 
obtain the desired dose and concentration and are administered by direct venous inoculation (DVI) 
without an adjuvant. The PfSPZ are attenuated by exposure to radiation during manufacture and are 
unable to cause parasitemia in humans or undergo transmission to mosquitoes that might bite an 
individual following immunization. 
 Nonclinical Investigations of PfSPZ Vaccine  
The safety and immunogenicity of PfSPZ Vaccine administered by subcutaneous (SC), intradermal, 
and IV routes were demonstrated in New Zealand White rabbits and mice. Three preclinical 
toxicology studies in rabbits given 5-7 doses of 1.35x105 PfSPZ by the ID, SC and IV routes were 
performed. In all 3, PfSPZ Vaccine was safe and well tolerated. More details are available in the 
PfSPZ Vaccine Investigator’s Brochure (IB).  
Formal biodistribution studies were performed, which examined the distribution and persistence in 
mouse tissues of Pf DNA following SC, ID, or IV inoculation of a single 1.35x105 PfSPZ dose of 
PfSPZ Vaccine. The studies showed that PfSPZ Vaccine was safe and well tolerated in mice and that 
there was no persistence of PfSPZ in the host tissue beyond 144 hours. The dose of sporozoites in 
these studies was equivalent to the highest dose administered in the first two Phase 1 clinical trials of 
PfSPZ Vaccine, a dose that has now been shown to be safe and well tolerated in adult and pediatric 
participants administered PfSPZ Vaccine by SC, ID, and/or IV routes. Details are available in the 
PfSPZ Vaccine IB.  
Recently, a reproductive toxicology study was performed in female New Zealand White rabbits. Fifty 
animals were dosed intravenously via marginal ear vein with phosphate-buffered saline (PBS) 
containing 1% human serum albumin (HSA) and 50 with 1.8×106 PfSPZ of PfSPZ Vaccine with 1% 
HSA in PBS. Animals received 4 vaccinations: 35, 28, and 7-10 days prior to mating (study days 1, 8 
and 29) and on gestation day 8. The study was interrupted prior to the planned fifth immunization due 
to HSA-induced anaphylaxis in vaccine and placebo recipients. However, pathological assessments 
showed no findings related to PfSPZ Vaccine or presence of maternally derived PfSPZ antibodies, 
which were developed by all vaccinees, including: 
1.  No redness or swelling observed at the injection site;  
2.  No effects on maternal body weight or food consumption;  
3.  No effect on maternal clinical pathology parameters (hematology, clinical chemistry, 
coagulation); 
4.  No effects on mating, fertility, or laparohysterectomy parameters, including the number of 
corpora lutea, implantations, live/dead fetuses, early/late resorptions, or pre- and post-
implantation loss; 

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Figure 4. PfSPZ-specific memory CD4 T-cell responses pre- and post-vaccination.  
Percent of memory CD4 T cells in the blood expressing interferon gamma (IFN-γ) interleukin 2 (IL-2) or tumor necrosis 
factor alpha (TNF-α) at pre-immunization or 2 weeks after the first and third doses of PfSPZ Vaccine (9.0x105). Results are 
the percentage of cytokine-producing cells after incubation with PfSPZ minus the percentage of cytokine-producing cells 
after incubation with vaccine diluent (medium with 1% human serum albumin). Bars indicate median values within each 
group. Differences within each age group between pre- and postvaccination groups were assessed by two-way ANOVA 
with Dunnett’s correction for multiple comparisons. *P < 0.05, **P < 0.01, ****P < 0.0001. Previously measured results 
from clinical trial VRC 314 conducted in the US with the same dosage regimen and the same assay conducted in the same 
laboratory as for the BSPZV2 trial assays are shown as a comparison. (3) 
 
 
The second pediatric trial was initiated six months later in western Kenya (#12 in Table 3) and 
addressed this hypothesis. After an initial age de-escalation / dose-escalation Phase 1 pilot study 
demonstrating that the vaccine was safe in all ages tested (10), a larger Phase 2 trial was performed in 
which more than 300 infants age 5-11 months were randomized to receive 3 doses of normal saline 
placebo or 4.5x105, 9.0x105, or 1.8x106 PfSPZ of PfSPZ Vaccine, which was administered weeks 1, 9 
and 17. Again, the vaccinations were safe, with no differences between vaccinees and normal saline 
placebo recipients in the rate or severity of solicited adverse events, unsolicited adverse events or 
laboratory abnormalities. The study subjects were followed over six months for incident malaria, and 
did not show statistically significant protection compared to controls over this period, although 
protection against both Pf malaria with symptoms and parasitemia was significant during the first 90 
days after immunization in the highest dose group (Oneko M and Steinhardt L, submitted). The early 
protection likely reflected vigorous antibody responses to the immunization as measured by PfCSP 
ELISA. It is surmised that as the antibody response faded, so did protection (Figure 5). This 
hypothesis was supported by the strong correlation between antibody responses to PfCSP and 
protection in this trial (see section on “Correlates of Protection” below). A more aggressive 
immunization regimen may be needed to induce T cell responses in infants that provide long-term 
protection.  
 
 

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A third pediatric study, EGSPV2, was conducted in Equatorial Guinea using a similar design to the 
Tanzanian study (addressing safety and immunogenicity, but not efficacy in children), and showed 
PfSPZ Vaccine to be safe and well tolerated in infants and children (same age groups as BSPZV2) 
(#14 in Table 3). Immunogenicity data from this trial are pending (Jongo S, unpublished data).  
A fourth pediatric study is now underway in 1-12-year-old Gabonese children, using a Day 1, 8 and 29 
regimen (9x105 PfSPZ/dose), measuring VE against naturally transmitted malaria (#17 in Table 3).  
The results of this study are expected in the next couple of months.  The blinded data (combining 
vaccinees and placebo recipients) do not indicate any concerning safety signal.   
In summary, trials of PfSPZ Vaccine in four African countries indicate that the vaccine is safe and 
well tolerated in all pediatric age groups.  This includes doses up to 1.8x106 PfSPZ in infants. As the 
dose planned for 6-to-10-year-olds in the current study is half that dose (9.0x105) and will be studied 
in 6-10-year-olds, we do not anticipate any safety issues.  Additional information on safety, including 
specific safety data for pediatric age groups as well as a meta-analysis of the pediatric trials, is 
presented below in the section on risks and benefits. 
Vaccine Efficacy: PfSPZ Vaccine administered intravenously at adequate doses has produced 80-
100% protection against both homologous and heterologous controlled human malaria infection 
(CHMI) in malaria-naive adults in multiple studies (11-13, 15). High level protection has also been 
induced in malaria-exposed African adults; in a study in Tanzania (the BSPZV2 clinical trial), 6/6 
(100%) adult volunteers undergoing homologous CHMI 3 to 10 weeks after receiving 3 doses of 
9.0x105 PfSPZ of PfSPZ Vaccine administered at 8-week intervals were protected (4) and in Mali, 
30/30 (100%) adults receiving 3 doses of 1.8x106 PfSPZ of PfSPZ Vaccine also at 8-week intervals 
were protected (Sissoko M and Healy S, unpublished). VE extended to protection against naturally 
transmitted malaria in the field: in three studies of PfSPZ Vaccine in Mali, immunization provided 51-
57% protection by time-to-event analysis (VE = 1 minus the hazard ratio) and 24-35% protection by 
relative risk analysis  (VE = 1 minus the risk ratio) against naturally transmitted malaria during a 
period of six months (Sissoko M and Healy S, unpublished)(9). In a similar trial in Burkina Faso, 
immunization provided 47% protection by time-to-event analysis and 38% protection by relative risk 
Figure 5. Vaccine efficacy at 3, 6, 9, and 12 months for the three vaccine groups (Oneko and 
Steinhardt, unpublished data) 

PfSPZ Vaccine in Children 
 
 
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V4.0, 01 December 2022 
 
 
 
analysis against naturally transmitted malaria during a period of six months (Sirima S and Lauren M, 
unpublished). In the same trial, VE was 46% at 18 months by time to event analysis (p=0.018).  In all 
these trials, VE in the field has been measured against malaria infection, not clinical disease, meaning 
that the protected research subjects remained free of parasitemia as detected by thick blood smear 
(TBS) over six months. These results indicated that PfSPZ Vaccine should be a powerful intervention 
for malaria control and elimination in Africa. 
Dose selection: In the US/EU and in some African studies, 9.0x105 PfSPZ appeared to be the optimal 
dose, and higher doses were less protective. For example, in the second CHMI assessment of VE in 
Tanzania (BSPZV2), when the dose was increase from 9.0x105 to 1.8x106 PfSPZ, VE fell from 100% 
to 33% (4); in the first CHMI trial in Equatorial Guinea (EGSPZV2), less than 50% of adults were 
protected after receiving three injections at 8-week intervals with a still higher dose, 2.7x106 PfSPZ of 
PfSPZ Vaccine (6). On the other hand, the 1.8x106 PfSPZ dose achieved 100% protection in Mali, 
performed in a location where naturally acquired immunity (NAI) was strongly developed (Sissoko M 
and Healy S, unpublished). In this case, NAI may have reduced the effective dose by neutralizing a 
proportion of the injected PfSPZ, such that the results reflected those of lower doses in other African 
countries where NAI is less fully developed. This finding was supported by the results of a study in 
Malian women of child-bearing potential (WOCBP, MLSPZV4): both 9.0x105 and 1.8x106 PfSPZ 
were tested head-to-head for protection against naturally transmitted Pf malaria, and the higher dose 
resulted in stronger VE (57% vs 41%), although the difference in VE between the two groups was not 
statistically significant (Sissoko M and Healy S, unpublished). Thus, it may be that higher doses are 
needed in those with heavy prior malaria exposure than for malaria-naïve individuals, for children 
living in endemic areas who have experienced less malaria exposure than their parents, or with adults 
with only limited exposure. At this point, our best estimate is that 9.0x105 PfSPZ will be an 
appropriate dose to assess in pre-school and school-age children in the current study. 
Vaccine intervals: Recent data indicate that accelerated vaccination schedules provide better VE, 
particularly when two or more priming doses are administered in quick succession. Until this effect 
was uncovered, earlier studies had assessed longer vaccination schedules, where 3 to 5 doses were 
administered over 16 to 20 weeks, with relatively even spacing. In the Warfighter 2 trial in the US, 
however, a dose of 4.5x105 PfSPZ of PfSPZ Vaccine was administered on Days 1, 3, 5 and 7, with a 
boost at week 17. This condensed priming regimen produced an excellent humoral immune response 
and 40% VE against a heterologous strain of malaria (heterologous = different from the vaccine strain) 
at three months (14). In this case, CHMI was administered by the bite of mosquitoes infected with a 
strain of malaria from Brazil, while the vaccine strain, NF54, originates from West Africa (16, 17). A 
heterologous CHMI at 3 months is highly stringent. Yet the 40% protection achieved was twice the 
VE achieved against CHMI by three other regimens studied in the Warfighter 2 trial, each of which 
followed a traditional week 1, 9 and 17 immunization schedule and with one of these regimens 
utilizing three doses of 9.x105 PfSPZ, the dose considered optimal in malaria-naive adults. Likewise, 
in the MAVACHE study in Germany, 9.0x105 PfSPZ administered on Days 1, 8 and 29 (weeks 1, 2 
and 4) protected 14/17 (78.8%1) of research subjects against a homologous strain of malaria 
(homologous = the same strain as the vaccine) at 3 and 10 weeks and 10/12 (83.3%) of research 
subjects against a heterologous strain of malaria at 3 and 10 weeks (Mordmüller B, unpublished). This 
demonstration of cross-strain protection indicated that the condensed regimen may be effective against 
the diverse parasites transmitted in nature. In MLSPZV3 trial in Mali, condensed and traditional 3 
dose regimens were compared head-to-head, and VE trended higher (without a statistically significant 
difference) following the 3-dose condensed regimen from MAVACHE (weeks 1, 2 and 4) than a 
 
1 Only 5/6 controls became positive 

PfSPZ Vaccine in Children 
 
 
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V4.0, 01 December 2022 
 
 
 
widely-spaced interval regimen (weeks 1, 7 and 15) (22% vs.14%2) (Sissoko M and Healy S, 
unpublished).  
The potency of condensed regimens may relate to the multi-dose priming series – Days 1, 3, 5 and 7 in 
Warfighter 2, and Days 1 and 8 in the MAVACHE, MLSPZV3 and MLSPZV4 trials. It has been 
hypothesized that two or more doses in succession may better recruit CD8+ T cells than more widely 
spaced doses. Another advantage is that condensed regimens improve the ease and practicality of 
immunization. Completing primary immunization in four weeks will benefit travelers, pregnant 
women and others at risk of malaria where rapid induction of immunity is an advantage. After testing 
over 16 different regimens, Sanaria (the sponsor of PfSPZ Vaccine) has down-selected the 1, 8, and 29 
day condensed regimen for testing in the planned Phase 3 program leading to licensure. 
Correlates of protection: The level of antibodies binding to the main surface protein of PfSPZ, the Pf 
circumsporozoite protein (PfCSP), as measured by ELISA, or to whole PfSPZ, have correlated with 
protection in both malaria-naïve and malaria exposed individuals (Erreur ! Source du renvoi 
introuvable.) (Oneko submitted, Sirima submitted)(9, 12, 15). The availability of these standardized 
assays and their association with protection suggest that it may be possible to develop a marker of 
“vaccine take” as a surrogate for the tissue (liver) resident cellular immune mechanisms thought to 
underlie protection induced by whole PfSPZ-based vaccines. Data collected to date, however, show a 
large degree of overlap between the PfCSP antibody levels of protected and non-protected subjects. 
Although the correlate holds true at the population level, there is no clear cut-off for individuals that 
indicates protective immunity. Measuring PfCSP antibodies by ELISA will be an important objective 
in the current trial. 
Median and interquartile range of net OD 1.0 for IgG antibodies to PfCSP two weeks after the last dose of 1.8x106 PfSPZ 
Vaccine efficacy at 3,in participants who were uninfected (protected) and infected during 6, 9 and 12 months follow up. P 
value calculated by Wilcoxon-Mann-Whitney test for the three vaccine groups (Oneko and Steinhardt, unpublished data). 
 
 
2 VE in the MLSPZV3 trial was not statistically significant – the only trial among 4 similar trials conducted in the Sahel where this was 
the case.  The poor result likely resulted from (1) failure to clear parasitemia prior to the first dose and (2) initiating the trial during the 
rainy season when transmission had already begun. 
Figure 6. Anti-CSP responses in vaccinees in the KSPZV1 clinical trial (Oneko, unpublished). 

PfSPZ Vaccine in Children 
 
 
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The underlying mechanism of protection is thought to be liver resident CD8 T cells that recognize 
liver-stage Pf antigens expressed on the surface of infected hepatocytes. However, it has not been 
possible to show a correlation between CD8 T cells responses and protection, likely because peripheral 
CD8 T cells populations do not reflect the tissue resident populations.  However, the frequency of the 
Vδ2 subset of γδ T cells measured post immunization has correlated with protection in field studies 
and is thought to be critical to the induction phase of protective immunity (18).  Interestingly, the 
infants studied in the BSPZV2 trial in Tanzania were assessed for Vδ2 γδ T cells in samples taken 
prior to immunization, and these cells were absent, as shown in Figure 7.  This may explain why the 
infants in both the BSZPV2 trial in Tanzania and in the larger KSPZV1 trial in Kenya did not develop 
measurable CD4 or CD8 T cell responses following vaccination.  Indeed, cellular immunity is now 
being analyzed in the Kenyan infant population, and no infants have shown measurable peripheral T 
cell responses (Oneko, submitted).  
 
 
 
 
 
 
 
 
 
 
 
 
 
Long-term development plan: The long-term objectives for PfSPZ Vaccine in malaria-exposed populations are 
1) seasonal malaria prophylaxis such as will be tested in the current trial; 2) protection of pregnant women; and 
3) deployment for use in mass vaccination programs (MVPs), in which entire populations in defined geographic 
areas will be immunized, excluding infants (based on the minimal protection seen in the Kenya study). The 
strategy for malaria elimination campaigns will be to combine mass drug administration (MDA) with MVPs; 
MDA will clear existing parasitemia and the MVP will prevent reinfection. This approach has the potential to 
halt malaria transmission and eliminate malaria from the target population.  
The current trial is a key step on the pathway to licensing PfSPZ Vaccine for the planned indications. It will 
measure vaccine safety and VE in children using a randomized, double-blind, placebo-controlled design, 
generating critical data to support the Phase 3 program and eventual licensure in the US, EU, Mali and other 
malaria-endemic countries.  
Figure 7. Frequency of Vδ2 γδ T cells prior to immunization in the BSPZV2 trial in Tanzania 
(Oneko, submitted). 

