Clinical trial NCT02942277
Conditions: Malaria
Keywords: Malaria Reactogenicity Antibody Direct Skin Feed Mosquito
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)
Start date: 2016-10-21; Completion date: 2020-07-15
Enrollment: 301 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Background:

Researchers are looking for new ways to control and eradicate malaria. They want to test vaccines to block malaria transmission in adults in Mali. These vaccines work by inducing antibodies in a person. The antibody is then taken up with blood by a mosquito that bites the person. This blocks parasite development in the mosquito. This stops malaria transmission to another person.

Objective:

To test the safety, reactogenicity, immunogenicity, and transmission-blocking activity of the vaccines Pfs25M-EPA and Pfs230D1M-EPA with AS01 in Malian adults.

Eligibility:

Healthy Malians ages 18-50 living in certain areas in Mali who:

Are not pregnant or breastfeeding

Are not infected with HIV, Hepatitis B and Hepatitis C

Do not have evidence of immunodeficiency

Do not have history of severe allergic reaction or anaphylaxis

Design:

Participants will be screened with:

Medical history

Physical exam

Malaria Comprehension Exam

Blood and urine tests

Electrocardiogram (for participants in certain study groups)

Participants will be randomly assigned to a study group.

Participants will be monitored for 12-16 months. For the first 7 months, they will have between 1 and nine visits a month. The number depends on the month and on what group they are in. For the rest of the months, they will have 1 monthly visit.

Each visit includes a physical exam. Most include blood tests.

Participants will get 3 doses of a study or comparator vaccine. They get the vaccine through an injection in the upper arm. This occurs at their first visit, then 1 month later, and then 5 months later.

Participants will be followed for at least 6 months after the last vaccine.

If participants develop an injection site rash or reaction, photographs may be taken of the site.

Detailed description:
A vaccine to interrupt malaria transmission (VIMT) would be a valuable tool for local elimination or eradication of this disease, and may contain components that block transmission to mosquitoes (such as Pfs25 or Pfs230) or that prevent human infection (such as the vaccine RTS,S). Pfs25 and Pfs230, surface antigens of zygotes and ookinetes (and gametocytes for Pfs230) in the mosquito stage of P. falciparum, are the lead candidates for a malaria transmission blocking vaccine (TBV). Recombinant Pfs25M and recombinant Pfs230D1M have each been conjugated to P. aeruginosa ExoProtein A (EPA) and adjuvanted with AS01. Our ongoing experience with Pfs25M-EPA and Pfs230D1M-EPA in Malian adult trial participants, and the extensive experience with the AS01 adjuvants in African children and adults, justify conducting the first-in-human trial of Pfs25M-EPA and Pfs230D1M-EPA with AS01 in Malian adults. This dose-escalating phase 1 study will determine safety, immunogenicity, and functional activity of these vaccines in Malian adults. Pfs25M-EPA + Pfs230D1M-EPA in AS01 will be assessed by mosquito feeding assays in Malian adults for evidence that they may reduce the number of malaria transmission events in study subjects.

A total of 305 subjects will be enrolled at multiple sites in Mali, West Africa to receive escalating doses of a malaria transmission blocking vaccine (s): Pfs25M-EPA/AS01, Pfs230D1M-EPA/AS01, or simultaneous administration of Pfs25M-EPA/AS01 and Pfs230D1M-EPA/AS01; or a comparator vaccine (ENERGIX-B). Enrollment within each group will be staggered for additional safety and subjects will only be enrolled into the co-administration group once each individual dose has been administered. Subjects will be followed for at least 6 months after the last vaccination. Safety outcomes will be local and systemic adverse events and serious adverse events. Immunogenicity outcomes will be antibody responses as measured by ELISA against recombinant Pfs25, Pfs230, EPA, CSP, and B cell and T cell responses. Functional activity of the induced antibodies will be assessed in TBV arms by standard membrane feeding assays conducted at the National Institute of Allergy and Infectious Diseases, and activity that interrupts malaria transmission will be measured in all arms by direct skin feeding assays in Mali.

Subjects in the open label safety cohorts (Arms 1a, 1b, 2a, 2b, 3a, 3b, 4a, 4b) will be offered reenrollment for follow-up laboratory assessment to explore the duration of immunogenicity and functional activity at approximately 9, 12 months post vaccination. Following scheduled, intentional unblinding, subjects enrolled in Arms 2c, 2d, and 4c will be provided the opportunity to re-enroll for a fourth vaccination (Arm 2c and 2d with 40 microgram dose of Pfs230D1M-EPA/AS01; Arm 4c with Menatctra) approximately 1 year post vaccination #3. Subjects in these arms will also be eligible to re-enroll for follow up of duration of immunogenicity and functional activity at approximately 9, 12 months post vaccination if they choose not to enroll to recive the booster vaccination. Subjects will be followed similarly to the previous year for safety, immunogenicity, and functional activity.

Arms: arm 1: (n=5), to receive 16 microgram Pfs25M-EPA/AS01 on D0, D28, D168 arm 2: (n=10), to receive 47 microgram Pfs25M-EPA/AS01 on D0, D28, D168 arm 3: (n=5), to receive 13 microgram Pfs230D1M-EPA/AS01 on D0, D28, D168 arm 4: (n=10), to receive 40 microgram Pfs230D1M-EPA/AS01 on D0, D28, D168 arm 5: (n=60), to receive 40 micrograms Pfs230D1M-EPA/AS01 on D0, D28, D168; all subjects will undergo antimalarial drug treatment with Coartem on D-7 (prior to vaccination #1); 4th vaccination on D476 arm 6: (n=60), to receive 40 micrograms Pfs230D1M-EPA/AS01 (500 (Micro)L TBV + AS01) on D0, D28, then 8 micro liters Pfs230D1M- EPA/AS01 (100 (Micro)L TBV + AS01;fractional dose) on D168; all subjects will undergo antimalarial drug treatment with Coartem on D-7 (prior to vaccination #1); 4th vaccination on D476 arm 7: (n=5), to receive 16 microgram Pfs25M-EPA/AS01 and 13 microgram Pfs230D1M-EPA/AS01 on D0, D28, D168 arm 8: (n=10), to receive 47 microgram Pfs25M-EPA/AS01 and 40 microgram Pfs230D1M-EPA/AS01 on D0, D28, D168 (Bamako) arm 9: (n=10), to receive ENGERIX-B on D0, D28, and D168 arm 10: (n=10), to receive ENGERIX-B on DO, D28, and Dl68 arm 11: (n=120), to receive ENGERIX-B on D0, D28, and D168 (start study with Arm 2c and 2d); all subjects will undergo antimalarial drug treatment with Coartem on D-7 (prior to vaccination #1); vaccination with Menactra on D476
Interventions: intervention 1: Pfs25M-EPA intervention 2: Pfs230D1M-EPA intervention 3: AS01 intervention 4: Engerix-B intervention 5: Menactra intervention 6: Normal Saline intervention 7: Coartem
Intervention descriptions: intervention 1: The PpPfs25M and EcEPA lots, both manufactured at Walter Reed Bioproduction facility (Silver Spring, Maryland) in cGMP compliance, were used to manufacture the conjugate. PpPfs25M is a Pichia- expressed recombinant Pfs25 with a molecular mass of 18,713 Daltons.The Pfs25M- EPA was formulated as conjugated Pfs25M in 4 mM PBS to a 2X dilution of the high dose (188 (Micro)g/ml in 0.5 ml volume) in cGMP compliance at Walter Reed Bio-production facility in April 2016 and will be provided as a single use vial. intervention 2: The Pfs230D1M-EPA was formulated as conjugated Pfs230D1M in 4 mM PBS to a 2X dilution of the high dose (160 (Micro)g/ml in 0.5 ml volume) in cGMP compliance at Walter Reed Bio-production facility in April 2016 and will be provided as a single use vial. intervention 3: AS01B adjuvant will also be provided as a single use vial by GSK, 100 microgram/mL MPL and 100microgram/mL QS21 in a liposomal formulation in a volume of 0.625 mL. One dose injected of AS01B corresponds to 50 μg QS21 and 50 μg MPL. intervention 4: ENGERIX-B (hepatitis B vaccine; recombinant): is a sterile suspension of noninfectious hepatitis B virus surface antigen (HBsAg) for intramuscular administration. It contains purified HBsAg obtained by culturing genetically engineered Saccharomyces cerevisiae cells, which carry the surface antigen gene of the hepatitis B virus. Each 1-mL adult dose contains 20 g of HBsAg adsorbed on 0.5 mg aluminum as aluminum hydroxide. FDA approved for persons 20 years of age and older for a series of 3 doses on a 0-, 1-, 6-month schedule. intervention 5: Menactra is FDA approved for active immunization to prevent invasive meningococcal disease caused by Neisseria meningitidis serogroups A, C, Y, and W-135 (but does not protect against serotype B) for use in individuals 9 months through 55 years of age intervention 6: Sterile isotonic (0.9%) normal saline will be commercially procured in the US and shipped to Mali at ambient temperature. Normal saline will be administered in a 0.5 mL dose as an intramuscular injection. Normal saline will also be used for diluting Pfs25M-EPA and Pfs230D1M-EPA prior to formulation with AS01B for the lower dose groups in Bamako, Mali. intervention 7: Artemether/lumefantrine (Coartem(R)) is a licensed antimalarial in the US and Mali for treatment of uncomplicated malaria. It has an excellent safety profile and is widely used to treat malaria. Subjects who may have any contraindications to the use of these drugs will be excluded at screening. Coartem will be dosed with food and administered over 3 days for a total of 6 doses, as per package insert and standard adult dosing