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3.3 
RISK/BENEFIT ASSESSMENT 
 Known Potential Risks 
Venipuncture 
Risks occasionally associated with venipuncture including the needle stick associated with DVI 
include pain, bruising, swelling, induration, erythema and bleeding at the site of venipuncture. 
Lightheadedness, and rarely, syncope can also occur. A possible but rare side effect is infection at the 
venipuncture site, which, if not treated promptly, could lead to more generalized symptoms such as 
limitation of arm movement, lymphadenopathy, fever, chills, headache, malaise, myalgia and joint 
pain.  
The side effects associated with venipuncture are generally mild and self-limiting but will be 
monitored if they occur and are clinically concerning.  
When children undergo needle stick, they may cry or struggle.  Gentle restraint a child during 
venipuncture may be required and can add to the discomfort, both physical and emotional, experienced 
by the child.  
The total amount of blood collected with venipuncture for blood drawing is within the American 
Association of Blood Banks recommendations and the current NIH guidelines, and will not 
compromise these otherwise healthy subjects (19). Any minor bruising, local tenderness or pre-
syncopal symptoms, or rarely, infection associated with venepuncture, will be documented as AEs.  
DVI Immunizations (either PfSPZ Vaccine or Normal Saline)  
Possible local site reactions resulting from the injection of 0.5 mL of PBS with HSA/PfSPZ Vaccine 
or normal saline, in addition to those listed above relating to the needle stick, include additional pain, 
bruising, swelling, induration, erythema and bleeding should the vein be missed or should some of the 
injectate extravasate during injection. When the injectate is placed into the lumen of the vein as 
intended, local reactions are unlikely because the injectate is disbursed by blood flow.    
As with any investigational product, 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 
PfSPZ Vaccine  
As described in the background section, PfSPZ Vaccine has been exceptionally safe and well tolerated 
in malaria-naïve adults in the USA and Europe, and in malaria-exposed adults, children and infants in 
Africa. As of 15 March 2021, 5606 doses of PfSPZ Vaccine (over 5.2 billion PfSPZ) have been safely 
injected into 1726 subjects aged 5 months to 65 years in Tanzania, Kenya, Mali, Burkina Faso, Gabon, 
Equatorial Guinea, Germany, the Netherlands and the US, including 873 doses administered by DVI to 
330 infants.  
An early safety concern with PfSPZ Vaccine was breakthrough malaria infections due to inadequate 
attenuation. However, there have been no observed breakthroughs following injection with over 5,000 
doses of PfSPZ Vaccine administered to date. A second concern had been local side effects at the 
injection site, given the reactogenicity of mosquito bites and the possibility that sporozoites may 
contribute. Interestingly, there appears to be little or no local reactogenicity associated with PfSPZ 
injection: in trials of attenuated sporozoite immunization by mosquito bite, the papules, edema, 
erythema and pruritus induced were equivalent between true immunization using infected mosquitoes 
and mock immunization using non-infected mosquitoes, indicating that mosquito saliva must be the 
cause (20). Additional information comes from trials of PfSPZ Vaccine and PfSPZ Challenge injected 

PfSPZ Vaccine in Children 
 
 
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V4.0, 01 December 2022 
 
 
 
ID and SC. These routes of administration leave a depot of PfSPZ in or under the skin, but do not 
cause local inflammation at the injection site, even after repeated dosing (12). The manufacturing 
process at Sanaria removes nearly all mosquito components, explaining the apparent absence of 
reactogenicity. 
The third concern has been that PfSPZ Vaccine might cause systemic reactogenicity, as commonly 
occurs with vaccines. However, even large numbers of PfSPZ (the equivalent of perhaps thousands of 
infectious mosquito bites) have been remarkably well tolerated. There have been no clearly 
documented immediate type hypersensitivity reactions. Doses as high as 2.7x106 PfSPZ have been 
given by DVI and as high as 2.2x106 PfSPZ have been given intramuscularly (IM) without apparent 
side effects. In all trials so far, the majority of vaccine recipients reported no or mild systemic AEs, 
with only a few classified as moderate in severity. Fever has only rarely been reported as a solicited 
AE and the few reports to date were generally attributed to other causes.  
An example of AE profiles in a relevant pediatric population is provided by the BSPZV2 trial in 
Tanzania, the first pediatric trial of PfSPZ Vaccine.  There were no systemic AEs recorded during the 
seven days after immunization in either the 1-5-year-olds or 6-10-year-olds (Table 4). There were 6 
Grade 2 and no Grade 3 laboratory abnormalities recorded post vaccination; 5/6 laboratory 
abnormalities occurred in the placebo group (Table 5). 
Table 4. Solicited adverse events recorded days 0-7 post-all immunizations. 
Participants received three doses weeks 1, 9, and 17 of 4.5.x105, 9.0x105 or 1.8x106 PfSPZ of PfSPZ Vaccine. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Group 3 (6-10 years) 
9x105 
(N=6) 
1.8x106 
(N=6) 
Placebo 
(N=6) 
Arthralgia 
0 
0 
0 
Chills 
0 
0 
0 
Fatigue 
0 
0 
0 
Headache 
0 
0 
0 
Malaise 
0 
0 
0 
Myalgia 
0 
0 
0 
Rash, urticaria, pruritus, edema 
0 
0 
0 
Subjective fever 
0 
0 
0 
Temperature (°C) 
0 
0 
0 
Group 4 (1-5 years) 
4.5x105 
(N=6) 
9x105 
(N=6) 
Placebo 
(N=6) 
Drowsiness 
0 
0 
0 
Elevated Temperature 
0 
0 
0 
Fever /history of fever 
0 
0 
0 
Inability/refusal to eat or drink 
0 
0 
0 
Irritability/fussiness 
0 
0 
0 

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V4.0, 01 December 2022 
 
 
 
Children age 7-12 were questioned with respect to solicited symptoms. There were no recorded instances of malaise. In all 
three age groups, there were no findings suggestive of an allergic reaction, including rash, urticaria, pruritis, or edema.  
Table 9. Laboratory abnormalities Grade 2 or higher by age group, days 1 to 28 post-
immunization. 
Tests were scheduled 1 and 7 days after each vaccine dose and prior to V3. The table also includes any unscheduled lab 
tests. Data from vaccine and normal saline placebo groups combined (trial remains blinded). Data are the number of 
participants experiencing the abnormalities and also the number of abnormalities experienced (no child had more than one 
lab abnormality of a given type). 
 
Laboratory abnormality Severity Grade 
1-2 years 
(n=36) 
3-6 years 
(n=101) 
7-12 years 
(n=63) 
ALT elevation 
Grade 2 or higher 
0 
0 
0 
Creatinine elevation 
Grade 2 
0 
0 
1 
 
Grade 3 or higher 
0 
0 
0 
Hemoglobin decrease 
Grade 2 or higher 
0 
0 
0 
WBC decrease 
Grade 2 or higher 
0 
0 
0 
Platelet count decrease* 
Grade 2 
0 
0 
1 
 
Grade 3 
1 
2 
0 
 
Grade 4 
0 
0 
1 
*All Grade 3 and 4 platelet count abnormalities occurred 1 day after immunization (two episodes with 
dose 1, 1 with dose 2, 1 with dose 3). Preceding values normal in all cases. Follow-up tests also 
normal in all cases, performed after 4 days (Grade 3), 5 days (Grade 3 and Grade 4), 6 days (Grade 3) 
and 21 days (Grade 2). There were no repeat episodes of decreased platelet counts with subsequent 
dosing. Toxicity scale criteria: Grade 2 creatinine elevation: > 88-141 µmol/L; Grade 2 platelet count: 
51-75,000/µL Grade 3 platelet count: 25-50,000/µL; Grade 4 platelet count: < 25,000/µL 
These findings in pediatric studies are illustrative of the findings in all trials of PfSPZ Vaccine, 
including all nine randomized, double-blind, normal saline placebo-controlled trials that have been 
completed and unblinded. None of these trials has shown any differences in the rates of any AEs 
between vaccine recipients and controls after unblinding (#5, 7, 8, 9, 10, 12, 14, 15 in Table 3) (Jongo 
S, unpublished; Sissoko M and Healy S, unpublished; Oneko M and Steinhardt L, unpublished)(3-10) 
except for one study where there was an excess of myalgia in the vaccine group (#10 in Table 3). 
Most importantly, there were no age or dose effects on the rates of AEs with doses as high as 1.8x106 
PfSPZ in infants and young children (3, 10). There also has not been an increase in AEs with repeated 
dosing (i.e., the rates of AEs after second or third doses have been similar to the rates after first dose). 
It is thus not clear that PfSPZ Vaccine has consistently caused any adverse reactions at the doses tested 
to date. In addition, there have been no allergic reactions linked to the vaccine. A few volunteers have 
developed urticarial rashes in the days following immunization, but in no cases have these rashes been 
clearly linked to the vaccine.  
A meta-analysis of AE data is shown for children using forest plots of total solicited AEs in vaccinees 
and placebos in the randomized, double-blind, placebo-controlled trials analyzed to date (Figure 8). In 
all cases, 95% confidence intervals cross a ratio of 1, indicating no differences between vaccinees and 
controls. 

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Figure 8. Effect of vaccine on incidence of any solicited systemic adverse events per study subject in randomized, double-blind, 
placebo-controlled trials of PfSPZ Vaccine in infants and children aged 5 months to <18 years in sub-Saharan Africa. 
Trial sites included: Bagamoyo, Tanzania (BSPZV2); Siaya, Kenya (KSPZV1b); and Bioko Island, Equatorial Guinea (EGSPZV2). #Vac. is the number of 
subjects enrolled in the vaccine arm in each study; # Plac. is the number of subjects enrolled in the NS control arm of each study. Numbers in parentheses are the 
number of subjects experiencing the AE after any of the injections administered.  V = vaccine recipients, NS = normal saline recipients, OR = odds ratio, LCL = 
lower confidence limit, UCL = upper confidence limit. Weight is based on sample size and is represented by the width of the blue bar.  
 

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Against this background of favorable safety data, two SAEs have occurred that were assessed as 
possibly related to the administration of PfSPZ Vaccine. In each case the potential association 
was based on timing without a clear pathophysiological link. Both of these SAEs occurred in the 
EGSPZV2 trial in Equatorial Guinea (#14 in Table 3) and led to halting of the study while the 
SAEs were reviewed by the Data and Safety Monitoring Board (DSMB). Direct causality could 
not be established, and in each case the DSMB, all associated IRBs, the regulatory agency in 
Equatorial Guinea and the US FDA approved resumption of the trial.  
Seizure in 15-year-old boy: In the first case, a 15-year-old boy with no known history of seizures 
experienced a seizure 3½ hours after receiving a third dose of 1.8x106 PfSPZ of PfSPZ Vaccine. 
The boy had a normal head CT scan and a normal head MRI. However, an electroencephalogram 
(EEG) revealed abnormalities consistent with generalized epilepsy. A consulting neurologist 
posited that the general inflammatory response to the vaccine may have lowered the seizure 
threshold in this individual, whose EEG was consistent with a predisposition to seizures, and 
whose sister had a history of a complex febrile seizures. Given that the boy experienced only one 
seizure, even though he received three identical doses of PfSPZ Vaccine, it is not possible to 
know whether the PfSPZ non-specifically affected the seizure threshold in this boy, as can occur 
with inebriation or sleep loss, or whether the timing of the seizure was coincidental. The boy was 
not treated with anti-epileptics and was interviewed one year after the seizure and reported good 
health without any additional seizures.   
This is the only seizure occurring in temporal proximity to the injection of PfSPZ Vaccine. The 
same 1.8x106 PfSPZ dose of PfSPZ Vaccine has been administered by DVI to 150 other children 
ranging in age from 5 months to 17 years, including 92 infants, most of whom received three 
injections with this dose, without any seizures linked by timing to the vaccine. Three infants in 
the KSPZV1 trial of PfSPZ Vaccine (#12= in Table 3) conducted in Kenya developed epilepsy 
during their 16-month participation in the trial, but the seizures did not occur at the time of 
vaccination, and the rate of new epilepsy diagnoses in the trial was not different from the 
background rate in this population. All three infants are now seizure-free and off medication, but 
one remains with developmental delay. Although 23 febrile seizures and 7 non-febrile seizures 
occurred in the 296 infants receiving injections with PfSPZ Vaccine in the KSPZV1 trial (versus 
5 febrile seizures in 105 placebo recipients), none occurred proximally to the injection of PfSPZ 
Vaccine as had happened with the boy in Equatorial Guinea. However, 10 of the 23 febrile 
seizures occurred in the 1.8x106 PfSPZ dose group, while only 5 occurred in the placebo group  
suggesting a trend of increased frequency in the vaccine group. Most of these seizures occurred 
during acute malaria infections spaced out during the 12 months of surveillance following 
immunization, and their frequency may have been increased in vaccinees secondary to the 
rebound malaria seen in the vaccine groups in that study (see below). Parents were contacted a 
year after the study ended, and asked about seizures, and the rates were no different among the 
three vaccine and one placebo groups. 
Given these considerations, as a precaution until more data are available, Sanaria recommends 
excluding any individual with a history of seizures other than simple febrile seizures as a young 
child from participation in clinical trials of PfSPZ-based products. It is expected that with further 
clinical experience this restriction may be lifted.  
Fetal loss in 19-year-old woman: In the second possibly related SAE, a 19-year old woman 
became pregnant at about the same time as she received a first dose of 2.7x106 PfSPZ of PfSPZ 

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Vaccine. An ultrasound at 9 weeks gestational age by last menstrual period showed a 6-week 
sized embryo without cardiac activity. Because about a quarter to a third of all pregnancies end 
in spontaneous abortion, it is unclear whether the administration of PfSPZ was related to the loss 
of this embryo. As was the case in the EGSPZV2 trial, avoidance of pregnancy remains an 
important inclusion criterion for trials of PfSPZ Vaccine.  
Rebound malaria: A finding from the KSPZV1 trial in 5-11-month-old Kenyan infants was that 
as the efficacy of the vaccine waned, the frequency of malaria infections increased in vaccinees 
relative to the controls. This evolution, called “rebound,” has been seen with the RTS,S malaria 
vaccine and with malaria chemoprophylaxis (21-23). The hypothesis proposed to explain 
rebound is that vaccine- or drug-induced protection delays the onset of naturally acquired 
immunity (NAI), which depends upon repeated exposure to malaria. While the placebo group is 
developing NAI, experiencing a progressively diminishing risk of clinical disease, the vaccinees 
lag behind based on their lower infection rates and reduced exposure to parasitemia. As a result, 
they may subsequently experience rates of parasitemia and Pf malaria with symptoms that 
exceed those in controls once vaccine-induced protection has waned. A pattern consistent with 
this hypothesis was seen in the KSPZV1 trial in Kenyan infants, where vaccinees had less 
exposure to parasitemia than placebo recipients during the first 3 to 6 months after vaccination 
(Oneko M and Steinhardt L, unpublished). Despite the excess rates of Pf malaria with symptoms 
in vaccinees toward the end of the 12-month follow-up, the total number of clinical episodes was 
about the same in placebo and vaccine groups over the full year that malaria parasitemia was 
monitored.  In Mali, an area of highly seasonal malaria transmission, it is very unlikely that 
rebound malaria cases will occur during the dry season after the scheduled surveillance period. 
In addition, if significant efficacy is seen on this trial it is likely that the study will be extended 
for an additional malaria transmission season to assess the protective efficacy of a booster dose, 
and monitoring for malaria cases would also occur for participants who re-enroll.    
Epstein-Barr virus reactivation: US FDA requested that serological studies of EBV capsid 
antigen be performed in previous pediatric trials of PfSPZ Vaccine. The concern was based on 
the association between Pf malaria, latent EBV reactivation, and endemic Burkitt lymphoma 
(24). Since PfSPZ Vaccine contains malaria parasites, exposure following injection might 
reactivate EBV, even though this reactivation is thought to be precipitated by blood stage 
parasites, not by sporozoites. If this were to occur, it should be reflected in increasing antibodies 
to EBV capsid antigen following immunization. The data from the BSPZV2 trial did not show 
any effect of immunization on antibody levels in any of the 5 age groups studied in that trial 
(adults, teenagers, older children, younger children and infants). Serology assays are currently 
being performed to test the same hypothesis using samples from EGSPZV2 and KSPZV1. 
Summary: The side effect profile of PfSPZ Vaccine recorded in 19 Phase 1 and 2 clinical trials 
has been benign, and not been different from that of NS placebo in trials including this control in 
those studies that have been unblinded. However, the side effect profile could evolve with the 
performance of trials which have increased power to detect small differences in the rates of AEs 
between vaccinees and placebo recipients, and which have a higher likelihood of revealing 
uncommon adverse effects due to larger sample sizes. Until such studies can definitively rule out 
side effects, the study Sponsor, Sanaria continues to describe the signs and symptoms that are 
typically caused by less well tolerated vaccines, or by malaria, as theoretical concerns. These 
include:  

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Local reactions: injection site pain, tenderness, erythema, swelling, induration, pruritus, 
bruising, bleeding, lymphangitis, regional lymphadenopathy, and arm motion limitation. 
Systemic reactions: measured fever, subjective fever, headache, malaise, fatigue, chills, 
rigors, sweats, myalgia, arthralgia, dizziness, nausea, vomiting, abdominal discomfort, 
anorexia, asthenia, insomnia, anxiety, confusion, cough, shortness of breath, chest pain, 
palpitations, and allergic reactions such as rash, urticaria, pruritus, angioedema, and 
anaphylaxis, the latter potentially resulting in death, and unexpected AEs that have not 
previously been reported. 
Although it has never happened, there is also the theoretical risk of breakthrough infection due to 
inadequate attenuation.  
Normal Saline  
The amount of normal saline used in this study is small (< 1 mL) and has been well tolerated, 
including in previous PfSPZ Vaccine studies. Overall, most AEs reported with the use of normal 
saline have been reactions that may have occurred because of administration technique and have 
been local injection reactions as noted above in Section 2.3.1.2.  
Antimalarial Medications (AL)  
Subjects will be treated with a registered, oral, proven, and highly efficacious treatment during 
the course of this study per standard clinical practice. These medications will be tracked closely, 
similar to study specific products. 
Artemether/lumefantrine (AL) is the first-line anti-malaria drug regimen currently recommended 
for use by the Malian Ministry of Health to treat cases of acute malaria disease in adults and 
children. 
AL has an acceptable safety profile. Individuals who may have any contraindication for the use 
of this drug (e.g., prolonged QTc or taking other medications that can prolong QTc, history of 
myocardial infarction) will be excluded at screening. The most common side effects (i.e., >10%) 
in children less than 16 years of age are headache, anorexia, fever, and cough. Discontinuation of 
artemether/lumefantrine due to AE is rare (0.2%) in adults. Rare but serious hypersensitivity 
reactions (urticarial and angioedema) and skin reactions (bullous eruption) have been reported 
post marketing.  
The frequency of dosing on this trial (two treatment courses spaced approximately one month 
apart) is not expected to increase the risk of side effects. 
For complete AL safety information, including less commonly reported side effects, please refer 
to the Package Insert for Coartem® that is provided. 
SARS-CoV-2 Transmission 
This trial will likely be conducted under the conditions of ongoing SARS-CoV-2 transmission in 
the local community.  As of 10 April 2021 there have been 11,393 confirmed cases and 404 
deaths due to COVID-19 in all of Mali.  However, these numbers could underestimate the true 
COVID-19 infection rate and it is assumed that by bringing community members together at a 
clinical trial site to receive immunizations and check-ups, the conduct of a clinical trial could in 
theory augment local transmission.  To prevent this, and to protect clinical staff members and 

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study participants, the clinical team will strictly follow mask wearing, hand-washing and social 
isolation measures in accordance with local public health guidelines and site Standard Operating 
Procedures. 
Risks to Privacy 
Subjects will be asked to provide personal health information (PHI). All attempts will be made to 
keep this PHI confidential within the limits of the law. However, there is a chance that 
unauthorized persons will see the subject’s PHI. All study records will be kept in a locked file 
cabinet or maintained in a locked room at the site. Electronic files will be password-protected. 
Only people who are involved in the conduct, oversight, monitoring, or auditing of this trial will 
be allowed access to the PHI that is collected. Any publications from this trial will not use 
information that will identify subjects by name. Organizations that may inspect and/or copy 
research records maintained at the site for quality assurance (QA) and data analysis include 
groups such as the local or central IRB, NIAID, and the Food and Drug Administration (FDA). 
A description of this clinical trial will be available on http://www.ClinicalTrials.gov, as required 
by US Law. This web site will not include information that can identify subjects. 
Unknown Risks 
There may be other risks, discomforts, or side effects that are unknown at this time. 
 Known Potential Benefits 
PfSPZ Vaccine has been shown in previous studies to provide some protection to the individual 
from malaria infection over a 24-week period during the malaria transmission season. For 
children who receive the study vaccine, there is a potential of direct benefit either from 
protection from patent parasitemia and/or Pf malaria with symptoms or from decreased poor 
outcomes related to secondary anemia.  
Subjects who receive placebo will not have personal, direct benefits from participation during the 
course of the trial ascribable to receiving an investigation vaccine. However, the risk to subjects 
receiving placebo is minimal, and all participants will have close follow up during their study 
participation and have study evaluations, which may provide some benefit. Even without any 
personal direct benefits, subjects may contribute to a societal benefit through the improvement of 
our understanding of these vaccines and how they can be utilized to improve maternal and 
neonatal malaria outcomes.  
Administration of artemether/lumefantrine on this trial to clear parasitemia has the potential to 
directly benefit participants, similarly to seasonal malaria chemoprevention.   
 Assessment of Potential Risks and Benefits 
The risks and benefits described above will be reviewed by the relevant Investigational Review 
Boards for an ethical determination. It is anticipated that this protocol will be classified as 
greater-than-minimal risk for vaccine recipients and minimal risk (or a minor increase over 
minimal risk) for placebo recipients. The protocol’s acceptability is enhanced by strategies for 
risk mitigation, which are described here. 
To increase safety during the trial, subjects will be monitored closely, including the measurement 
of vital signs, the performance of focused physical examinations and hematology assessments, 

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and the collection of AEs. SAEs will be monitored throughout the trial. Any medical conditions 
that develop will be evaluated and treated to the extent possible. Study doctors will be available 
24 hours a day, 7 days a week to provide care.  
Because the study will be performed in a malaria-endemic area, malaria infection is a risk during 
the trial, and subjects will be told to contact research personnel if malaria symptoms develop. 
During every encounter with clinical staff, subjects will be asked actively about the symptoms of 
malaria. If symptoms are present, a blood sample for TBS will be obtained (if not already 
scheduled) and will be examined for the presence of parasites. Those found positive will be 
promptly treated. Study subjects will be followed during the three-day administration of 
antimalarial treatment to assure clinical resolution. 
To minimize the risks associated with skin puncture, immunizations will be preferentially given 
in a cleaned and disinfected area of the upper extremity via a sterile 25-gauge needle and syringe 
by a skilled medical provider using aseptic technique, thus minimizing any chance of 
extravasation or infection. Blood drawing will likewise be performed with a sterile needle and 
syringe after cleaning and disinfecting the phlebotomy site. 
Emergency equipment and supplies to allow urgent endotracheal intubation, administration of 
oxygen, epinephrine, antihistamines, corticosteroids and other drugs, will be available at any site 
where immunization is performed to treat any acute allergic reactions that might occur. Staff will 
be available who are trained in pediatric advanced cardiac life support or equivalent. A 
defibrillator and cardiac monitor will be available for use.  
In order to decrease the risk of a breach of subject confidentiality, all study procedures will be 
conducted per GCP guidelines. Personal identifiers will not be used in publications or 
communications from this research study. Rather, a coded and unique study number will identify 
specific details related to each study subject in an effort to maintain their confidentiality. All 
study-related information will be stored securely in locked areas with access limited to study 
staff. All databases will be secured with a password-protected access system, and subjects’ study 
information will not be released without their written permission, except as necessary for 
monitoring compliance with legal or regulatory requirements or obtaining medical consultations 
to provide medical care. 
According to Malian Law No 09-059, when a research participant is a minor with either direct 
individual benefit or without direct individual benefit, their legal representative(s) and/or 
guardian(s) must provide consent. In addition, the research must not present a serious foreseeable 
risk to participants who are minors and, if the study is to be conducted without direct benefit to 
participant(s) who are minors, the research must be useful to people with the same age, illness or 
disability characteristics and provide results that cannot be achieved otherwise.  We believe this 
study satisfies those criteria. 
Summary:  
We propose that for vaccine recipients, participation in this trial involves greater than minimal 
risk but a well-justified prospect of direct benefit from the vaccine while for placebo recipients 
participation in this trial constitutes no greater than minimal risk based on the benign nature of 
injection of normal saline through a small-gauge needle.  

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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
against all episodes of Pf 
malaria with symptoms 
and/or Pf malaria rather than 
against the first episode only, 
or using proportional 
analysis, or using ITT or 
ATP populations, or severity 
of malaria infections or 
hemoglobin concentration. 
 
- VE against all episodes 
(rather than first episode) of 
Pf malaria with symptoms and 
Pf malaria by Cox regression 
analysis 
- VE computed by 
proportional (binary) analysis 
(1 – risk ratio) for first 
episode of Pf malaria 
detected by TBS, from 2 
weeks after V3 to 26 weeks 
after V3 in the ITT, mITT and 
ATP populations (whole 
group and subgroups) 
- VE computed as one minus 
the estimated HR for first 
episode of Pf malaria 
detected by TBS, from 2 
weeks after V3 to 26 weeks 
after V3 in the ITT and ATP 
populations (whole group and 
subgroups) 
- VE for Pf malaria measured 
by and nucleic acid detection 
in children from 2 weeks after 
V3 to 26 weeks after V3 (time 
to event, proportional/binary) 
- VE by both time-to-event  
and proportional/binary 
analyses for preventing and 
delaying and by relative risk 
analysis for preventing first 
episodes of Pf malaria with 
symptoms with  
- 
parasite density > 
500 parasites/µL, 
or  
- 
parasite density > 
5000 parasites/µL 
important to understand the 
spectrum of VE. 

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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
- or of Pf malaria with 
symptonms characterized by: 
- 
grade 3 signs or 
symptoms,  
- 
fever and 
convulsions, or  
- 
hospitalization, or 
- 
other clinical 
measures in 
vaccinees vs. 
controls. 
- Rates of anemia (grade 1, 
grade 2, grade 3) in vaccinees 
vs. controls. 
 
To explore the relationship 
between detectable 
parasitemia prior to 
vaccination and VE. 
Retrospective malaria qPCR 
values taken prior to 
vaccination 
 
Analyzing quantitative 
malaria PCR results across 
several pre-vaccination 
timepoints may allow for 
assessment of the impact on 
VE of low-level parasitemia 
that would otherwise be 
below the threshold of 
detection at a single 
timepoint. 
 
5. STUDY DESIGN 
5.1 
OVERALL DESIGN 
This phase 2 study will enroll healthy Malian children between 6 and 10 years of age residing in 
Bancoumana and surrounding villages to participate in a randomized, double blind, placebo-
controlled study to assess the safety, immunogenicity and protective efficacy of PfSPZ Vaccine. 
Participants will be immunized with a 3-dose series of 9.0 x105 PfSPZ of PfSPZ Vaccine or 
normal saline (placebo) at 1, 8, and 29 days. Subjects will be screened for eligibility for 
enrollment. Enrollment will begin with AL dosing approximately 4 weeks prior to their first dose 
of vaccine. Volunteers will be randomized into two arms (1 vaccine arm, 1 control arm) in a 1:1 
ratio. 

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Vaccinated subjects and controls will then be followed for safety and assessment for malaria 
infection during the subsequent malaria transmission season.   
~268 children between the ages of 6 and 10 years old inclusive will be enrolled as follows: 
 
 
 
Arm 1 (PfSPZ Vaccine): (n = ~134) children ages 6 – 10 will receive three doses of 
PfSPZ Vaccine (9.0x105 PfSPZ) via direct venous inoculation (DVI) at 1, 8, and 29 days  
 
Arm 2 (normal saline): (n = ~134) children ages 6 – 10 will receive normal saline via 
DVI at 1, 8, and 29 days  
 
All subjects will receive artemether-lumefantrine (AL) approximately 1-4 weeks before the first 
and third dose of PfSPZ Vaccine or normal saline for clearance of Pf parasitema.  
 
NB.: the second round of AL clearance prior to third vaccinations may or may not proceed 
depending on the timing of the beginning of the rainy season and the timing of malaria 
transmission in the community and will be decided by the Mali PI. 
Vaccinated participants and non-vaccinated controls will be monitored for development of Pf 
malaria with symptoms and Pf malaria (parasitemia) through the natural malaria transmission 
season to estimate vaccine efficacy (VE).  During the surveillance period, both active and 
passive surveillance will be used to identify Pf malaria with symptoms (see section 9.7). Blood 
smears will be made at any time a participant presents with a clinical syndrome consistent with 
malaria and read in real time, with all infections treated. Twenty-eight days later, these subjects 
will resume surveillance to make it possible to count all episodes of malaria with symptoms 
(exploratory objective). Parent/guardians will be encouraged to report to the clinic any time that 
a child is ill. 
In addition, blood smears will be made every four weeks in all participants as active surveillance 
for Pf malaria (parasitemia).  However, to avoid confounding the primary clinical endpoint, 
these blood smears will be reported retrospectively at the end of the primary surveillance period. 
This study will not interfere with routine standard-of-care for children. Participants will receive 
medical care through the local health clinic, including standard immunizations and seasonal 
malaria chemoprophylaxis if indicated, per the standard of care in Mali. The study will support the 
clinics by providing acute care assessments, ensuring supply of RDT and blood smears for malaria 
diagnosis, drugs for malaria treatment and prevention, and neurodevelopmental screening. During 
the study visits, the team will encourage families to continue consistent follow-up with their 
pediatric providers and will coordinate any acute care in consultation with the local pediatrician as 
needed. 
Indoor residual spraying (IRS) is performed in only about 3 of the 75 districts in Mali, and this 
does not include Bancoumana.  Therefore, it is not expected that IRS will affect outcomes in this 
study.  In 2018, bednet use was 82.2% in the Koulikoro region of Mali where Bancoumana is 
located. It is expected that randomization will balance out bednet use between the study groups.  
However, participants will be evaluated at routine visits with regard to bednet use, so that a 
subgroup analysis can be performed taking bednet use into consideration. 

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5.2 
SCIENTIFIC RATIONALE FOR STUDY DESIGN 
 Justification for Dose 
Multiple trials in US and African adults and in African children (as summarized in Table 3) have 
been completed to date with reassuring safety results at different PfSPZ Vaccine doses (up to 
2.7x106 PfSPZ) and dosing regimens. These trials include 7 trials in Europe and the United 
States as well as 13 in malaria exposed adults, children and infants in Africa. Clinical trials have 
assessed as few as two doses and as many as six doses. Numerous regimens have been carefully 
assessed, including widely spaced (weeks 1, 9, 17), condensed (days 1, 8, 29), and combination 
regimens (multi-dose prime with late boost). Ten different dose strengths have been tested, 
starting at 7.5x103 and increasing to 2.7x106 PfSPZ per injection.  
In 4 of the field trials conducted in Mali and Burkina Faso, PfSPZ Vaccine administered after 
antimalarial pretreatment using various dosages and schedules (including a 28-day regimen) has 
demonstrated efficacy against new Pf infection of up to 56% in time-to-event analyses over 
periods ranging from 20-24 weeks during the malaria season. These findings are discussed in 
more detail in Section 2.2.1.1 above. In a trial from Burkina Faso, PfSPZ Vaccine showed 
significant efficacy when subjects were followed over two years. 
A major finding has been that an accelerated 28-day regimen (Days 1, 8 and 29) provides 
protective efficacy which is as good as or better than any of the more widely-spaced regimens. 
Key findings from the 20 trials conducted with PfSPZ Vaccine include: 1) two doses are not 
sufficient (maximum VE 67% against homologous CHMI), 2) three doses are as good as 5 doses, 
3) multi-dose priming is important, 5) priming with Days 1 and 8 appears to be as good as or 
better than priming with Days 1, 3, 5 and 7, and 6) dose escalation after a certain threshold 
appears to have no additional benefit. The dose of 9.0x105 PfSPZ administered on Days 1, 8 and 
29 has been down-selected by Sanaria as the dose to be brought into Phase 3 testing and is the 
dose and regimen that will be used in this trial.  
 Rationale for Age Groups Selected 
Naturally acquired immunity (NAI) to malaria develops in those living in endemic areas as a 
result of repeated exposures to malaria parasitemia (28). It includes an anti-disease component 
(ability to sustain a given density of parasitemia without signs or symptoms) and an antiparasite 
component (ability to suppress parasitemias to lower densities)(29). The rate with which 
individuals develop NAI varies with the level of exposure across a range extending from high 
exposure rates (multiple infections per individual per year) where malaria-related morbidity and 
mortality are concentrated in the youngest age groups (e.g., < 5 years of age) because all older 
individuals have developed some level of NAI, and low exposure rates (multiple years between 
infections) where malaria-related morbidity and mortality are spread across all age groups 
including adults because most individuals fail to develop functional NAI.  
A cohort study of the community dynamics of malaria transmission (including the incidence of 
clincal malaria) was recently conducted in Bancoumana, Mali, and enrolled a total of 830 
volunteers aged 6 months to 65 years of age (Sissoko et al, unpublished data). Study participants 
were seen monthly and at the time of any illness by study clinicians. Malaria blood smears 
and/or rapid diagnostic tests (RDTs) were performed upon observation of clinical symptoms to 
confirm clinical malaria before treatment initiation. Incidence data on malaria with symptoms 
collected from February 2018 to January 2020 are presented in Table 10.  

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in the same trial when the injection day was shifted forward by two days to avoid immunizing 
during parasitemia (Jackson L, unpublished data). Furthermore, immunosuppression has also 
been demonstrated in Kenyan children (30), and has been mechanistically explored in murine 
studies.  
The requirement for pre-treatment has been shown in field studies of PfSPZ Vaccine and PfSPZ-
Cvac. Sanaria and collaborators have measured VE against naturally transmitted Pf malaria 
infection in 6 field trials of PfSPZ Vaccine (#4, 9, 10, 12, 16 and 19 in Table 2) and 2 field trials 
of PfSPZ-Cvac in Africa (#7 and 10 in Table 5 in the next section).  In the five trials where 
parasitemia was cleared prior to the first vaccine dose, including four trials of PfSPZ Vaccine 
(MLSPZV1, MLSPZV2, BFSPZV1, MLSPZV4) and one trial of PfSPZ-Cvac (MLSPZCV3), 
there was statistically significant VE during the 6 months after the last dose of vaccine. In the 
three trials in which clearance prior to the first vaccine dose was not done, including two trials of 
PfSPZ Vaccine (MLSPZV3 and KSPZV1) and one trial of PfSPZ-Cvac (MLSPCV1), there was 
no statistically significant VE during the 6 months after the last dose of vaccine. We believe that 
this is due to the immunosuppressive effects of asexual blood stage parasites on the immune 
response to pre-erythrocytic stage parasites discussed above. The most striking comparison is 
between MLSPZV3 and MLSPZV4 – both trials assessing the identical regimen in Malian adults 
in two consecutive rainy seasons (Table 12): 
Table 12. VE against Pf infection in MLSPZV3 and MLSPZV4   
  
PfSPZ 
Vaccine 
dose 
Regimen 
Study 
population 
Time-to-event 
VE (1 – odds 
ratio) 
Proportional 
VE (1 – risk 
ratio) 
MLSPZV3  9.0 x 105 
PfSPZ 
Days 1, 8, 29 
(no clearance 
before V1) 
Men and 
women 
14% 
P = 
0.521  
8.3% 
P = 
0.732 
MLSPZV4 
9.0 x 105 
PfSPZ 
Days 1, 8, 29 
(clearance 
before V1) 
Women 
41% 
P = 
0.005  
23% 
P < 
0.001 
In the first trial, MLSPZV3, conducted without pre-vaccination clearance, there was no 
significant protection, whereas when the identical regimen was administered after parasite 
clearance in MLSPZV4 during the next transmission season, there was highly significant 
protection (Diawara and Healy, unpublished). 
When all eight trials are considered together, the difference in protection between trials with and 
without pre-treatment is statistically significant (Fisher’s exact test p=0.018). For this reason, all 
field trials of PfSPZ vaccines, if taking place in endemic areas where study subjects may be 
chronically infected with malaria, include treatment with a curative antimalarial drug regimen 
prior to the first and perhaps third vaccinations. As further confirmation, Sanaria will be 
conducting a trial in 2-10 year-old children in Equatorial Guinea in 2022 in which participants 
will be randomized to receive or not receive pre-treatment using a randomized, double-blind 
design (Richie, personal communication).  

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Therefore, to reduce the risk of immunosuppression, all study subjects will be cleared with a full 
six-dose, three-day treatment regimen of AL prior to V1. 14 days is about 2 to 4 half-lives of the 
longer acting drug, lumefantrine, which has a half-life of 3-6 days. This gap should be sufficient 
because lumefantrine is not known to have activity against sporozoites or liver stage parasites. 
The 14-day gap will also provide some time for the immune system to recover from the effects of 
pre-existing parasitemia, although the actual time required is not known. 
When PfSPZ Vaccine immunization is done when there is ongoing malaria transmission risk, the 
three-day treatment course with AL is administered a second time prior to the third vaccine to 
avoid the immunosuppressive effects of parasitemia just described, in case any participants may 
have acquired a naturally transmitted malaria infection during the immunization period. 
However, because immunizations will be done during the dry season, when the risk of malaria 
transmission is essentially nil in this region of Mali, a second AL administration will not be done.  
 