Protocol documents:
Prot_SAP_000.pdf: 
PHASE 1 STUDY OF PFS25M-PFS230D1M/AS01  
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Phase 1 Dose Escalating, Double-Blind, Randomized Comparator Controlled Trial of the 
Safety and Immunogenicity of Pfs25M-EPA/AS01 and Pfs230D1M-EPA/AS01 Vaccines, 
Transmission Blocking Vaccines against Plasmodium falciparum, at Full and Fractional 
Dosing in Adults in Mali 
 
 
 
NIAID Protocol Number: 17-I-N006 
Project Assurance:  FWA #00005897 
Multi-institution:  Yes 
 
FMPOS Protocol Number: N°2016/133/CE/FMPOS 
Project Assurance:  FWA #00001769 
 
Sponsored by: 
Laboratory of Malaria Immunology and Vaccinology (LMIV) 
National Institute of Allergy and Infectious Diseases (NIAID) 
National Institutes of Health (NIH) 
 
IND Sponsor: 
Office of Clinical Research Policy and Regulatory Operations (OCRPRO), Division of Clinical 
Research (DCR), NIAID, NIH 
 TBV IND No:  17130 
 
Version:  11.0 
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CONFIDENTIAL 
                                
 
 
 
 


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Team Roster 
 
LMIV Staff (NIH/NIAID) 
 
NIAID Principal Investigator: 
Patrick Duffy, MD (D, E, F) 
LMIV/NIAID/NIH 
Building 29B, Room 4NN0629 Lincoln Drive 
Bethesda, Maryland 20892 
Tel: (301) 761-5089 
Patrick.Duffy@nih.gov  
 
NIAID Associate Investigators: 
Jen Hume, DPhil (D, E, F) 
 
 
Jacquelyn Lane, PA-C (D, E, F) 
 
 
David Cook, MD (D, E, F) 
 
 
Sara Healy, MD, MPH (D, E, F) 
 
Biostatistician: 
 
Bruce Swihart, PhD (E, F) 
 
 
NIAID/NIH/BRB 
 
 
Tel: (240) 669-5254 
 
 
Mali Staff (University of Sciences, Techniques, & Technologies of Bamako 
[USTTB]) 
  
Principal Investigator: 
 
 
 
 
Issaka Sagara MD, MSPH, PhD 
 
 
MRTC/DEAP/FMPOS  
USTTB 
 
 
BP 1805, Point G 
 
 
Bamako, Mali 
 
 
Tel:  +223-2022-8109 
 
 
Email: isagara@icermali.org 
 
 
Clinical Associate Investigators:    
Mahamadou S. Sissoko, MD, MSPH  
 
 
Mahamadoun Hamady Assadou, MD, MPH 
 
 
Abdoulaye Katile, MD, MPH 
 
 
Mamady Kone, MD 
 
 
Kourane Sissoko, MD 
 
 
Bourama Kamate, MD 


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Sibiri Sissoko, MD 
 
 
Amagana Dolo, PharmD, PhD 
 
 
Amatigue Zeguimé, PharmD 
 
 
 
 
Mamadou B. Coulibaly, PharmD, PhD  
 
 
Cheick F. Traore, PhD 
 
 
Daman Sylla, MD 
 
 
Key Study Personnel Roles: 
 
A. Obtain information by intervening or interacting with living individuals for research purposes 
B. Obtaining identifiable private information about living individuals 
C. Obtaining the voluntary informed consent of individuals to be subjects 
D. Makes decisions about subject eligibility 
E. Studying, interpreting, or analyzing identifiable private information or data/specimens for 
research purposes 
F. Studying, interpreting, or analyzing coded (linked) data or specimens for research purposes 
G. Some/all research activities performed outside of NIH 
 
 
Safety Oversight 
 
Data Safety Monitoring Board: 
NIAID DSMB 
 
Sponsor Medical Monitor: 
Shirley Jankelevich, MD  
Clinical Monitoring Research Program 
Frederick National Laboratory for Cancer 
Research  
Leidos Biomedical Research, Inc.  
5705 Industry Lane, Room 248  
Frederick, Maryland  21704 USA 
Tel: (301) 846-7322 
Fax: (301) 846-6224 
BlackBerry: (301) 401-3426 
Email: Shirley.jankelevich@nih.gov 
 
Mali Site Medical Monitor: 
Mamadou Dembele, MD 
 
 
 
Service Medicine Interne 
 
 
Centre Hospitalo-Universitaire du Point G 


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Tel: + 223 2022 5003  
 
 
Mobile: +223 7604 9387 
 
 
Email: hassiramadydembele@yahoo.fr 
 
 
 
 
Institutional Review Boards 
 
Institutional Review Board (IRB):  
NIH IRB 
 
 
FMPOS Ethics Committee (EC) 
 
 
Participating Sites 
 
Clinical Trial Sites:  
 Mali, West Africa  
Bancoumana Malaria Vaccine Center 
MRTC/DEAP/FMPOS 
 