 Rationale for Parasitemia Threshold Selected 
To comply with a recommendation by the FDA, we have used a data collected in a survey of 
Community Dynamics of Malaria Transmission and Mosquito Feeding in Bancoumana, Mali 
(NCT03304704) conducted from February 2018 to September 2020 which should be applicable 
to the proposed study population.  This Community Survey encompassed three dry seasons 
(2018, 2019 and 2020) and the full rainy seasons of 2018 and 2019. The available data set ended 
early in the rainy season of 2020. In the Community Survey study, participants ranging in age 
from 2 months to 87 years were seen in scheduled visits at the clinic once a month, at which time 
they were questioned about symptoms, examined for axillary temperature, finger-pricked for a 
malaria thick blood smear, classified according to diagnosis if clinically ill, and treated as 
needed. Participants were also encouraged to report to the clinic outside of scheduled visits in 
case of acute illness, in which case the same assessments were made. Any density of parasitemia 
identified, if associated with clinical signs or symptoms consistent with malaria, was classified as 
“malaria.” Note that fever was not a requirement for a diagnosis of clinical malaria. This 
followed the Malian Ministry of Health directive for identifying a case of clinical malaria:  
Mali Ministry of Health Case Definition for uncomplicated clinical malaria requiring treatment: 
1. Parasitemia confirmed by a biological examination, either microscopy or rapid diagnostic 
test plus 
2. Fever (axillary temperature ≥ 37.5 ° C) / history of fever and/or3 headache, myalgia, joint 
weakness, digestive disturbances, chills, body aches. 
If a participant was positive for parasitemia as confirmed by thick blood smear and had clinical 
signs or symptoms but these were not consistent with malaria, the individual was not classified 
as having clinical malaria but was given an alternate diagnosis.  If a participant was positive for 
parasitemia, did have clinical signs and symptoms consistent with malaria, but additionally had 
signs or symptoms referrable to a second diagnosis, the individual was assigned both diagnoses, 
e.g., malaria + rhinitis.  If parasitemia was present in the absence of signs or symptoms, the 
diagnosis “asymptomatic parasitemia” was assigned. All of these assignments were collected as 
 
3 French language version uses “avec” (with) but this is interpreted in practice as “and/or” meaning that fever is not 
required for a diagnosis. 

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“adverse events (Aes)” and visits to the clinic where an AE was identified were called “AE 
visits.” 
To address the Agency’s recommendation for selecting a parasitemia threshold, we examined the 
subset of data from the Community Survey study restricted to 6- to 10-year-olds living in 
Bancoumana, our proposed study site.  This included 277 children who were seen on average 
20.3 times (range: 1 to 43) totaling 5626 clinical visits. Of those, 221 children had at least one 
AE visit, where they were either positive for parasites or classified as having some diagnosis, 
including clinical malaria (“malaria”). On average, each AE child had 7.3 AE visits (range: 1 to 
23).  There were 21 cases where one AE visit was assigned two diagnoses. In 15 of those cases, 
one of the diagnoses was clinical malaria. The most common presentation of two diagnoses was 
malaria plus rhinitis. Thus, this dataset is appropriate to assess parasitemia thresholds and their 
potential impact on mis-identifying acute malaria based on symptoms that may arise from other 
non-malarial illnesses. 
For the 277 children in the 6- to 10-year-old age group, 5990 blood smears were collected, of 
which 964 smears showed detectable parasitemia in 195 unique individuals. Of the 964 positive 
smears, 582 (60.4%) occurred without reported symptoms, 382 (39.6%) were associated with at 
least one symptom, 370 were reported as malaria, 10 were reported as another diagnosis, and 15 
were reported as malaria + another diagnosis. Additionally, 5026 child visits were negative for 
parasitemia by RDT and/or smear. Of those, 4419 (87.9%) occurred without any reported 
symptoms, and 607 (12.1%) were associated with at least one symptom.  
The association between parasitemia density (x-axis) and axillary temperature (y-axis) in the 370 
AE visits (165 children) classified with a diagnosis of malaria is shown in Figure 9. Although 
temperature was weakly but significantly associated with parasite density in these children 
(Pearson R=0.18, p=0.001), many children with malaria did not have fever at both high and low 
parasite densities. Fever was relatively less common in these non-malaria cases. A significant 
number of children with no parasitemia also had fever. 

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Figure 9: Pattern of Fever and Parasites 
 
As described below, we used these data to identify a parasitemia threshold selected to increase 
the specificity with which clinical malaria could be diagnosed in this population without 
significant loss of sensitivity. Two approaches were used. The first was to generate a Receiver 
Operator Characteristics (ROC) Curve using data from all clinic visits with a diagnosis including 
the diagnosis of asymptomatic parasitemia. The second was to calculate the fraction of fevers 
that could be attributed to the presence of parasitemia, using data from all clinic visits. We 
selected fever because in our proposed clinical trial, the presence of fever will be a component of 
the case definition for clinical malaria. 
ROC Curve: Clinical malaria diagnoses and parasitemia measurements from 1604 AE visits over 
the entire study period were inputs to the R ROC function (package ROC [1]) to generate an 
ROC curve (Figure 10).  By definition of ROC, the parasitemia threshold corresponding to the 
point on the curve that is farthest from the X=Y diagonal represents the optimal yes/no cut point 
discriminant value. The optimal yes/no cut point was calculated to be 1740 parasites/uL, 
suggesting that if no criteria other than parasitemia is available for making a clinical malaria 
diagnosis, then only children presenting with at least 1740 P/uL should be called clinical malaria, 
and those with lower levels should be considered to be asymptomatic. The area under this curve 
was calculated to be 0.944 indicating that the 1740 P/uL threshold will correctly identify 94.4% 
of visits. The ROC analysis was repeated separately for each of the 2018 and 2019 malaria 

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seasons (July-December), using 421 and 492 AE visits respectively, and the cut points were 2500 
and 1060 P/uL, respectively. 
Figure 10: Acute Malaria vs Parasite Density 
 
Fever Attributable Fraction (AF): Fever occurred in child visits with and without parasitemia, 
and parasitemia occurred in child visits with and without fever, but fever was most commonly 
observed in visits diagnosed as clinical malaria, allowing the calculation of what fraction of 
fevers could be attributed to malaria. To investigate the relationship between parasitemia and 
clinical malaria, a series of Attributable Fraction (AF) calculations were performed, using a 
range of parasitemia thresholds (densities of 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 
20,000, 50,000, 100,000, 200,000 and 500,000 parasites/uL), to assess how changing the 
parasitemia threshold changes the fraction of fevers attributable to clinical malaria. The AF was 
calculated as from [2]: AFe = (CIe – CIu) / CIe where CIe is the cumulative incidence of the 
exposed group and CIu is the cumulative Incidence of the unexposed group. For both groups, the 
cumulative incidence is calculated as the proportion of child visits with fever given the number 
of child visits in that group.  Each child visit is grouped as being either exposed or unexposed, 
using the visit parasitemia relative to the threshold value – visits with parasite density at or above 
the threshold were called exposed, while visits with no parasites or density below the threshold 
were called unexposed. 

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Figure 11: Attributable Fraction using Fever as a Symptom of Diseases 
 
AF peaked at 5000 parasites/uL but was relatively flat from about 1000 to 100,000 parasites/uL.  
Based on this plateau shape, a threshold of 1000 parasites/uL appeared to be a reasonable cut-off, 
as higher thresholds did not significantly improve AF yet would significantly diminish the 
number of malaria cases and weaken the power of a clinical study.  
Selection of Parasite Density Threshold for the Proposed Study: The RTS,S Phase 3 clinical trial 
used a threshold of 5000 parasites/uL, which appeared optimal to increase the specificity of the 
case definition for malaria in the infants and young children enrolled in that study.  Because the 
densities of parasitemia in malaria endemic areas decrease progressively with duration of 
exposure and increasing age, due to the acquisition of naturally acquired immunity, the densities 
recorded in older children are lower, and in adults lower still.  For example, in a cohort of 
Ghanaian adults who were followed longitudinally for new Plasmodium falciparum infection 
following drug clearance of parasitemia, the geometric mean parasite density in P/uL on first 
symptomatic parasitemia was only 451 for men (range 171 to 1190) and 347 for women (range 
131 to 921). The geometric mean parasite density for asymptomatic parasitemia was 2- to 3-fold 
lower – 129 (range 96–169) for men and 162 (range 118–223) for women. These data would 
suggest a cut-off in the range of 200-300 would be appropriate for Ghanaian adults.  
Our study is enrolling 6- to 10-year-olds in Mali. It is thus not surprising that our ROC curves 
suggest an optimal cut-off between 1000 and 2500, midway between 5000 (appropriate for 
infants) and 200-300 (appropriate for adults). Our AF analysis yielded similar results and 

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provided a rationale for moving the threshold “to the left” on Figure  in order to reduce the false 
negative rate without significantly increasing the false positive rate. Thus, we propose to use a 
density of 1000 parasites/uL as a cut off in our study for the primary case definition of clinical 
malaria.  
Relative Independence of Vaccine Efficacy from Parasite Density Thresholds: Although parasite 
density thresholds have been used in the past as described for RTS,S – to optimize sensitivity 
and specificity of the primary case definition – key field trials of malaria vaccines have shown 
that VE may not be significantly affected by the threshold selected. The data supporting this 
contention come from the RTS,S Phase 3 trial conducted at 11 sites across Africa [3] and the 
recently published Phase 2b trial of R21 conducted in Burkina Faso [4].  The outcome variable 
for both trials was clinical malaria, defined as presence of axillary temperature of 37·5°C or 
higher and Pf parasite density of more than 5000 asexual forms per μL. A secondary analysis 
was also performed in each trial using any density of parasitemia identified on thick blood smear 
(>0 P/uL) rather than >5000 P/ul as the threshold for diagnosing clinical malaria.  The data show 
that whichever density was selected, VE was essentially identical. This finding is perhaps not 
surprising when one considers that VE is calculated by comparing the rate of clinical malaria in 
vaccine and placebo groups, a relative difference that might not be affected by changes in the 
specificity of the case definition as long as the likelihood of false positives and false negatives is 
the same in both vaccine and placebo groups. 
In summary, although this trial is following FDA’s recommendation and has a primary case 
definition that requires a parasite density >1000 P/uL in the 6-10-year-old target population, we 
will also assess a secondary case definition for parasite density >0 P/uL, and based on the results 
just presented, we are expecting that the outcome will be similar. 
 
6. STUDY POPULATION 
6.1 
INCLUSION CRITERIA 
In order to be eligible to participate in this study, an individual (or their legal guardians as 
applicable) must meet all of the following criteria: 
1. Parent(s) or guardian(s) willing and able to provide consent prior to initiation of any 
study procedures 
2. Stated willingness of parent(s) or guardian(s)  to comply with all study procedures and 
availability for the duration of the study  
3. Malaria comprehension exam completed by parent(s) or guardian(s) and passed with a 
score of ≥80% or per investigator’s discretion 
4. Healthy children 6-10 years of age at enrollment (inclusive) 
5. Parent(s) or guardian(s) are able to provide proof of identity to the satisfaction of the 
study clinician completing the enrollment process 
6. Willing to have blood samples stored for future research 
6.2 
EXCLUSION CRITERIA 
An individual who meets any of the following criteria will be excluded from participation in this 
study: 

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1. Medical, behavioral, cognitive, or psychiatric disease that in the opinion of the 
investigator affects the ability of the participant’s parent and/or legal guardian to 
understand and comply with the study protocol 
2. Menstruating females (in order to avoid cultural implications of further assessing 
pregnancy potential i.e. sexual activity in this age group) 
3. Hemoglobin (Hgb), WBC, absolute neutrophils, and platelets outside the local 
laboratory-defined limits of normal and ≥ Grade 2 (subjects may be included at the 
investigator’s discretion for ‘not clinically significant’ abnormal values) 
4. Alanine transaminase (ALT) or creatinine (Cr) level above the local laboratory-defined 
upper limit of normal and ≥ Grade 2 (subjects may be included at the investigator’s 
discretion for ‘not clinically significant’ abnormal values) 
5. Infected with human immunodeficiency virus (HIV), hepatitis B, or hepatitis C 
6. Sickle cell disease by history 
7. Taking or planning to take seasonal malaria chemoprophylaxis 
8. Clinically significant abnormal electrocardiogram (ECG) such as abnormal QTc 
9. History of receipt of the following: 
o Investigational malaria vaccine in the last 2 years 
o Immunoglobulins and/or blood products within 6 months of enrollment 
o Investigational product within 3 months of enrollment 
o Chronic (≥14 days) oral or IV corticosteroids (excluding topical or nasal) at 
immunosuppressive doses (i.e., prednisone ≥20 mg/day or equivalent) or 
immunosuppressive drugs within 30 days of enrollment 
o Live vaccine within 30 days of enrollment 
o Killed vaccine within 14 days of enrollment or planned receipt of a killed vaccine 
within 14 days of scheduled vaccination 
10. Known medical problems: 
o Pre-existing autoimmune or antibody-mediated diseases (e.g. systemic lupus 
erythematosus, rheumatoid arthritis, multiple sclerosis, Sjögren’s syndrome, or 
autoimmune thrombocytopenia) 
o Severe asthma (defined as asthma that is unstable or required emergent care, 
urgent care, hospitalization, or intubation during the past two years, or that has 
required the use of oral or parenteral corticosteroids at any time during the past 
two years) 
o Immunodeficiency disorder 
o Asplenia or functional asplenia 
o Diabetes 
o Deep venous thrombosis or thromboembolic event 
o Seizures (exception is simple febrile seizures during childhood) 
11. Evidence of clinically significant neurologic, cardiac, pulmonary, hepatic, endocrine, 
rheumatologic, autoimmune, hematological, oncologic, or renal disease by history, 
physical examination, and/or laboratory studies  
12. History of a severe allergic reaction or anaphylaxis following previous vaccinations or 
medications 
13. Known allergies or other contraindications against: artemether-lumefantrine, PfSPZ 
Vaccine, or human serum albumin  

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14. Current or planned participation in an investigational vaccine study through the last 
required protocol visit. 
15. Other condition(s) that, in the opinion of the investigator, would jeopardize the safety or 
rights of a participant participating in the trial, interfere with the evaluation of the study 
objectives, or would render the subject unable to comply with the protocol 
6.3 
INCLUSION OF VULNERABLE PARTICIPANTS 
 Participation of family members of study team members 
Family members of study team members may be enrolled in this study as this population meets 
the study entry criteria.  Neither participation nor refusal to participate as a subject in the 
research will have an effect, either beneficial or adverse, on the participant’s family members 
employment. 
Every effort will be made to protect participant information, but such information may be 
available in medical records and may be available to authorized users outside of the study team 
in both an identifiable and unidentifiable manner. 
6.4 
INCLUSION OF PREGNANT WOMEN, FETUSES OR NEONATES 
This trial will not include pregnant women, fetuses, or neonates as they are outside the targeted 
population age range. 
6.5 
LIFESTYLE CONSIDERATIONS 
Not applicable.  
6.6 
SCREEN FAILURES 
Screen failures are defined as participants who consent to participate in the clinical trial but are 
not subsequently enrolled in the study due to failure to satisfy eligibility criteria. A minimal set 
of screen failure information is required to ensure transparent reporting of screen failure 
participants, to meet the Consolidated Standards of Reporting Trials (CONSORT) publishing 
requirements and to respond to queries from regulatory authorities. Minimal information 
includes demography, screen failure details, eligibility criteria, and any serious adverse event 
(SAE). 
Individuals who do not initially meet the criteria for participation in this trial (screen failure) 
because of an abnormal vital sign, laboratory, or ECG finding that may be transient (e.g., 
secondary to acute illness) may be rescreened at the principal investigator’s (PI’s) discretion. 
Rescreened participants will maintain the same participant number as for the initial screening. 
6.7 
STRATEGIES FOR RECRUITMENT AND RETENTION 
 Costs 
There is no cost to subjects for taking part in this trial. 
 Compensation 
Subjects will be given in kind (such as rice and/or millet) or cash equivalent, that can be given in 
multiple installments if preferred (e.g. for screening, at each vaccination visit, and periodically 
through the natural human malaria infection [NHMI] period), to compensate for the time taken to 
come to the study clinic for study-related visits. Preferred compensation will be decided in 

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is dispensed into vials that are stored in liquid nitrogen vapor phase (LNVP) at -150°C to -
196°C. All the procedures are described in detail in the cross-referenced IND 
. 
Diluent 
The diluent for PfSPZ Vaccine is composed of phosphate-buffered saline (PBS) and about 1% 
human serum albumin (HSA). HSA is a licensed product which is approved for parenteral, IV 
administration to humans and is purchased by Sanaria Inc. Diluent is released with a Certificate 
of Analysis (CoA), and is placed on a stability program. Vials of diluent will be shipped to the 
clinical site.  
PBS that will be used was manufactured in compliance with GMP by Sanaria Inc. (9800 Medical 
Center Dr., Rockville, MD, USA). A Certificate of Analysis (CoA) is generated for each lot of 
PBS that is released for use in clinical studies. In addition, the PBS lots are placed on stability.  
HSA is a licensed product which is approved for parenteral, IV administration to humans and is 
purchased by Sanaria Inc. The HSA is repackaged at Sanaria. Repackaged HSA is released with 
a CoA. 
0.9% Sodium Chloride Normal Saline (NS) (Placebo) 
Sterile isotonic 0.9% sodium chloride (normal saline (NS)) will be procured in the US and 
shipped to Mali at ambient temperature. Like the vaccine product, NS is a clear liquid, making it 
indistinguishable when drawn up into a syringe. NS will be used as a placebo, rather than a 
comparator vaccine, as currently there are no licensed vaccines available as IV formulations. 
Artemether Lumefantrine (AL) 
Artemether-lumefantrine (AL) will be administered approximately 1-2 weeks prior to the first 
and perhaps third dose of PfSPZ Vaccine or normal saline. AL will be purchased from a licensed 
pharmacy in Mali from a single manufacturer and lot number.  
 Dosing and Administration 
PfSPZ Vaccine + Diluent 
The dose of PfSPZ Vaccine administered to vaccinees is 9x105 PfSPZ in a range 0.25 to 0.5 mL 
of diluent. The dose of NS is likewise 0.5 mL. Each product is a clear, non-viscous liquid that 
has no perceptible color or odor. Each product is received by the blinded clinical vaccine 
administration team in syringes that appear identical in every way with labels and documents that 
ensure blinding of the clinical vaccine administration team but clear tracking of volunteers and 
syringes. Each is administered by DVI. PfSPZ Vaccine, diluent and NS will be shipped by 
Sanaria to the study site in Mali, where it will be received by Pharmaceutical Operations staff. 
The syringes are prepared by the unblinded Pharmaceutical Operations team following SOPs. 
The preparation area is not accessible by clinical staff during syringe preparation or at any time 
when the unblinded randomization list is being reviewed. After preparation, the filled syringes 
are provided to the clinical team for DVI. The Pharmaceutical Operations team does not interact 
with study participants. The maximum allowable time between thawing of a PfSPZ Vaccine vial 
and injection is 30 minutes.  
To perform DVI, a superficial vein in the arm or hand is located, prepped for venipuncture, a 
restricting band is placed on the arm, the needle is inserted into the vein, the plunger is 

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withdrawn slightly to cause blood flashback thereby confirming successful venous entry, the 
restricting band is removed, and the contents of the syringe are injected over a few seconds. 
Hemostasis is achieved by immediate direct pressure after withdrawing the needle. The syringe 
and needle are discarded into a sharps/biohazard container. The subject is then transferred to the 
observation area. 
During administration of PfSPZ Vaccine, advanced-life-support drugs and resuscitation 
equipment will be immediately available for the management of anaphylaxis. The study staff 
administering PfSPZ Vaccine will wear gloves. The subject will stay in the clinical area for at 
least 30 minutes after vaccine administration. 
0.9% sodium chloride Normal Saline (NS) (Placebo) 
NS will be injected by needle and syringe into a peripheral vein by a qualified member of the 
clinical team. NS will be prepared such that a defined volume will appear indistinguishable from 
PfSPZ Vaccine.   
During administration of NS, advanced-life-support drugs, trained personnel, and resuscitation 
equipment will be immediately available for the management of anaphylaxis. The study staff 
administering NS will wear gloves. The subject will stay in the clinical area for at least 30 
minutes after vaccine administration. 
Artemether Lumefantrine 
All antimalarial medications used for the study will be maintained at the study site and 
administered by direct observational therapy (first dose) by a study team member.  AL (and other 
antimalarials) will be purchased from commercial sources as detailed above and provided by the 
MRTC study team to subjects for dosing around vaccination only. 
7.2 
PREPARATION/HANDLING/STORAGE/ACCOUNTABILITY  
 Acquisition and Accountability. 
PfSPZ Vaccine 
PfSPZ Vaccine is manufactured and released for use by Sanaria Inc. Sanaria will supply the 
investigational product, diluent and NS placebo to the clinical site. Syringes will also be supplied 
by Sanaria Inc. The pharmacist at the study site will ensure that all investigational products are 
inventoried and stored as indicated in the relevant SOP. Accurate records will be maintained 
regarding receipt of PfSPZ Vaccine, diluent and NS, as well as syringes which include: date 
received, lot number, amount received and disposition.  
Sanaria will receive copies of document records in parallel regarding disposition of product, 
which include:  
• Participant identification number  
• Date and time PfSPZ Vaccine vial is thawed and time PfSPZ Vaccine is inoculated.  
• Volume injected 
• Lot number 
• Signature of the person preparing the syringe 
• Signature of the verifier 
• Signature of the person administering the injection  

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• Signature of the verifier 
Any partly used vials of PfSPZ Vaccine, NS, as well as empty vials, will be destroyed according 
to the applicable SOP and as determined by Sanaria. An inventory of the number of vials shipped 
versus used will be recorded. Unthawed and unopened vials of PfSPZ Vaccine and unused and 
unopened diluent vials and NS vials will be inventoried and returned to Sanaria. 
PfSPZ Vaccine is not a licensed product and must be distributed under an Investigational New 
Drug Application (IND) in accordance with FDA regulations. The product must be administered 
by or under the supervision of the principal investigator or a subinvestigator of the clinical study, 
with the exception of syringe preparation which is delegated to the Pharmaceutical Operations 
team. Sanaria personnel will provide oversight of the syringe preparation work performed by the 
Pharmaceutical Operations team. 
The clinical site must confirm that the vials of PfSPZ Vaccine have been transported and stored 
below -150°C according to SOP. Immediately prior to use, the cryovials will be thawed by 
trained Pharmaceutical Operations staff. 
All PfSPZ Vaccine vials that are used will be documented on inventory forms as well as 
documented disposition forms according to SOP. All unused PfSPZ Vaccine vials will be 
returned to Sanaria Inc. 
Diluent  
The Diluent is phosphate buffered saline (PBS) containing human serum albumin (HSA). 
Diluent will be provided to the clinical sites by Sanaria Inc. The clinical site must confirm and 
document that the vials of diluent have been transported and stored within specified ranges. All 
diluent vials that are used will be documented on inventory forms as well as documented 
disposition forms according to SOP. 
Diluent vial accountability will be maintained to document chain of custody from Sanaria Inc., to 
study site. An inventory to account for number of vials used will be recorded and kept in the 
study file. All unused diluent vials will be returned to Sanaria Inc. 
0.9% Sodium Chloride Normal Saline (NS) (Placebo) 
The clinical site will confirm and document that the vials of NS have been transported and stored 
within specified ranges.  
NS accountability will be maintained to document chain of custody from Sanaria Inc., to study 
site. An inventory to account for number of vials dispensed for each subject injection will be 
recorded and kept in the study file. All unused NS vials will be returned to Sanaria Inc. 
Artemether Lumefantrine 
Dosing of artemether lumefantrine will be used according to the package label.  
 Formulation, Appearance, Packaging, and Labeling 
PfSPZ Vaccine 
PfSPZ Vaccine is a suspension of aseptic, purified, metabolically active, non-replicating (live, 
radiation attenuated) cryopreserved Pf sporozoites (PfSPZ) formulated in cryoprotectant and 

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temperature probe according to Sanaria standard operating procedure (SOP). Receipt of the 
products will be documented on a tracking log by trained study staff according to Sanaria SOP. 
Diluent  
The Diluent is stored at ambient temperature within specifications (between 15°C to 30°C) that 
is monitored by a continuous data logger in a controlled access room. Receipt of the products 
will be documented on a tracking log by trained study staff according to Sanaria SOP. 
0.9% Sodium Chloride Normal Saline (NS) (Placebo) 
The NS is stored at room temperature in a controlled room per product standards. Each NS vial 
can either be single use or multi-use if used in a biological safety cabinet to maintain asepticity 
over a period of a few hours (i.e. the duration of vaccine preparation on a given day). 
Artemether Lumefantrine 
AL tablets will be maintained in the manufacturer’s original packaging and stored at the clinic 
under recommended storage conditions until prepared for dispensing. 
7.3 
MEASURES TO MINIMIZE BIAS: RANDOMIZATION AND BLINDING 
 Randomization 
Randomization will occur in blocks at 1:1 into vaccine or placebo arms. Randomization will be 
assigned at the time of first artemether/lumefantrine dose 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 (PfSPZ Vaccine or 
control) will be made available only to the study statistician and associated team members, 
pharmaceutical operations team/syringe preparers (at the start of the study), independent safety 
monitor (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 head of pharmaceutical operations. 
If a subject is given the wrong treatment at V1 due to an allocation error, the same vaccine will 
be administered at V2 and V3 so long as the error is identified before the timing of the next 
injection. In the situation where an eligible participant already assigned a randomization number 
becomes ineligible during the window between randomization number assignment and 
administration of V1 (e.g., as a result of new information about the participant, withdrawal from 
participation, or other factors), a replacement will not be assigned, and the randomization 
number will not be assigned to another participant. This occurrence is anticipated to be a rare 
event.  
 Blinding 
The study is double-blind (clinical staff and participants). Blinding extends to the parents and/or 
guardians of pediatric participants, to laboratory staff conducting clinical chemistry, hematology, 
and parasitology tests, and to laboratory staff 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. 

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 Unblinding 
Details of unblinding procedures are provided in the unblinding SOP. An overview is provided 
here.  
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 local safety monitor (LSM), 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.   
If pausing criteria are met based on blinded data, the following procedure will be followed, as 
described in Section 7.1.1. The principal investigator (who remains blinded) will provide the list 
of research subjects contributing to pausing criteria to the head of Pharmaceutical Operations or 
their designee, and will also inform the head of Pharmaceutical Operations how many of these 
research subjects have to be vaccine recipients (as opposed to NS recipients) in order to know if 
pausing criteria have truly been met. The head of Pharmaceutical Operations will then privately 
identify the treatment assignment of each participant on the list, and then inform the principal 
investigator if the pausing criteria have been met, but will not unblind the principal investigator 
with respect to individual participants.  
7.4 
CONCOMITANT THERAPY 
For this protocol, a prescription medication is defined as a medication that can be prescribed only 
by a properly authorized/licensed clinician. Medications to be reported in the Case Report Form 
(CRF) are concomitant prescription medications, over-the-counter medications and supplements. 
8. STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL  
8.1 
SUSPENSION OF STUDY INTERVENTION 
In this section, study “pause” indicates that immunizations will be briefly stopped for a particular 
group to allow consultation with the Sponsor and DSMB. Immunizations may resume 
immediately based on (1) the decision of the Sponsor, taking into account any recommendations 
of the DSMB and (2) concurrence by the principal investigator. IRBs and regulatory authorities 
will be notified but need not be involved in the decisions. Alternatively, if a study has been 
paused, the Sponsor may decide to impose a “safety hold,” implying the need for wider 
consultation, potentially including IRBs and regulatory agencies, depending on the 
circumstances. A pause or a safety hold may be restricted to a particular subgroup, depending on 
the circumstances.  
The study intervention will be paused if specified criteria in Section 7.1.1 are met (see below). 
Discontinuing vaccinations does not mean discontinuation of study visits or procedures related to 
safety. Study procedures related to research assays, such as collecting samples for immunology 
tests, may be continued if participants and their parents/guardians agree.  
7.1.1 Pausing Criteria, Unblinding and Safety Hold 

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As described in Background and Risk sections above, PfSPZ Vaccine has been safe and well 
tolerated in three randomized, double-blind, normal saline placebo-controlled trials in African 
children of the same or similar ages as planned for enrollment in this trial: BSPZV2 in Tanzania 
(3), KSPZV1 (Part 1) in Kenya (31), and EGSPZV2 in Equatorial Guinea (Jongo et al, 
unpublished data).  In these trials, there was no difference in the rate or severity or nature of AEs 
or laboratory abnormalities compared to the normal saline controls. PfSPZ Vaccine also 
appeared to be safe and well tolerated in a fourth trial in 1-to-12 year olds underway in 
Lambaréné Gabon based on blinded data (LaSPZV1). Finally, it was safe and well tolerated in a 
large trial of Kenyan infants, KSPZV1 (Part 2), where approximately 80 infants received three 
doses of 1.8x106 PfSPZ at 8 week intervals (Oneko et al, submitted) – twice the dose planned for 
7-to-10-year-olds in the current trial –and where similar numbers of infants received three doses 
of 9.0x105 or 4.5x105 PfSPZ. Thus, it appears unlikely that safety issues will arise in the current 
trial. One note of caution, however, is that this will be only the second study where children have 
been administered a multi-dose priming regimen – two doses one week apart – the first being the 
Lambaréné trial, which enrolled about 128 children to receive PfSPZ Vaccine. It will therefore 
be important to continue careful recording of adverse events and to check safety labs after the 
second dose of vaccine. 
Our pausing rules are established against this background.  Since no adverse events are expected 
to occur at a rate higher than the background rate in the population, a small cluster of adverse 
events in vaccinees could signal a safety issue. For this reason, we have established the following 
two criteria. If either criterion is met, the clinical team will contact the Sponsor, which will 
contact the DSMB to review the events in question: 
• Five percent or more of the vaccinees enrolled at any time point in one arm (calculated as 
5% of the vaccinees in the full study roster enrolled to date, as long as there are at least 
three subjects with the AE) experience the same severe (grade 3) AE during the periods 
of AE collection deemed possibly, probably or definitely related to study product 
administration (PfSPZ Vaccine or NS), or experience the same grade 3 laboratory 
abnormality on day V2 + 14 or day V3+14. Grade 3 AEs deemed possibly, probably or 
definitely related to malaria (as opposed to PfSPZ Vaccine or to NS) will not count 
toward meeting this criterion. The site, where clinical staff are blinded, can only monitor 
total populations (vaccine plus placebo recipients) and if a 5% threshold is reached, 
unblinded study staff (e.g., the head of Pharmaceutical Operations) will need to confirm 
that the total indeed constitutes 5% of vaccinees. Note that we have considered using 
Grade 2 rather than Grade 3 laboratory abnormalities, but drops in hemoglobin, 
neutrophils, platelets and sometimes other blood count indices meeting Grade 2 criteria 
for severity are common in African pediatric populations. 
• One participant experiences an SAE deemed possibly, probably or definitely related to 
study product. This criterion will not be met if it turns out that the participant received 
NS. SAEs related to malaria or to antimalarial treatment which are expected, will be 
reported expeditiously but will not fulfill this criterion. If the SAE is unexpected with 
respect to malaria or antimalarial treatment, it will fulfill the criterion.  
If either of these criteria appear to be met by ongoing surveillance at the site, the principal 
investigator will engage the head of Pharmaceutical Operations on site, or his/her designee, to 

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follow unblinding procedures (see Section 6.3.3 for a description). To meet the criteria, the 
subjects must actually have received PfSPZ Vaccine and not NS placebo.   
After confirmation that a pausing criterion has been met, the Sponsor will contact the DSMB to 
describe the circumstances of the event(s). After an email discussion or, if recommended by the 
DSMB, an ad hoc teleconference, the DSMB, which will include the local safety monitor as a 
voting member, will make a written recommendation about whether or not the study should be 
unpaused, remain paused pending more data and further review, or placed on full safety hold, 
and whether or not the affected participants or their caregivers should be unblinded and notified 
of their treatment assignment. Based on the DSMB recommendation, a decision will be made by 
the Sponsor and principal investigator to either unpause the study and continue administration of 
investigational product in the affected group, to pause the study temporarily in the affected group 
to allow collection of more data about the triggering event(s) or other safety data, or to institute a 
full safety hold for the affected group or groups. IRBs (excepting IRBs providing administrative 
review) and regulatory authorities will be promptly informed of a study pause or of a full safety 
hold. Their permission will not be required to un-pause the study, but will be to reverse a safety 
hold.  
Irrespective of the pausing criteria, the Sponsor, local safety monitor, principal investigator or 
applicable ethical committees or regulatory agencies may decide to pause the study, or to put the 
study on safety hold based on any constellation of AEs even if the 5% threshold is not reached 
and even if the AEs do not reach Grade 3 severity, or based on any other legitimate reason, such 
as AEs occurring in another study, product quarantine, manufacturing issues, expiry of 
permissions, out-of-specification stability test results, etc. For a full safety hold to be lifted, there 
must be written concurrence by the entity or entities that initiated the hold that the study can 
resume, and this may include regulatory agencies in addition to the two IRBs of record.  
7.1.2 Temporary Hold of Vaccination of Individual Participants 
The administration of PfSPZ Vaccine or NS to a study participant may be temporarily put on 
hold, based on clinical judgement, for the following reasons:  
1. The participant is ill on the day of vaccination (administration may proceed in the case of 
a minor illness, based on the investigator's clinical judgment). 
2. The participant has an abnormal laboratory value determined to be clinically significant 
and meriting delay in immunization, based on the investigator’s clinical judgment.  
3. The participant has malaria or has taken medication for malaria or for another acute 
illness that may affect response to PfSPZ Vaccine. Participants with malaria will be 
treated with an antimalarial medication and administration of PfSPZ Vaccine will be 
postponed for a period of time after termination of treatment that is appropriate to allow 
sufficient washout of the drug.  
4. The participant has received a non-live vaccine during the previous 14 days or a live 
vaccine during the previous 28 days (other than the investigational product) or three or 
more of any type of vaccine in the preceding four months.  
5. At the request of the participant. 

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6. On the advice of the principal investigator, local safety monitor, IRB, regulatory 
authority, DSMB or sponsor. 
7. On the advice of one of the investigators, as long as the principal investigator concurs. 
Vaccination will be rescheduled at the earliest possible time within the acceptable window of the 
originally scheduled vaccination, or outside that window if there is no other option.  
8.2 
DISCONTINUATION OF STUDY INTERVENTION 
Discontinuation from receipt of study products (AL, PfSPZ Vaccine or normal saline) for an 
individual does not mean discontinuation from the study, and remaining study procedures should 
be completed as indicated by the study protocol.  If a clinically significant finding is identified 
after enrollment (including, but not limited to changes from baseline), the investigator or 
qualified designee will determine if any change in participant management is needed. Any new 
clinically relevant finding will be reported as an adverse event (AE). 
The data to be collected at the time of study intervention discontinuation will include continued 
study procedures as outlined in the protocol. If no further vaccinations are to be administered, 
safety labs related to vaccination schedules may be deferred at the investigator’s discretion.  
Participants are free to withdraw from participation in the study at any time upon request. 
An investigator may discontinue or withdraw a participant from the study for the following 
reasons: 
• Completion of study intervention 
• New diagnosis which requires discontinuation of the study intervention 
• If any clinical adverse event (AE), laboratory abnormality, or other medical condition or 
situation occurs such that continued participation in the study would not be in the best 
interest of the participant 
• Investigator discretion 
• Participant unable to receive AL dosing or subsequent vaccinations more than 7 days 
from proposed study window.  
• Significant study intervention non-compliance  
• If the participant meets an exclusion criterion (either newly developed or not previously 
recognized) that precludes further study participation 
• Subject has completed the study follow-up period 
• Death 
• Screen Failure 
8.3 
PARTICIPANT DISCONTINUATION/WITHDRAWAL FROM THE STUDY 
Participants are free to withdraw from participation in the study at any time upon request.  
The reason for participant discontinuation or withdrawal from the study will be recorded on the 
End of Study Case Report Form (CRF). Replacement of a subject may occur in the case that a 
parent/guardian signs the informed consent and the subject is randomized but the subject does 
not go on to receive the study intervention. In the case where a parent guardian/signs the 
informed consent, the subject is randomized, receives the first dose of PfSPZ Vaccine and the 

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subject is withdrawn or the subject or parent/guardian decides to withdraw, no subject 
replacement may occur. 
8.4 
LOST TO FOLLOW-UP 
A participant will be considered lost to follow-up if he or she fails to return for scheduled visits 
and the parent/guardian is unable to be contacted by the study site staff.  
The following actions must be taken if a participant fails to return to the clinic for a required 
study visit: 
• The site will attempt to contact the parent/guardian and reschedule the missed visit within 
the specified visit window and counsel the parent/guardian on the importance of 
maintaining the assigned visit schedule and ascertain if the parent/guardian (and if 
applicable, the subject) wishes to and/or should continue in the study. 
• Before a participant is deemed lost to follow-up, the investigator or designee will make 
every effort to regain contact with the parent/guardian (where possible, 3 telephone calls 
and, if necessary, a home visit to the participant’s last known address). These contact 
attempts should be documented in the participant’s study file.  
• Should the parent/guardian continue to be unreachable, he or she will be considered to 
have withdrawn the subject from the study with a primary reason of lost to follow-up. 
 