Bamako/Sotuba, Mali 
Bancoumana and surrounding villages, Mali 
Doneguebougou and surrounding villages, 
Mali 
 
 
Laboratories 
Immunology Laboratories: 
 LMIV/NIAID/NIH 
 
 
 Bldg 29B, 29 Lincoln Drive 
 
 
 Bethesda, Maryland 20892 USA 
 
 
  
 
 
 
MRTC/DEAP Immunology Laboratory 
 
 
FMPOS, University of Sciences, Techniques 
& Technologies, Bamako 
 
 
BP 1805, Point G 
 
 
Bamako, Mali  
 
 
 
 
Clinical Laboratory:  
 MRTC/DEAP Clinical Laboratory 
 
 
 FMPOS, USTTB 
 
 
 BP 1805, Point G 
 
 
 Bamako, Mali 
 


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 Rodolphe Merieux Laboratory 
 
 
   Bamako, Mali 
 
Entomological Laboratory: 
 MRTC Medical Entomological Laboratory 
 
 
 FMPOS, USTTB 
 
 
 BP 1805, Point G 
 
 
 Bamako, Mali 
 
Collaborating Laboratories: 
 NIH Intramural Sequencing Center 
National Human Genome Research Institute 
(NHGRI) 
5625 Fishers Lane  
 
 
 Room 5S-16C MSC9400 
Bethesda, MD 20892 USA 
(301) 435-6156  
 
 
 
 
 NIH Research Technologies Branch 
 
 
 Genomic Technologies Section 
 
 
 Building 50, Room 5509 
 
 
 50 South Drive, MSC 8005 
 
 
 Bethesda, MD 20892 USA 
 
 
 (301) 496-0502 
 
 
 
 Laboratory of Malaria and Vector Research 
 
 
 12735 Twinbrook Parkway, Twinbrook 3 
 
 
 Rockville, Maryland  20852 USA 
 
 
 
 Laboratory of Parasitic Diseases 
 
 
 NIAID/NIH 
 
 
 Building 4, Room B1-03 
 
 
 4 Memorial Drive 
 
 
 Bethesda, MD 20892 USA 
 
 
 
 Center for Human Immunology, 
Autoimmunity, and Inflammation 
 
 
 National Heart, Lung and Blood Institute 
(NHLBI) 
 
 
 Building 10 - Magnuson CC, 7N110A 
 
 
 10 Center Dr 
 
 
 Bethesda, MD 20892-1655 USA 
 


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 Adaptive Biotechnologies 
 
 
 1551 Eastlake Ave E, Ste 200 
 
 
 Seattle, WA 98102, USA 
 
 
 
 Atreca, Inc. 
 
 
 500 Saginaw Drive, 1st Floor 
 
 
 Redwood City, CA 94063 USA 
 
 
 
 PATH Malaria Vaccine Initiative (MVI) 
 
 
 455 Massachusetts Avenue NW, Suite 1000 
 
 
 Washington, DC 20001 USA 
 
 
 
 GlaxoSmithKline (GSK) Vaccines 
 
 
 Rue de l'Institut 89, BE-1330  
 
 
 Rixensart, Belgium.  
 
 
 
 IAVI-Human Immunology Laboratory 
 
 
 Imperial College London, 
 
 
 Faculty of Medicine 
 
 
 2nd Floor Lift Bank D 
 
 
 Chelsea and Westminster Hospital 
 
 
 369 Fulham Road, London, UK 
 
 
 SW10 9NH 
 
 
 
 Precision Medicine 
 
 
 8425 Precision Way, Suite M 
 
 
 Frederick, MD 21701 USA 
 
 
Funder 
 
 
 NIAID Intramural Program 
 
 
Collaborators 
 
 
 PATH Malaria Vaccine Initiative 
 
 
 455 Massachusetts Avenue NW, Suite 1000 
 
 
 Washington, DC 20001 USA 
 
 
 
 GlaxoSmithKline (GSK) Vaccines 
 
 
 Rue de l'Institut 89, BE-1330  


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 Rixensart, Belgium.  
 
 
 
 Thomas Churcher, PhD 
 
 
 Imperial College London 
 
 
 St. Mary’s Campus, School of Public Health 
 
 
 Infectious Disease Epidemiology 
 
 
 Norfolk Place, London W21PG, UK 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
TABLE OF CONTENTS 
TEAM ROSTER ........................................................................................................................... 2 
TABLE OF CONTENTS ............................................................................................................. 7 
LIST OF FIGURES .................................................................................................................... 13 
LIST OF ABBREVIATIONS .................................................................................................... 15 
PROTOCOL SUMMARY ......................................................................................................... 18 