9. STUDY ASSESSMENTS AND PROCEDURES 
9.1 
SCREENING PROCEDURES 
 Screening activities performed prior to obtaining informed consent 
Minimal risk activities that may be performed before the subject has signed a consent include the 
following:  
• Email, written, in person or telephone communications with the parents/guardians of 
prospective subjects.  
• Review of existing medical records. 
 Screening activities performed after a consent for screening has been signed 
The following activities will be performed only after the parent/guardian of the subject has 
signed the consent form for this study. Assessments performed at outside facilities or on another 
MRTC or NIH protocol within the timeframes below may also be used to determine eligibility 
once a participant has signed the consent. 
All screening tests and procedures must be performed within 56 days prior to enrollment (AL 
dosing), unless a time period is specifically mentioned.  
• Malaria comprehension exam completed by the parent/guardian, wrong questions 
reviewed, and confirmed passing of ≥80% 
• Demographic information will be obtained from interview of the subject’s parent/guardian 
• Complete past medical history will be obtained by interview of subjects’ parent/guardian 
• Eligibility criteria will be reviewed with subjects’ parent/guardian 

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• All concomitant medications and vaccinations taken within one month prior to screening 
will be reviewed.  
• Planned vaccinations and expected timing  
• Result of laboratory tests will be reviewed and recorded 
• Complete physical exam will be completed 
• Vital signs will be obtained, including height, weight, temperature, heart rate, and blood 
pressure 
• Blood collected for baseline laboratory evaluation, including malaria assessment 
• Electrocardiogram completed  
9.2 
ENROLLMENT AND ARTEMETHER LUMEFANTRINE VISIT 
• Prior to enrollment, all screening procedures will be confirmed completed 
• Confirmation of the willingness to participate of the subject and the subject’s 
parent/guardian  
• Interim clinical (focused physical exam, medical) history will be obtained with updates 
on any changes since previous clinic visit or contact 
• Eligibility criteria will be reviewed and confirmed by study PI 
• Blood draw for research and clinical labs, including malaria assessment 
• AL dosing started (first dose in clinic by directly observed administration) 
9.3 
VACCINATION VISITS 
• Confirmation of the willingness to participate of the subject and the subject’s 
parent/guardian  
• Interim clinical (focused physical exam, medical) history will be obtained with updates 
on any changes since previous clinic visit or contact 
• Eligibility criteria will be reviewed and confirmed by study PI 
• Blood draw for research and clinical labs, including malaria assessment 
• Vaccination completed  
• Post vaccination observations and evaluation completed 
9.4 
SAFETY FOLLOW-UP VISITS 
• Confirmation of parent/guardian of subject’s continued willingness to participate 
• Interim clinical (focused physical exam, medical) history will be obtained with updates 
on any changes since previous clinic visit or contact 
• Blood draw for research and clinical labs, including malaria assessment 
• Solicited local and systemic reactogenicity completed (through 7 days post vaccination) 
9.5 
EFFICACY ASSESSMENTS 
• Confirmation of parent/guardian of subject’s continued willingness to participate 
• Interim clinical (focused physical exam, medical) history will be obtained with updates 
on any changes since previous clinic visit or contact 
• Blood draw for research and clinical labs, including malaria assessment 

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9.6 
UNSCHEDULED VISITS 
• Interim clinical (focused physical exam, medical) history will be obtained with updates 
on any changes since previous clinic visit or contact 
• Blood draw for clinical labs, as indicated, and research labs (with unscheduled blood 
smear positive visit) 
9.7 
CLINICAL EVALUATIONS 
Complete physical examination: (e.g., height and weight, heart rate, organ systems, or other 
functional abilities).  
Electrocardiograms (EKGs): Electrocardiograms (12-lead ECGs) will be performed during 
screening and as needed throughout the study by the study site team in Mali and read by a Mali 
cardiologist if needed. Subjects with QT interval (QTc) > 460 ms will be excluded as AL may 
prolong QTc. Subjects with clinically significant abnormalities will also be excluded from the 
study. 
Malaria blood smears: The gold standard for malaria diagnosis and evaluation of VE endpoints 
is the detection of malaria parasites on Giemsa-stained thick blood films. Blood smears are 
prepared in duplicate according to standard malaria challenge procedures and evaluated by 
trained study microscopists, and the results reported to the study PI. At least 0.5 µL are scanned 
for the presence of malaria parasites. This method allows for detection of a parasite density of 
approximately 2 parasites/µL and early diagnosis, often before subjects become symptomatic for 
malaria. Slides are considered positive if at least one unambiguous P. falciparum parasite per 
slide are identified and confirmed by a second microscopist.  
When venipuncture is performed, thick blood smears will be prepared from the blood remaining 
in the IV cannula or needle, or (at time points when no IV blood collection is planned) from a 
finger prick sample. The smears will be examined microscopically. 
Thick blood smears will be used for diagnosis throughout the study. 
Passive and active case detection: Contact information for the study clinic and principal 
investigator will be provided to the parent/legal guardian of all participants so they will be able 
to easily schedule a clinical evaluation for any symptoms of concern experienced by the study 
participant.  Study participants are also welcome as walk-ins at the study clinic. Parents or 
guardians will be actively encouraged throughout the trial to report to the clinic whenever a 
participant is ill.  
In addition, active outreach to study subjects will occur no less frequently than every two weeks 
during the malaria surveillance period. Parents / participants will be questioned at these visits for 
history of malaria symptoms including subjective fever, and body temperature will be assessed at 
each encounter (see Table 13).  
Blood smears will be collected and examined in the case of any participant presenting with signs 
or symptoms consistent with malaria. Blood smears will additionally be collected every four 
weeks. However, the results of blood smears collected from asymptomatic participants will be 
reported in the clinical database after surveillance is complete so as not to interfere with the 
detection of malaria with symptoms (clinical malaria episodes).   

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A short questionnaire about bednet use will be filled out at each clinic visit to document this 
variable, that could affect study results. 
 
9.8 
UNSCHEDULED BLOOD SMEAR POSITIVE VISITS 
After receipt of at least one vaccination, if a subject has detected Pf malaria parasites on 
peripheral thick blood smear (scheduled or unscheduled visits), they will be asked to the clinic to 
provide an additional blood sample within 48 hours for the following: 
• 0.5 mL EDTA microtainer for whole blood ex-vivo assays if sample not already 
obtained within the last two days (i.e. at the time of the positive blood smear) 
• 4 mL EDTA tube to obtain RNA and DNA for the study of host and parasite 
transcriptome (RNA) and parasite genotype (DNA) and to obtain plasma for 
proteomics studies  
o If 4mL EDTA sample has been collected in the last 28 days for this purpose, it 
can be deferred at the investigator’s or subject’s discretion.  
• 0.5 mL for qPCR if sample not obtained within the last two days with the positive 
blood smear 
N.B.: scheduled smears on asymptomatic during the NHMI phase will be read and/or 
reported retrospectively and smear positive visits will not apply  
The unscheduled blood smear positive blood draw does not need to be completed with every 
positive blood smear and subjects have the right, as with every blood draw, to refuse to return for 
this additional blood draw.  While subjects may be asked back to the clinic after at least one 
vaccination, preference for unscheduled blood smear positive blood draws are for first positive 
blood smear post vaccination #3, subsequent positive blood smear after treatment with anti-
malarial treatment (>14 days after treatment), and new positive blood smear after having a 
negative blood smear >28 days.  If repeat blood smears are done to monitor the clearance of 
parasitemia following treatment, these additional labs will not be drawn. 
9.9 
MALARIA QPCR 
While detection of parasites on thick blood smears remains the most common primary endpoint 
in human challenge trials, both PCR- and nucleic acid sequence-based amplification (NASBA)- 
based methods have been increasingly used to support blood smear data in malaria vaccine trials 
(32, 33). These research molecular assays have significantly increased sensitivity for detection of 
P. falciparum blood-stage infection approaching 20 parasites/mL, often resulting in diagnosis 2-
4 days earlier than by paired thick blood smears (32, 34, 35). Quantification of parasite density 
by these methods allows evaluation of parasite growth curves for assessing the utility of partially 
effective vaccine candidates. LMIV has also developed a research qPCR that detects 18s of Pf 
with a detection limit of at least 20 parasites/mL that will be used during the study for 
comparison to traditional thick blood smears.  
Pf qPCR may be performed from all scheduled visits with a malaria blood smear noted to capture 
infections that remain below the detection limit for microscopy. For subject convenience, a 

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finger prick sample can be used for both preparation of the microscopy slide and for DNA 
preservation. 
In the case of positive blood smears that constitute a primary or secondary endpoint for a 
participant, an FDA qualified qPCR assay will be performed by the laboratory of Dr. Sean 
Murphy, University of Washington, as confirmatory. 
Immunological Assays 
As indicated in the objectives, assays will be conducted to assess immunogenicity in addition to 
safety as described above. Laboratory assays to assess immune response to PfSPZ Vaccine will 
be performed at the MRTC, LMIV, NIAID, NIH, and Sanaria Inc according to standard 
laboratory procedures. 
These assays include: 
1. Binding ELISA for antibodies to Pf liver stage and blood stage antigens (which 
includes but not limited to: CSP, sporozoite surface protein 2, liver stage antigen 1, 
erythrocyte binding antigen 175, merozoite surface protein 1, merozoite surface 
protein 5, and exported protein 1 [PfCSP, PfLSA-1, PfEBA-175, PfMSP-1, PfMSP-
5, malaria protein EXP-1] 
2. (IFN-γ) ELISPOT assay and multi-parameter flow cytometry with intracellular 
cytokine staining on peripheral blood mononuclear cells in Pf liver- stage antigens 
(CSP, LSA-1) and PfSPZ  
3. B and T cells studies to analyze immunologic responses  
Sanaria may also assess antibodies to whole PfSPZ by immunofluorescence assay (IFA) and 
inhibition of sporozoite invasion assay (ISI) and to asexual erythrocytic stage parasites by IFA as 
described (36, 37). 
Laboratory assays to assess immune responses to novel pre-erythrocytic antigens may be 
performed in the laboratories at the LMIV, NIAID, and NIH according to standard laboratory 
procedures. The target proteins are novel antigens that confer protection against liver stage 
malaria in rodent malaria models (P. yoelii, P. berghei) according to vaccination studies 
conducted by Seattle BioMed (part of Seattle Children’s Research Institute) and LMIV. The 
novel antigens to be used for these laboratory assays include PFL1995c, PFE0305w, LISP1 
(PF14_0179), SAP1 (PF11_0480), MAL7P1.164, PF14_0113, using the identifiers in the 
PlasmoDB database (www.plasmodb.org). These antigens were initially selected on the basis of 
their gene expression during early liver stage development of Pf, and preliminary testing shows 
that these antigens are immunologically recognized by individuals previously exposed to Pf. The 
potential utility of these antigens as pre-erythrocytic vaccines has been supported by animal 
studies, wherein orthologues of these genes incorporated in DNA vaccines induce protective 
immunity in mice that significantly reduces the liver stage development of P. berghei and P. 
yoelii parasites. The assays included in this study can confirm that individuals receiving PfSPZ 
Vaccine develop immune responses to pre-erythrocytic antigens and can provide additional data 
by which to assess the potential for these antigens to be developed as subunit vaccines to prevent 
infection.  

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 Samples for Genetic/Genomic Analysis 
Description of the scope of genetic/genomic analysis 
Whole genome transcriptional profiling will be performed to explore possible gene expression 
profiles or pathways that predict optimal responses to vaccination. Gene expression profiling 
following vaccination will allow the predictive capacity of eventual protected and unprotected 
vaccinees, and thus will assist in defining the correlates of protection induced by vaccination. 
Transcriptional analyses will be performed on whole blood. Blood will be collected via venous 
puncture and placed in PAXGene (or similar) tubes to preserve RNA integrity until the RNA is 
extracted. 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. 
Description of how privacy and confidentiality of medical information/biological specimens will 
be maximized 
Subjects will not be identified in any publicly released reports of this study. 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. The investigator will inform the subjects that the above-
named representatives will review their study-related records without violating the 
confidentiality of the subjects. All laboratory specimens, evaluation forms, reports, and other 
records that leave the site will be identified only by a coded number in order to maintain subject 
confidentiality. All records will be kept locked and all computer entry and networking programs 
will be done with coded numbers only. Clinical information will not be released without written 
permission of the subject, except as necessary for monitoring by IRB, FDA, NIAID, OHRP, or 
the sponsor’s designee. 
9.10 SAFETY AND OTHER ASSESSMENTS 
9.11 ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS 
 Definition of Adverse Event 
Adverse event means any untoward medical occurrence associated with the use of an 
intervention in humans, whether or not considered intervention-related (21 CFR 312.32 (a)). 
 Definition of Serious Adverse Events (SAE) 
An adverse event (AE) or suspected adverse reaction is considered "serious" if, in the view of 
either the investigator or sponsor, it results in any of the following outcomes:  
• death,  
• a life-threatening adverse event, 
• inpatient hospitalization or prolongation of existing hospitalization,  
• a persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions, or  
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered serious when, based upon appropriate medical judgment, they 
may jeopardize the participant and may require medical or surgical intervention to prevent one of 

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the outcomes listed in this definition. Examples of such medical events include allergic 
bronchospasm requiring intensive treatment in an emergency room or at home, blood dyscrasias 
or convulsions that do not result in inpatient hospitalization. 
 Classification of an Adverse Event 
Severity of Event 
For adverse events (AEs) not included in the protocol defined grading system (see Appendix A), 
the following guidelines will be used to describe severity.  
• Mild – Events require minimal or no treatment and do not interfere with the participant’s 
daily activities.  
• Moderate – Events result in a low level of inconvenience or concern with the therapeutic 
measures. Moderate events may cause some interference with functioning. 
• Severe – Events interrupt a participant’s usual daily activity and may require systemic 
drug therapy or other treatment. Severe events are usually potentially life-threatening or 
incapacitating.  Of note, the term “severe” does not necessarily equate to “serious”.] 
Relationship to Study Intervention 
All adverse events (AEs) must have their relationship to study intervention assessed by the 
investigator who examines and evaluates the participant based on temporal relationship and 
his/her clinical judgment. The degree of certainty about causality will be graded using the 
categories below. In a clinical trial, the study product must always be suspect.  
• Definitely Related – There is clear evidence to suggest a causal relationship, and other 
possible contributing factors can be ruled out. The clinical event, including an abnormal 
laboratory test result, occurs in a plausible time relationship to study intervention 
administration and cannot be explained by concurrent disease or other drugs or 
chemicals. The response to withdrawal of the study intervention (dechallenge) should be 
clinically plausible. The event must be pharmacologically or phenomenologically 
definitive, with use of a satisfactory rechallenge procedure if necessary. 
• Probably Related – There is evidence to suggest a causal relationship, and the influence 
of other factors is unlikely. The clinical event, including an abnormal laboratory test 
result, occurs within a reasonable time after administration of the study intervention, is 
unlikely to be attributed to concurrent disease or other drugs or chemicals, and follows a 
clinically reasonable response on withdrawal (dechallenge). Rechallenge information is 
not required to fulfill this definition. 
• Possibly Related – There is some evidence to suggest a causal relationship (e.g., the 
event occurred within a reasonable time after administration of the trial medication). 
However, other factors may have contributed to the event (e.g., the participant’s clinical 
condition, other concomitant events). Although an AE may rate only as “possibly related” 
soon after discovery, it can be flagged as requiring more information and later be 
upgraded to “probably related” or “definitely related”, as appropriate. 
• Unlikely related – A clinical event, including an abnormal laboratory test result, whose 
temporal relationship to study intervention administration makes a causal relationship 

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improbable (e.g., the event did not occur within a reasonable time after administration of 
the study intervention) and in which other drugs or chemicals or underlying disease 
provides plausible explanations (e.g., the participant’s clinical condition, other 
concomitant treatments). 
• Not Related – The AE is completely independent of study intervention administration, 
and/or evidence exists that the event is definitely related to another etiology. There must 
be an alternative, definitive etiology documented by the clinician.] 
Expectedness  
Principal investigators or designees will be responsible for determining whether an adverse event 
(AE) is expected or unexpected.  An AE will be considered unexpected if the nature, severity, or 
frequency of the event is not consistent with the risk information previously described for the 
study intervention. 
 Time Period and Frequency for Event Assessment and Follow-Up 
The occurrence of an adverse event (AE) or serious adverse event (SAE) may come to the 
attention of study personnel during study visits and interviews of a study participant presenting 
for medical care, or upon review by a study monitor. 
All AEs including local and systemic reactions not meeting the criteria for SAEs will be captured 
on the appropriate case report form (CRF). Information to be collected includes event 
description, time of onset, clinician’s assessment of severity, relationship to study product 
(assessed only by those with the training and authority to make a diagnosis), and time of 
resolution/stabilization of the event. All AEs occurring while on study must be documented 
appropriately regardless of relationship. All AEs will be followed to adequate resolution. 
Any medical condition that is present at the time that the participant is screened will be 
considered as baseline and not reported as an AE. However, if the study participant’s condition 
deteriorates at any time during the study after enrollment, it will be recorded as an AE.  
Changes in the severity of an AE will be documented to allow an assessment of the duration of 
the event at each level of severity to be performed. AEs characterized as intermittent require 
documentation of onset and duration of each episode.  
At each study visit, the investigator will inquire about the occurrence of AE/SAEs since the last 
visit.  Events will be followed for outcome information until resolution or stabilization 
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 12 below) and unsolicited AEs will be recorded through Day 7 after each 
injection. Injection site reactions will be assessed until Day 7 after injection (PfSPZ Vaccine or 
normal saline) or until resolved.  
After the periods specified above only unsolicited aEs, SAEs, UPs, and new onset of chronic 
illness (NOCI) will be recorded. 