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PRÉCIS ........................................................................................................................................ 24 
1 INTRODUCTION AND RATIONALE ................................................................................ 25 
1.1 BACKGROUND ................................................................................................................................ 25 
1.2 RATIONALE FOR STUDY ................................................................................................................. 27 
2 PREVIOUS PRECLINICAL EXPERIENCE WITH STUDY AGENTS ......................... 29 
3 PREVIOUS HUMAN EXPERIENCE WITH STUDY AGENTS ...................................... 31 
3.1 PFS25-EPA ..................................................................................................................................... 31 
3.1.1 
Pfs25H-EPA/Alhydrogel®  in Healthy US Adults ................................................................... 31 
3.1.1.1 
Safety of Pfs25H-EPA/Alhydrogel® in Healthy US Adults ................................ 32 
3.1.1.2 
Immunogenicity of Pfs25H-EPA/Alhydrogel® in Healthy US Adults ................ 33 
3.1.2 
Pfs25H-EPA/Alhydrogel® in Healthy Malian Adults ............................................................. 35 
3.1.2.1 
Safety of Pfs25H-EPA/Alhydrogel® in Healthy Malian Adults ........................... 36 
3.1.2.2 
Immunogenicity of Pfs25H-EPA/Alhydrogel® in Healthy Malian Adults .......... 38 
3.1.3 
Pfs25M-EPA/Alhydrogel® in Healthy US Adults ................................................................... 40 
3.1.3.1 
Safety of Pfs25M-EPA/Alhydrogel® in Healthy US Adults ................................ 41 
3.1.3.2 
Immunogenicity of Pfs25M-EPA/Alhydrogel® in Healthy US Adults ................ 42 
3.1.4 
Pfs25M-EPA/Alhydrogel® in Healthy Malian Adults ............................................................ 44 
3.1.4.1 
Safety of Pfs25M-EPA/Alhydrogel® in Healthy Malian Adults.......................... 45 
3.1.4.2 
Immunogenicity of Pfs25M-EPA/Alhydrogel® in Healthy Malian Adults ......... 46 
3.2 PFS230-EPA ................................................................................................................................... 51 
3.2.1 
Pfs230D1M-EPA/Alhydrogel® in Healthy US Adults ............................................................ 51 
3.2.1.1 
Safety of Pfs230D1M-EPA/Alhydrogel® in Healthy US Adults ......................... 51 
3.2.1.2 
Immunogenicity of Pfs230D1M-EPA/Alhydrogel® in Healthy US Adults ......... 52 
3.2.2 
Pfs230D1M-EPA/Alhydrogel® in Healthy Malian Adults ..................................................... 53 
3.2.2.1 
Pfs230D1M-EPA/Alhydrogel® in Healthy Malian Adults .................................. 53 
3.2.2.2 
Immunogenicity of Pfs230D1M-EPA/Alhydrogel® in Healthy Malian Adults .. 54 
3.3 PFS25-EPA AND PFS230-EPA ....................................................................................................... 55 
3.3.1 
Pfs25M-EPA/Alhydrogel® and Pfs230D1M-EPA/Alhydrogel® in Healthy US Adults .......... 55 
3.3.1.1 
Safety of Pfs25M-EPA/Alhydrogel® and Pfs230D1M-EPA/Alhydrogel® in 
Healthy US Adults ......................................................................................................................... 55 
3.3.1.2 
Immunogenicity of Pfs25M-EPA/Alhydrogel® and Pfs230D1M-
EPA/Alhydrogel® in Healthy US Adults ....................................................................................... 56 
3.3.2 
Pfs25M-EPA/Alhydrogel® and Pfs230D1M-EPA/Alhydrogel® in Healthy Malian Adults .... 57 
3.3.2.1 
Safety of Pfs25M-EPA/Alhydrogel® and Pfs230D1M-EPA/Alhydrogel® in 
Healthy Malian Adults ................................................................................................................... 57 
3.3.2.2 
Immunogenicity of Pfs25M-EPA/Alhydrogel® and Pfs230D1M-
EPA/Alhydrogel® in Healthy Malian Adults ................................................................................. 58 
3.4 RATIONALE FOR FRACTIONAL DOSING .......................................................................................... 58 
3.5 RATIONALE FOR LONG TERM IMMUNOGENICITY FOLLOW-UP (PILOT/SOTUBA) AND EVALUATION 
OF FOURTH VACCINATION (MAIN/BANCOUMANA-DONEGUEBOUGOU) ................................................ 60 
3.5.1 
Long Term Immunogenicity Follow-up (Pilot/Sotuba) .......................................................... 60 
3.5.1.1 
Safety Results ...................................................................................................... 60 


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3.5.1.1.1 
Pfs25M-EPA/AS01 alone .................................................................................... 60 
3.5.1.1.2 
Pfs230D1M-EPA/AS01 alone ............................................................................. 60 
3.5.1.1.3 
Pfs25M-EPA/AS01 and Pfs230D1M-EPA/AS01 ............................................... 60 
3.5.1.1.4 
Engerix-B (comparator) ....................................................................................... 61 
3.5.1.2 
Immunogenicity and Functional Activity ............................................................ 61 
3.5.2 
Evaluation of Fourth Vaccination (Main/Bancoumana-Doneguebougou) ........................... 64 
3.5.2.1 
Safety ................................................................................................................... 64 
3.5.2.2 
Immunogenicity and Functional Activity ............................................................ 64 
3.6 CLINICAL DEVELOPMENT PLAN..................................................................................................... 67 
4 STUDY OBJECTIVES ........................................................................................................... 68 
4.1 PRIMARY OBJECTIVE ..................................................................................................................... 68 
4.2 SECONDARY OBJECTIVE ................................................................................................................ 69 
4.3 EXPLORATORY OBJECTIVES .......................................................................................................... 69 
5 STUDY DESIGN ..................................................................................................................... 69 
5.1 OVERALL DESIGN .......................................................................................................................... 69 
5.1.1 
Sotuba/Bamako Pilot Study ................................................................................................... 70 
5.1.2 
Bancoumana and Doneguebougou Main Study ..................................................................... 71 
5.2 STUDY ENDPOINTS ......................................................................................................................... 71 
5.2.1 
Primary Endpoint .................................................................................................................. 71 
5.2.2 
Secondary Endpoints ............................................................................................................. 71 
5.2.3 
Exploratory Endpoints ........................................................................................................... 71 
5.3 SAMPLE SIZE AND ESTIMATED DURATION OF STUDY ................................................................... 71 
6 STUDY POPULATION ......................................................................................................... 73 
6.1 DESCRIPTION OF POPULATION AND SITES ..................................................................................... 73 
6.1.1 
Rationale for Research Subject Selection .............................................................................. 73 
6.2 CLINICAL SITES .............................................................................................................................. 73 
6.2.1.1 
Sotuba/Bamako Site ............................................................................................. 74 
6.2.1.2 
Bancoumana and Doneguebougou Site ............................................................... 74 
6.3 RECRUITMENT ................................................................................................................................ 74 
6.4 INCLUSION CRITERIA (INITIAL ENROLLMENT AND RE-ENROLLMENT) .......................................... 75 
6.5 EXCLUSION CRITERIA (INITIAL ENROLLMENT AND RE-ENROLLMENT) ......................................... 75 
6.6 JUSTIFICATION FOR EXCLUSION OF PREGNANT WOMEN ............................................................... 77 
6.7 JUSTIFICATION FOR EXCLUSION OF CHILDREN .............................................................................. 77 
7 STUDY AGENTS ................................................................................................................... 77 
7.1 TRANSMISSION BLOCKING VACCINES (PFS25M-EPA/AS01, PFS230D1M-EPA/AS01) .............. 77 
7.1.1 
Pfs25M-EPA/AS01 ................................................................................................................. 77 
7.1.2 
Pfs230D1M-EPA/AS01 .......................................................................................................... 78 
7.1.3 
Disposition and Dispensation ................................................................................................ 79 
7.1.4 
Formulation, Packaging, and Labeling ................................................................................. 79 
7.1.5 
Study Agent Storage and Shipping ......................................................................................... 79 
7.1.6 
Preparation, Administration, and Dosage of Study Agents ................................................... 80 


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7.2 COMPARATOR VACCINES ............................................................................................................... 81 
7.2.1 
ENGERIX-B (Hepatitis B Vaccine) ........................................................................................ 81 
7.2.2 
Menactra® .............................................................................................................................. 81 
7.3 NORMAL SALINE ............................................................................................................................ 82 
7.4 ADMINISTRATION ........................................................................................................................... 82 
7.4.1.1 
Contraindications to Vaccination ......................................................................... 82 
7.4.1.2 
Indications for Deferral of Vaccination ............................................................... 83 
7.5 COARTEM® ..................................................................................................................................... 83 
7.5.1 
Preparation and Administration ............................................................................................ 84 
7.5.2 
Storage and Handling ............................................................................................................ 84 
7.5.3 
Return of Study Product ......................................................................................................... 84 
7.6 PROHIBITED MEDICATIONS AND PROCEDURES ............................................................................. 84 
7.7 VACCINE ACCOUNTABILITY .......................................................................................................... 85 
8 STUDY SCHEDULE .............................................................................................................. 85 
8.1 SCREENING PROCEDURES .............................................................................................................. 85 
8.1.1 
For Initial Enrollment (2016/2017) ....................................................................................... 85 
8.1.2 
Rescreening for Re-enrollment (2018) .................................................................................. 86 
8.2 ASSIGNMENT TO GROUPS .............................................................................................................. 87 
8.3 STUDY SUBJECT SCHEDULE ........................................................................................................... 88 
9 STUDY PROCEDURES/EVALUATIONS .......................................................................... 88 
9.1 PHOTOGRAPHS OF RASH OR INJECTION SITE REACTIONS.............................................................. 88 
9.2 CLINICAL LABORATORY TESTING ................................................................................................. 88 
9.3 MALARIA DIAGNOSTICS ................................................................................................................ 89 
9.3.1 
Blood Smears ......................................................................................................................... 89 
9.3.1.1 
Symptomatic Malaria ........................................................................................... 89 
9.3.2 
Rapid Diagnostics .................................................................................................................. 89 
9.3.3 
Malaria PCR .......................................................................................................................... 90 
9.3.4 
Malaria Clinical Management ............................................................................................... 90 
9.4 IMMUNOLOGIC LABORATORY TESTING ......................................................................................... 90 
9.4.1 
Antibody Assay ....................................................................................................................... 90 
9.4.2 
B-Cell and T-Cell Assays ....................................................................................................... 91 
9.4.3 
Transcriptional Profiling ....................................................................................................... 91 
9.4.4 
Transmission-Blocking Assays/Evaluations .......................................................................... 92 
9.4.4.1 
Standard Membrane-Feeding Assay .................................................................... 92 
9.4.4.2 
Direct Skin Feeds ................................................................................................. 93 
9.4.4.2.1 
Withdrawal from DSFs ........................................................................................ 95 
9.5 ENTOMOLOGICAL PROCEDURES AND INSECTARY USED IN DSF ................................................... 95 
9.5.1 
Mosquito Collection ............................................................................................................... 95 
9.5.2 
Mosquito Rearing at the Insectary ......................................................................................... 95 
9.5.3 
Mosquito Dissection .............................................................................................................. 96 
9.6 SCHISTOSOMIASIS AND HELMINTHES INFECTION TESTING ........................................................... 96 
9.7 OTHER LABORATORY ASSAYS ....................................................................................................... 97 