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documentation of the event may be requested by the study sponsor and should be provided as 
soon as possible. 
All SAEs (regardless of relationship and whether or not they are also UPs) will be reported and 
sent to the IND sponsor by fax (SAE fax line: 240-306-0596) or email attachment. Deaths, 
immediately life threatening and all possibly, probably or definitely related SAEs will be 
communicated by telephone, fax, email or automated report via the data management system by 
the PI within 24 hours of site awareness of occurrence to the IND sponsor. All other SAEs will 
be reported to the IND sponsor within three business days after the site becomes aware of the 
event. The PI must document that the communication is received and acknowledged. 
Sanaria Inc.  
SAE Fax: 240-306-0596 
 
Individuals: 
1. Stephen L. Hoffman, M.D. 
  
 
    
  
Email: slhoffman@sanaria.com 
 
  2. Thomas L Richie, M.D., Ph.D. 
 
  
 
    
 
  Email: trichie@sanaria.com 
 
3. L.W. Preston Church, M.D. 
  
 
  
 
  Email : lwpchurch@sanaria.com  
 
4. Tooba Murshedkar, MS 
  SAE Fax: 240-306-0596 
Email: tmurshedkar@sanaria.com 
In Mali – the clinical site investigator will also notify the LMIV PI and the site medical monitor 
in Mali by email, fax, or telephone within one working day of notification of an SAE occurrence. 
LMIV Contact Information: 
PI: Patrick Duffy, MD 
Tel: 301-761-5089 
Fax: 301-480-1962 
Email: patrick.duffy@nih.gov 
 

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AI: David Cook, MD 
Tel: 240- 627-3066 
Fax: 301-82 7-1661 
Email: david.cook@nih.gov 
 
 
Independent Safety Monitor: 
Prof. Abdoul Aziz Diakite, MD 
Head, Department of General Pediatrics 
Gabriel Touré University Hospital 
Bamako, Mali 
Phone : + 223 66 74 49 56 
Email: doc abdela@yahoo.fr 
 
The study sponsor will be responsible for notifying the Food and Drug Administration (FDA) of 
any unexpected fatal or life-threatening suspected adverse reaction as soon as possible, but in no 
case later than 7 calendar days after the sponsor's initial receipt of the information.  In addition, 
the sponsor must notify FDA and all participating investigators in an Investigational New Drug 
(IND) safety report of potential serious risks, from clinical trials or any other source, as soon as 
possible, but in no case later than 15 calendar days after the sponsor determines that the 
information qualifies for reporting. 
 
 Events of Special Interest  
Seizures of any severity will be considered an event of special interest and will be reported to the 
IND sponsor and FMPOS on the NIH Reportable Events Form sent by fax or email attachment 
no later than 7 calendar days of site awareness of the event. 
All UPs that are also adverse events will be reported to the IND sponsor on the NIH Reportable 
Events Form sent by fax or email attachment no later than 7 calendar days of site awareness of 
the event.  
UPs that are not AEs will also be reported to the IND sponsor. 
 Reporting of Pregnancy  
Not applicable given all enrolled children will not be of childbearing potential.  
9.12 UNANTICIPATED PROBLEMS 
 Definition of Unanticipated Problems (UP) 
Any incident, experience, or outcome that meets all of the following criteria: 
• Unexpected in terms of nature, severity, or frequency given (a) the research procedures 
that are described in the protocol-related documents, such as the Institutional Review 

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Board (IRB)-approved research protocol and informed consent document; and (b) the 
characteristics of the participant population being studied; and 
• Related or possibly related to participation in the research (“possibly related” means there 
is a reasonable possibility that the incident, experience, or outcome may have been 
caused by the procedures involved in the research); and 
• Suggests that the research places participants or others (which may include research staff, 
family members or other individuals not directly participating in the research) at a greater 
risk of harm (including physical, psychological, economic, or social harm) than was 
previously known or expected. 
  Reporting Procedures to the FMPOS EC  
The USTTB PI is responsible for reporting per FMPOS EC reporting requirements. All UPs, 
major protocol deviations, all non-compliance, new information that might affect the willingness 
of a subject to enroll or remain in the study, and any suspension or termination of research 
activity will be reported to the FMPOS EC within 7 calendar days of investigator awareness. 
Any death of a research subject that is possibly, probably, or definitely related to the research 
will be reported within 24 hours of investigator awareness. 
The following events will be reported to the FMPOS EC as a high-level summary at the time of 
continuing review:  
• AEs and SAEs that are not UPs, as a narrative summary indicating whether these events 
were within the expected range. 
• Minor and major protocol deviations. 
• UPs reported to the FMPOS EC. 
• Non-compliance reported to the FMPOS EC that is not related to a protocol deviation. 
 
10. STATISTICAL CONSIDERATIONS 
This section describes the statistical analyses of primary and secondary study objectives. Further 
details, including methods for assessing exploratory objectives, are described in a separate 
statistical analysis plan to be generated prior to unblinding. Any changes to analyses described in 
the protocol or statistical analysis plan will be fully disclosed in the clinical study report.  
10.1 STATISTICAL HYPOTHESIS 
Primary Endpoints 
Safety: The primary safety endpoint is the proportion of vaccinees experiencing related SAEs 
from V1 to 26 weeks after V3. There is no formal hypothesis test associated with this primary 
safety endpoint. Rather, the proportion of subjects experiencing related SAEs will be presented 
for each arm separately using frequencies, percentages, and descriptive 95% confidence intervals 
(CIs).  

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Efficacy: the primary efficacy endpoint is the vaccine efficacy against malaria with symptoms. 
For this protocol, there is a primary and a secondary case definition of Pf malaria with 
symptoms.   
The primary case definition is as follows: 
Pf malaria with symptoms is defined as a positive thick blood smear at a denisity of >1000 P/uL 
plus: 
 Measured axillary temperature ≥ 37.5 degrees Celsius or history of fever 
(subjective or objective) in the last 24 hours, or,  
 Symptoms of malaria – 
o Verbal individual (individual able and willing to answer questions): A 
verbal individual is considered symptomatic if reporting at the time of 
evaluation at least two of the following symptoms/symptom groups: 
headache, chills and/or rigors, malaise and/or fatigue, dizziness and/or 
light-headedness, myalgias and/or arthralgias; or 
o Non-verbal individual (small child or any individual unable or unwilling 
to answer questions): A non-verbal individual is considered symptomatic 
if manifesting at the time of evaluation at least two of the following 
signs/sign groups: drowsiness, irritability and/or fussiness, inability and/or 
refusal to eat or drink, prostration; or 
 Any individual: Signs of severe malaria (e.g. impairment of consciousness, severe 
anemia, hemoglobinuria, acute kidney injury, etc.). 
The secondary case definition is as follows:  
Pf malaria with symptoms is defined as a positive thick blood smear at a density of > 0 P/uL 
plus: 
 Measured axillary temperature ≥ 37.5 degrees Celsius or history of fever 
(subjective or objective) in the last 24 hours, or,  
 Symptoms of malaria as defined in the primary case definition; or 
 Meeting criteria for severe malaria 
The formal hypothesis associated with the primary efficacy endpoint is a superiority test of 
H0: VE = 0 versus HA: VE < > 0 
(equivalently, H0: HR = 1 vs. HA: HR <> 1). The primary efficacy hypothesis test will be 
conducted at the two-sided α=0.05 significance level, restricted to subjects in the mITT 
population (defined below in Section 9.3). 
The primary efficacy endpoint will be assessed over all participants, the vaccinated versus 
controls.   
Secondary Endpoints 
Safety: The secondary safety endpoints include the related SAEs as well as all other measures of 
safety and tolerability – unsolicited AEs, laboratory abnormalities, and solicited AEs.  

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Efficacy: The secondary efficacy endpoint is the vaccine efficacy against Pf malaria.   
Pf malaria is defined as: 
 At least one unambiguous asexual parasite on thick blood smear identified by two 
independent microscopists after each examining 0.50 µL of blood in a study 
participant 
10.2 SAMPLE SIZE DETERMINATION 
Power calculations for safety analysis: 
The goal of the safety evaluation for this study is to identify safety concerns associated with 
product administration. The ability of the study to detect serious adverse events (SAEs) can be 
expressed by the true event rate above which at least 1 SAE would likely be observed and the 
true event rate below which no events would likely be observed. Specifically, within an arm of 
size n=120, there is a 90% chance of observing no event if the true rate is no more than 0.08%, 
and a 90% chance of observing at least 1 event if the true rate is no more than 1.9%.  
Probabilities of observing 0, 1, and 2 or more events within an arm of size 120 are presented in 
Table 13, for a range of possible true adverse event rates. These calculations provide a more 
complete picture of the sensitivity of this study design to identify potential safety problems with 
the vaccine.  
Table 13. Probability of observing 0, 1 and 2 or more events, within an arm of size 120 for 
different true event rates. 
True event rate (%) Pr(0/120) 
Pr(1/120) 
Pr(2+/120) 
1 
0.299 
0.363 
0.338 
3 
0.026 
0.096 
0.878 
5 
0.002 
0.013 
0.984 
10 
<0.001 
<0.001 
>0.999 
 
An alternative way of describing the statistical properties of the study design is in terms of the 
95% confidence interval for the true rate of an adverse event based on the observed data. Table 
14 shows the 2-sided 95% confidence intervals for the probability of an event based on a 
particular observed rate. If none of the 120 participants in an arm in the main study experience a 
safety event, the 95% 2-sided upper confidence bound for the true rate of such events in the total 
vaccinated population is 0.030.  

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Table 14. Two-sided 95% confidence intervals based on observing a particular rate of 
safety endpoint arms for an arm of size 120. 
Observed event rate 
95% Confidence interval (%) 
0/120 
(0.000, 0.030) 
1/120 
(0.000, 0.046) 
2/120 
(0.002, 0.059) 
3/120 
(0.005, 0.071)  
4/120 
(0.009, 0.083) 
5/120 
(0.014, 0.095) 
6/120 
(0.019, 0.106) 
 
The sample size determination is mainly based on the assessment of efficacy, which will be 
shown below.   
Power calculations for efficacy analysis: 
The primary efficacy endpoint is the efficacy against Pf malaria with symptoms. The vaccine 
efficacy will be assessed by comparing the vaccinated versus the controls.  
Based on recent data from Bancoumana, the symptomatic malaria rate under the control is very 
likely above 0.35 for children of age 6-10. We estimate a sample size of 120 per arm will allow 
reasonable power to detect the between-arm difference.  
Table 15 presents the power calculation for detecting vaccine efficacy based on a two-sided log-
rank test with the familywise type I error of 0.05.  In the power calculation, vaccine efficacy is 1 
minus the probability of Pf malaria with symptoms among the vaccinated divided by the 
probability of Pf malaria with symptoms among the controls. A drop-out rate of 10% is assumed. 
With 240 subjects in the two arms, this study has over 80% power to detect the vaccine efficacy 
if the Pf malaria with symptoms rate is no less than 0.35 under the control and the vaccine 
efficacy is no less than 0.5.  
In order to allow a booster study to proceed during a second transmission season if VE is 50% or 
greater in the first year, an additional 10% dropout rate has been assumed and 14 participants 
added for a total sample size of approximately 134 in each arm in the first year. 
 

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Table 15. Power calculation for plausible Pf malaria with symptom rates for two-sided log-
rank test under type 1 error rate of alpha=0.05. 
Pf malaria with 
symptoms rate 
under control 
Vaccine 
efficacy 
Power(%) 
(90 per arm) 
Power(%) 
(100 per 
arm) 
Power(%) 
(110 per 
arm) 
Power(%) 
(120 per 
arm)  
Power(%) 
(130 per 
arm)  
Power(%) 
(140 per 
arm)  
0.3 
0.4 
43 
47 
50 
54 
57 
60 
0.5 
62 
66 
70 
74 
77 
80 
0.6 
79 
83 
86 
89 
91 
93 
0.35 
0.4 
50 
55 
59 
62 
66 
69 
0.5 
70 
75 
79 
82 
85 
88 
0.6 
86 
89 
92 
94 
95 
97 
0.4 
0.4 
58 
63 
67 
71 
74 
77 
0.5 
78 
82 
86 
88 
91 
93 
0.6 
91 
94 
96 
97 
98 
98 
0.5 
0.4 
73 
78 
81 
85 
87 
90 
0.5 
90 
92 
95 
96 
97 
98 
0.6 
97 
98 
99 
99 
100 
100 
0.6 
0.4 
86 
89 
92 
94 
96 
97 
0.5 
96 
98 
99 
99 
99 
100 
0.6 
99 
100 
100 
100 
100 
100 
 
10.3 POPULATIONS FOR ANALYSES 
The following analysis populations will be used when describing subject disposition and 
performing statistical analyses: 
• The Screened Population includes all subjects who are screened and provide informed 
consent, regardless of whether the subject is randomized or treated. This population will 
be used to fully account for subject disposition.  

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• The Intention to Treat (ITT) Population is a subset of the screened population that is 
deemed eligible to participate in the study, is randomized to a treatment group, receives 
at least one injection with PfSPZ Vaccine or placebo, and contributes evaluable person-
time to the risk set.  
• The Modified Intention to Treat (mITT) Population includes all subjects who are 
deemed eligible to participate, are randomized, receive all three injections of vaccine or 
placebo, and contribute evaluable person-time to the risk set, including subjects to whom 
V2 and/or V3 is administered out of the pre-specified time window or an incomplete 
injection is given, defined as an estimated injection volume of >10% and < 80%. 
• The According to Protocol (ATP) Population is a subset of the mITT Population, 
including subjects who complete the vaccination series per protocol and contribute 
evaluable person-time to the risk set.  
10.4 STATISTICAL ANALYSES 
 General Approach 
Missing Data 
Subjects who discontinue early or are lost to follow-up will be censored from applicable analyses 
at the last date where the study outcome can reliably be determined (e.g., date of last available 
TBS specimen without parasitemia).  
It is anticipated that some event dates required for safety analyses may be incomplete. If the 
event onset day is unknown, then the date will be imputed as the last date the subject could 
reliably be assumed to be event free (e.g., the previous study visit where the event was not 
reported). If the resolution day is incomplete, then the date will be imputed as the date that the 
event was reliably known to no longer be present. 
It is anticipated that some TBS results will be missing. In those instances where one TBS is 
missed, the result will be imputed to be the same as the result of the next TBS. Up to three such 
single missing TBS will be allowed in a research subject during the 26 weeks of follow-up 
without disqualifying the participant from the ATP population (as long as the individual meets 
other requirements for ATP, such as receiving all three immunizations within the correct time 
windows). If there are situations where two or more consecutive TBS are missed, the result will 
again be imputed to be the same as the result of the next TBS, and the research subject will be 
included in the m-ITT population (as long as the individual meets other requirements for m-ITT, 
such as receiving all three immunizations). If the only TBS missed is the last, there will be no 
imputation, the missing data point will be ignored, and the individual may be included in the 
ATP population. If the two or more TBS missed are the last, there will be no imputation, the 
missing data points will be ignored, and the individual may be included in the m-ITT population. 
However, if more than the last two TBS are missed, the individual will not be included in 
proportional efficacy analyses, although may be included in time-to-event efficacy analyses.  
Multiple Testing 
The primary VE endpoint is the VE against Pf malaria with symptoms. The overall vaccine 
efficacy will be assessed over all participants.  
No multiplicity adjustment will be made for secondary efficacy or safety endpoints.  

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 Safety Analyses 
The primary safety endpoint is the occurrence of an SAE between V1 and 26 weeks after V3 
which is at least possibly related to treatment. Serious AEs which meet this definition will be 
summarized in frequency tables and listings according to system organ class, preferred term, 
relatedness, and severity. The primary safety analysis will be performed using the ITT 
population and according to treatment received at V1, regardless of any randomization or 
allocation errors. All SAEs will be coded using the Medical Dictionary for Regulatory Activities 
(MedDRA) and summarized using frequencies, proportions, and exact 95% CIs for proportions. 
Rates of SAEs (overall and for any system organ class or preferred term with at least 5 events in 
either group) will be compared using two-sided 0.05 level Fisher’s Exact Tests.  
Secondary safety endpoints include the occurrence of unsolicited AEs, laboratory abnormalities, 
and solicited AEs in the ITT population. Unsolicited AEs documented from V1 to 14 days 
following V3 will be coded using MedDRA and summarized in frequency tables according to 
system organ class, preferred term, relatedness, and severity. Summaries will be provided overall 
and according to the vaccination number (1, 2, or 3) immediately prior to AE onset. Rates of 
unsolicited AEs (overall and for any system organ class or preferred term with at least 5 events 
in either group) will be compared using two-sided 0.05 level Fisher’s Exact Tests. Adverse 
events leading to premature discontinuation from the study will be listed separately. Clinically 
relevant laboratory abnormalities recorded in the 14 days after V3 will be summarized in 
frequency tables by type, grade, and clinical relevance.   
 Efficacy Analysis 
9.4.3.1 Primary: VE against Pf malaria with symptoms 
The primary efficacy analysis will be the modified Intention to Treat (mITT).  
The primary efficacy analysis will be based on time to the first Pf malaria with symptoms. Entry 
into the risk set begins on the latter of a) 14 days after completing the primary vaccination series 
(V3 + 14 days) or b) 28 days after initiating treatment for a parasitemia event that was detected 
on or before V3 + 14 days, whichever comes later. Time-to-event is defined as the number of 
days between entry into the risk set and date of first Pf malaria with symptoms; subjects who do 
not experience the event by 26 weeks after V3 will be censored from the primary analysis or 
earlier date of last negative TBS result. 
The survival patterns will be described by Kaplan-Meier curves for each arm and compared by 
the logrank test between two arms. The protective efficacy will be assessed from the Cox 
proportional hazards model with treatment arm as the regressor, and the vaccine efficacy will be 
estimated as one minus the hazard ratio.  
As a secondary efficacy analysis, the cumulative probability of Pf malaria with symptoms 
through 26 weeks post V3 will be presented for each treatment arm and compared across arms 
based on Kaplan-Meier estimates along with 95% confidence intervals. This estimation accounts 
for right censoring and the comparison should be equivalent to Fisher’s exact test in case of no 
censoring.  