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10 RESEARCH USE OF STORED HUMAN SAMPLES, SPECIMENS, OR DATA ......... 97 
11 DATA SHARING PLAN ........................................................................................................ 98 
12 ASSESSMENT OF SAFETY ................................................................................................. 99 
12.1 DOCUMENTING, RECORDING, AND REPORTING ADVERSE EVENTS ............................................... 99 
12.2 DEFINITIONS ................................................................................................................................... 99 
12.3 INVESTIGATOR ASSESSMENT OF ADVERSE EVENTS .................................................................... 101 
12.3.1 Severity ................................................................................................................................ 102 
12.3.2 Causality .............................................................................................................................. 103 
12.4 INVESTIGATOR REPORTING RESPONSIBILITIES TO THE SPONSOR ............................................... 104 
12.4.1 Adverse Events ..................................................................................................................... 104 
12.4.2 Serious Adverse Events ........................................................................................................ 104 
12.4.3 Unanticipated Problems ...................................................................................................... 105 
12.4.4 Pregnancy ............................................................................................................................ 105 
12.5 REPORTING PROCEDURES TO THE NIH IRB................................................................................. 106 
12.5.1 Expedited Reporting to the NIH IRB ................................................................................... 106 
12.5.2 Annual Reporting to the NIH IRB ........................................................................................ 107 
12.6 INVESTIGATOR REPORTING RESPONSIBILITIES TO THE LOCAL IRB ............................................ 107 
12.7 FOLLOW-UP OF ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS .......................................... 107 
12.8 SPONSOR’S REPORTING RESPONSIBILITIES .................................................................................. 107 
12.9 HALTING CRITERIA FOR THE STUDY ............................................................................................ 108 
12.9.1 Reporting of Study Halting .................................................................................................. 109 
12.9.2 Resumption of a Halted Study .............................................................................................. 109 
12.10 
HALTING CRITERIA FOR A SUBJECT .................................................................................... 109 
12.10.1 Reporting of Halting for a Subject ....................................................................................... 110 
12.10.2 Resumption of a Halted Subject ........................................................................................... 110 
12.11 
DISCONTINUATION OF STUDY ............................................................................................. 110 
12.12 
WITHDRAWAL OF AN INDIVIDUAL SUBJECT ....................................................................... 110 
12.12.1 Replacement of Withdrawn Subjects .................................................................................... 111 
12.13 
UNBLINDING ........................................................................................................................ 111 
12.13.1 Unblinding by Group for Interim Analysis .......................................................................... 112 
12.14 
SAFETY OVERSIGHT ............................................................................................................ 112 
12.14.1 Safety Review and Communications Plan ........................................................................... 112 
12.14.2 Sponsor Medical Monitor .................................................................................................... 112 
12.14.3 Independent Safety Monitor (ISM) ...................................................................................... 113 
12.14.4 Data and Safety Monitoring Board ..................................................................................... 113 
13 CLINICAL MONITORING ................................................................................................ 116 
13.1 SITE MONITORING PLAN .............................................................................................................. 116 
14 STATISTICAL CONSIDERATIONS ................................................................................ 116 
14.1 DESCRIPTION OF STATISTICAL METHODS .................................................................................... 116 
14.2 PRIMARY OBJECTIVE ................................................................................................................... 117 
14.3 SECONDARY OBJECTIVES............................................................................................................. 117 