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Sensitivity analyses will include an ITT and ATP analysis with entry into the risk set as with the 
mITT analysis, and ATP analyses with entries into the risk set at time of first vaccination and at 
V3.  
9.4.3.2 Secondary: VE against Pf malaria  
VE against Pf malaria (see definition in Section 9.1) will be assessed as for VE against Pf 
malaria with symptoms, using the mITT population and censoring participants who do not 
experience the endpoint by 6 months after V3.  
The cumulative probability of Pf malaria through 26 weeks post V3 will be similarly presented 
as for Pf malaria with symptoms.  
 Exploratory Analyses 
The trend of the immune response, more specifically the humoral and cellular immune 
responses, to PfSPZ Vaccine will be modelled and compared between the vaccine recipients and 
the controls. This will be achieved via a generalized linear regression which accounts for the 
within-subject correlation.   
To assess genetic relatedness of the PfSPZ Vaccine parasite strain to malaria infection parasites,  
a genotypic sieve analysis to analyze the sequences of peripheral blood Pf parasites from infected 
subjects.  The sieve analysis targets at differentiating protective efficacy against different 
genotypes of infection-inducing parasites with genotype defined by the number of mismatches to 
the PfSPZ footprint. 
 Planned Unblinded Analyses 
The study statistician will undertake an unblinded preliminary analysis of the safety and efficacy 
data from the trial after the data for all originally-planned primary and secondary endpoints have 
been gathered but before formal database lock.  This will allow for the timely planning of future 
trials in this study population.   
The results of these analyses will be transmitted by the study statistician to the LMIV Senior 
Investigator only.  This will allow the remainder of the study team, including the PI, to remain 
blinded in the case of a future extension of the trial such as for a booster vaccination, extended 
follow-up, or a combination of both.  The LMIV Senior Investigator may share the results of the 
unblinded analysis with the trial sponsor, Sanaria, the DSMB, and other unblinded parties as 
appropriate for the purposes of future planning. 
The analyses of the Primary Objectives will include measurement of VE against first episode of 
Pf malaria with symptoms by time-to-event analysis. 
The analyses of the Secondary Objectives will include measurement of VE against first episode 
of Pf malaria (parasitemia) with or without associated symptoms by time-to-event analysis. 
Other unblinded analyses of safety and efficacy may also be requested by the Senior LMIV 
Investigator.  For example, analyses of the Exploratory Objectives may include measurement of 
VE using other outcome measures such as against all episodes of Pf malaria with symptoms and 
Pf malaria rather than against the first episode, using proportional analysis, or using ITT or ATP 
populations, or using parasite density thresholds of > 5000 parasites/µL. 
 

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11. REGULATORY AND OPERATIONAL CONSIDERATIONS 
11.1 INFORMED CONSENT PROCESS 
 Consent Procedures and Documentation 
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 on-going 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 and their parents or guardians 
will be given the opportunity to ask questions and have them answered.  
Participants and their parents or guardians will be asked to read and review the consent 
documents. The study staff member will explain the research study to the participant and answer 
any questions. A verbal explanation will be provided in terms suited to the participant’s 
comprehension, and will include the purposes, procedures, and potential risks of the study. 
Participants will be informed of their rights, such as the fact that participation is voluntary and 
that they may withdraw from the study at any time, without explanation or prejudice.  
After the verbal explanation, participants (and/or their parents or guardians) will have the 
opportunity to carefully review the written consent form and ask questions prior to signing. They 
will also have the opportunity to discuss the study with family members or others they may 
choose, or to request additional time to consider prior to agreeing to participate.  
Assent, either written or verbal, is not typically obtained for minors under the age of 12 in Mali.  
Therefore, assent will not be documented on this trial enrolling only children under the age of 10.  
Nevertheless, consent will be obtained from participants’ parent(s) and/or guardian(s) in a 
location that is supportive of children.  
The participation of both parents in the consent process, and the signature of both parents will be 
encouraged. 
The informed consent process will be conducted and documented on the source document by 
signature and date, before any study procedures can begin. A copy of the informed consent 
document will be given to the participants’ parent(s) and/or guardian(s) for their records. 
Consent forms will be approved by all participating IRBs. The participants’ parent(s) and/or 
guardian(s) 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 ICF will be signed (or fingerprinted) and personally dated by the subject (or 
subject’s legal guardian) and the person who conducted the informed consent discussion. The 
original signed ICF 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. 

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Community Permission in Mali 
Community permission will be obtained from village elders, family heads, and other community 
members after explanation and discussion of the study (Diallo 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. 
• The study and the informed consent process are explained in detail to heads of families 
by study investigators/personnel.   
The individual informed consent process and relevant 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. 
Malaria Comprehension Exam 
Subjects or their parents/guardians will be encouraged to ask questions, and then the 
parents/guardians will take a multiple-choice questionnaire (true/false) to evaluate consent 
comprehension. All incorrect responses will be reviewed with the parents/guardians, and they 
must verbalize understanding of all incorrect responses. A score of ≥80% correct is required for 
enrollment. For parents/guardians scoring less than 80%, study staff may choose to review study 
details again with subject and parents/guardians and reassess comprehension with a repeat 
Malaria Comprehension Exam. At the discretion of the investigator, any subject or 
parent/guardian 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 parents/guardians 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 the parents/guardians. This will help ensure that the parents/guardians 
have sufficient understanding before the consent form is signed. The parents/guardians may 
either sign the consent form immediately or later after further consideration. parents/guardians 
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. 
 Consent for minors when they reach the age of majority  
We request a waiver of informed consent to continue to use data and/or specimens for those 
individuals who reach the age of majority after all study visits are complete.  Considering the 
length of time between the minor’s last contact with the research team and their age of majority, 
it will likely be very difficult to locate these subjects to complete the standard protocol consent. 
The retention of samples and data will not affect the welfare of these subjects. 

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 Considerations for Consent of family members of study team members 
Consent from staff members will be obtained by an individual independent of the staff member’s 
team whenever possible.   
 Consent of Subjects who are, or become, decisionally impaired 
Not applicable. 
11.2 STUDY DISCONTINUATION AND CLOSURE 
This study may be temporarily suspended or prematurely terminated if there is sufficient 
reasonable cause.  If the study is prematurely terminated or suspended, the principal investigator 
(PI) will promptly inform study participants, the Institutional Review Board (IRB), and sponsor 
and will provide the reason(s) for the termination or suspension.  Study participants will be 
contacted, as applicable, and be informed of changes to study visit schedule. 
 Circumstances that may warrant termination or suspension include, but are not limited to: 
• Determination of unexpected, significant, or unacceptable risk to participants 
• Demonstration of efficacy that would warrant stopping    
• Insufficient compliance to protocol requirements 
• Data that are not sufficiently complete and/or evaluable 
• Determination that the primary endpoint has been met 
• Determination of futility 
Study may resume once concerns about safety, protocol compliance, and data quality are 
addressed, and satisfy the sponsor, IRB and, as applicable, the Food and Drug Administration 
(FDA). 
11.3 CONFIDENTIALITY AND PRIVACY 
Participant confidentiality and privacy is strictly held in trust by the participating investigators, 
their staff, and the sponsor(s). This confidentiality is extended to cover testing of biological 
samples and genetic tests in addition to the clinical information relating to participants. 
Therefore, the study protocol, documentation, data, and all other information generated will be 
held in strict confidence. No information concerning the study or the data will be released to any 
unauthorized third party without prior written approval of the sponsor.  
All research activities will be conducted in as private a setting as possible. 
The study monitor, other authorized representatives of the sponsor, representatives of the 
Institutional Review Board (IRB), and/or regulatory agencies may inspect all documents and 
records required to be maintained by the investigator, including but not limited to, medical 
records (office, clinic, or hospital) and pharmacy records for the participants in this study. The 
clinical study site will permit access to such records. 
The study participant’s contact information will be securely stored at each clinical site for 
internal use during the study. At the end of the study, all records will continue to be kept in a 
secure location for as long a period as dictated by the reviewing IRB, Institutional policies, or 
sponsor requirements. 

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Study participant research data, which is for purposes of statistical analysis and scientific 
reporting, will be transmitted to and stored at the MRTC, NIH, and Sanaria Inc. This will not 
include the participant’s contact or identifying information. Rather, individual participants and 
their research data will be identified by a unique study identification number. The study data 
entry and study management systems used by clinical sites and by the study team research staff 
will be secured and password protected. At the end of the study, all study databases will be de-
identified and archived. 
To further protect the privacy of study participants, a Certificate of Confidentiality has been 
issued by the National Institutes of Health (NIH).  This certificate protects identifiable research 
information from forced disclosure. It allows the investigator and others who have access to 
research records to refuse to disclose identifying information on research participation in any 
civil, criminal, administrative, legislative, or other proceeding, whether at the federal, state, or 
local level. By protecting researchers and institutions from being compelled to disclose 
information that would identify research participants, Certificates of Confidentiality help achieve 
the research objectives and promote participation in studies by helping assure confidentiality and 
privacy to participants. 
11.4 FUTURE USE OF STORED SPECIMENS AND DATA 
All samples stored for use in the trial will be labeled with the participant’s study identification 
(ID) number, which cannot identify the study subject directly (not linkable to any personally 
identifiable information) but is linkable to other research databases (e.g., questionnaires, CRFs, 
logbooks) generated by the study. The subject ID log linking the study subject’s ID number to 
the name of the participant will be kept secure, with access limited to authorized research team 
members.  
At the completion of the clinical trial and the exploratory studies described in the protocol, 
samples may be destroyed, transferred to another subsequent trial, or stored, the latter only if 
permission has been given for future use and the IRBs are informed. If stored, they will be coded 
to allow linking with relevant clinical data without the possibility to link back to the study 
subject.  
Samples may be used in the future to learn more about protection from malaria, vaccine-induced 
immunity and confounders of vaccine take. The data generated in the study will form the basis 
for further studies using these specimens aimed at defining the immune responses to Pf infection 
that lead to protection.  For any studies that are not specifically established in the ICF, the 
investigators wishing to study these samples will need to obtain approval from the IRB.  
Participants may withdraw permission for future use of specimens at any time. If a participant 
withdraws his or her permission for future use of specimens, the investigator or designee will 
destroy all known remaining specimens and report this destruction to the study subject and the 
IRBs and make a note in the study subject’s chart. This decision will not affect the research 
subject’s participation in this trial or any other trials that are overseen by the IRBs. If specimens 
for future use have already been de-identified, their destruction may not be possible. 
Temperatures will be monitored continuously in freezers and refrigerators where samples are 
stored. 

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11.5 SAFETY OVERSIGHT 
As agreed with the Office of Clinical Research Policy and Regulatory Operations (OCRPRO), a 
DSMB chartered by the IND sponsor, Sanaria Inc. will be used for this study. Safety oversight 
will be under the direction of a Data and Safety Monitoring Board (DSMB) composed of 
individuals with the appropriate expertise, including malaria vaccine and pediatric experience. 
Members of the DSMB will be independent from the study conduct and free of conflict of 
interest, or measures should be in place to minimize perceived conflict of interest.   
The DSMB will review the study prior to initiation, after completion of the vaccination series 
and at study close. The board may convene additional reviews as necessary, and will issue 
recommendations concerning continuation, modification, or termination of the study. All SAEs 
will be reported by the PI to the sponsor immediately upon becoming aware of them. All SAEs 
that are possibly, probably or definitely related to the study agent, UPs, and safety reports (as 
available) will be reported by the sponsor to the DSMB. 
The DSMB will review the study data to evaluate the safety, efficacy, study progress, and 
conduct of the study. The sponsor will notify the DSMB of any cases of intentional or 
unintentional unblinding as soon as possible. The sponsor will notify the DSMB at the time 
pausing criteria are met and obtain a recommendation concerning continuation, modification, or 
termination of the study. The PI will submit the written DSMB summary reports with 
recommendations to the IRB(s). 
The DMSB will operate under the rules of an approved charter that will be written and reviewed 
at the organizational meeting of the DSMB. At this time, each data element that the DSMB needs 
to assess will be clearly defined. The DSMB will provide its input to Sanaria Inc. 
11.6 CLINICAL MONITORING 
As per International Council for Harmonisation of Technical Requirements for Pharmaceuticals 
for Human Use- Good Clinical Practice (ICH-GCP) guideline 5.18 and FDA 21 CFR 312.50, 
clinical protocols are required to be adequately monitored by the study Sponsor. OCRPRO will 
provide oversight and monitor the compliance of this trial and study monitoring will be 
conducted according to the “NIAID Intramural Clinical Monitoring Guidelines.” Monitors under 
contract to the NIAID/OCRPRO will visit the clinical site to monitor aspects of the trial in 
accordance with appropriate regulations. The objectives of a monitoring visit will be: 
• to verify the prompt reporting of all monitored data points, and prompt reporting of all 
SAEs 
• to check the existence of signed informed consent documents and documentation of the 
ICF process for each monitored subject 
• to compare individual subject’s records (e.g. CRFs, electronic data) to the source 
documents (supporting data, laboratory specimen records, clinical notes) 
• to ensure the investigators are in compliance with the protocol 
The monitors will also inspect the clinical site’s regulatory files to ensure that applicable 
regulatory requirements (FDA, Office for Human Research Protections [OHRP]) and ICH 
guidelines are being obeyed. During the monitoring visits, the PI and/or designated study staff 
will be available to discuss the study. The site PI will provide direct access and allow the study 

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monitors, LMIV, the IND Sponsor, and regulatory authorities to access all study-related 
documents. 
A specific monitoring plan will be discussed with the PI, study staff, and IND Sponsor 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. 
Quality control procedures will be implemented beginning with the data entry system, and data 
quality control checks that will be run on the database will be generated. Any missing data or 
data anomalies will be communicated to the site for clarification/resolution. 
11.7 QUALITY ASSURANCE AND QUALITY CONTROL 
The clinical site will perform internal quality management of study conduct, data and biological 
specimen collection, documentation and completion.  An individualized quality management 
plan will be developed to describe a site’s quality management. 
Quality control (QC) procedures will be implemented beginning with the data entry system and 
data QC checks that will be run on the database will be generated. Any missing data or data 
anomalies will be communicated to the site(s) for clarification/resolution. 
Following written Standard Operating Procedures (SOPs), the monitors will verify that the 
clinical trial is conducted and data are generated and biological specimens are collected, 
documented (recorded), and reported in compliance with the protocol, International Conference 
on Harmonisation Good Clinical Practice (ICH GCP), and applicable regulatory requirements 
(e.g., Good Laboratory Practices (GLP), Good Manufacturing Practices (GMP)).  
The investigational site will provide direct access to all trial related sites, source data/documents, 
and reports for the purpose of monitoring and auditing by the sponsor, and inspection by local 
and regulatory authorities. 
11.8 DATA HANDLING AND RECORD KEEPING 
 Data Collection and Management Responsibilities 
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 documentation will be used as source. Any type of 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.  
Clinical data (including adverse events (AEs), concomitant medications, and expected adverse 
reactions data) and clinical laboratory data will be entered into DFExplore, a 21 CFR Part 11-
compliant data capture system provided by OCICB.  The data system includes password 

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protection and internal quality checks, such as automatic range checks, to identify data that 
appear inconsistent, incomplete, or inaccurate. Clinical data will be entered directly from the 
source documents. 
Collection and Storage of Biometric Data 
In this study we are planning on using biometric data to be able to identify people who are study 
participants.  Following subject enrollment, all subjects will receive an identification card with 
their photo on it to present at the clinic with each study visit. Fingerprinting may also be 
implemented to ensure subjects are identified correctly during the visits. The biometric 
application that would be used is the Biometric Screening Log application developed by the data 
management unit of University Clinical Research Center (UCRC) of the University of Science, 
Techniques and Technology of Bamako (USTTB) in Mali.  This system utilizes FBI-certified 
fingerprint scanners to scan participant fingerprints.  In addition to providing identity verification 
during scheduled and unscheduled visits to the site at key stations (Identification Point, Clinic 
Exam Room, Lab/Phlebotomy, and Vaccination rooms), the software provides the following 
functionality: 
• 
ID card printing with QR code on a Zebra ID card printer 
• 
Participant-specific labels with 2 or 3 dimensional barcodes for samples, also printed 
from the Zebra printer 
• 
Dynamic visit calendar for full week 
• 
Daily report generated on visits that are completed, pending, missed, and scheduled 
• 
Weekly summary report available on study dashboard  
Particiants’ fingerprints may be collected and stored.  Neurotechnology’s VeriFinger SDK 
fingerprint compression algorithm will be used to create a secure template.  This algorithm meets 
accuracy requirements outlined in the Wavelet Scalar Quantization (WSQ) Gray-Scale 
Fingerprint Image Compression Specification developed by the FBI and NIST.  PII and 
biometric templates are saved on a SGDBR SQL Server database compliant with Title 21 of the 
Code of Federal Regulations.  The HF SQL database is installed on a local server.  The server is 
located in a secure area with limited access (physical and logical access security, temperature 
monitoring, fire and smoke detection, camera surveillance).  Regular backup will be done based 
on data changes that will be encrypted and stored on local servers to prevent any leak of PII to 
NIH servers and or to NIH personnel. 
 Study Records Retention 
Study documents should be retained for a minimum of 2 years after the last approval of a 
marketing application in an International Conference on Harmonisation (ICH) region and until 
there are no pending or contemplated marketing applications in an ICH region or until at least 2 
years have elapsed since the formal discontinuation of clinical development of the study 
intervention, and as per the NIH Intramural Records Retention Schedule. No records will be 
destroyed without the written consent of the sponsor, if applicable. It is the responsibility of the 
sponsor to inform the investigator when these documents no longer need to be retained. 

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