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14.4 EXPLORATORY OBJECTIVES ........................................................................................................ 118 
14.5 SAMPLE SIZE AND POWER CALCULATIONS ................................................................................. 118 
14.6 RANDOMIZATION ......................................................................................................................... 121 
15 HUMAN SUBJECT PROTECTIONS AND ETHICAL OBLIGATIONS ..................... 121 
15.1 INSTITUTIONAL REVIEW BOARD .................................................................................................. 122 
15.2 INFORMED CONSENT PROCESS .................................................................................................... 122 
15.2.1 Mali Site Community Permission and Individual Informed Consent Process ..................... 122 
15.2.1.1 
Community Permission ...................................................................................... 122 
15.2.1.2 
Individual Informed Consent ............................................................................. 123 
15.3 SUBJECT CONFIDENTIALITY ......................................................................................................... 124 
15.4 RISKS ............................................................................................................................................ 124 
15.4.1 Venipuncture ........................................................................................................................ 124 
15.4.2 Intramuscular Immunizations .............................................................................................. 124 
15.4.2.1 
Immunization with Pfs25M-EPA/AS01 and/or Pfs230D1M/AS01 ................... 125 
15.4.2.2 
Immunization with ENGERIX-B ...................................................................... 125 
15.4.3 Immunization with Menactra® ............................................................................................. 126 
15.4.4 Coartem® ............................................................................................................................. 126 
15.4.5 Direct Skin Feeds ................................................................................................................. 127 
15.4.6 Other risks ........................................................................................................................... 127 
15.5 BENEFITS ...................................................................................................................................... 128 
15.6 COMPENSATION ........................................................................................................................... 128 
16 DATA HANDLING AND RECORD KEEPING ............................................................... 129 
16.1 DATA CAPTURE AND MANAGEMENT ........................................................................................... 129 
16.2 TYPES OF DATA ............................................................................................................................ 130 
16.3 RETENTION OF STUDY RECORDS ................................................................................................. 130 
16.4 PROTOCOL REVISIONS ................................................................................................................. 130 
APPENDIX A: STUDY SCHEMA.......................................................................................... 131 
APPENDIX B:  SCHEDULE OF PROCEDURES/EVALUATIONS ................................. 133 
APPENDIX C:  DAY TO DAY SCHEDULE BY ARM ....................................................... 137 
APPENDIX D:  TOXICITY TABLE ...................................................................................... 169 
APPENDIX E: REFERENCES ............................................................................................... 174 
 
 


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LIST OF FIGURES 
Figure 1: Protein alignments of recombinant Pfs25M and Pfs230D1M to their respective native protein or 
protein fragment .......................................................................................................................................... 27 
Figure 2: Comparison of Transmission Blocking Activity of Pfs25M-EPA/AS01 and Pfs230D1M-
EPA/AS01 to Pfs25M-EPA/Alhydrogel® and Pfs230D1M-EPA/Alhydrogel®  in CD1 Mice. .................. 30 
Figure 3: Measurement of Pfs25-specific Antibody Decay from Mouse AS01 study Comparing 
Alhydrogel® versus AS01. .......................................................................................................................... 31 
Figure 4: Related Local Injection Site Reactogenicity and Systemic Adverse Events. .............................. 32 
Figure 5: Laboratory Adverse Events ......................................................................................................... 33 
Figure 6: Anti-Pfs25 Antibodies in Volunteers Immunized with Pfs25-EPA/Alhydrogel® ....................... 34 
Figure 7: Transmission Reducing Activities Induced by Pfs25-EPA/Alhydrogel® ................................... 35 
Figure 8: Phase 1 Study of Pfs25H-EPA Conjugates in Malian Healthy Adults ........................................ 36 
Figure 9: Anti-Pfs25 and Anti-EPA IgG ELISA Titers in Phase 1 Trial of Pfs25H-EPA/Alhydrogel® in 
Mali ............................................................................................................................................................. 38 
Figure 10: Serum Transmission Blocking Activity after Pfs25H-EPA/Alhydrogel® Dose 4 in Mali. ....... 39 
Figure 11: Confirmation that Human Transmission Blocking Activity is Mediated by Anti-Pfs25 IgG ... 40 
Figure 12: Pfs25–specific antibody responses in US naïve subjects receiving Pfs25M vaccinations (#15-I-
0044) ........................................................................................................................................................... 43 
Figure 13: Pfs25 and Pfs230 Functional Activity (SMFA) ........................................................................ 44 
Figure 14: Pfs25–specific antibody responses in Malian subjects receiving Low Dose Pfs25M, 
Pfs230D1M, and Pfs25M + Pfs230D1M (#15-I-0044) .............................................................................. 47 
Figure 15 : Pfs25 anf Pfs230 immunogenicity (ELISA) and functional activity (SMFA) in malaria-
exposed Malian adults for 3 and 4 vaccine doses ....................................................................................... 48 
Figure 16: Pfs230–specific antibody responses in US naïve subjects receiving Pfs230D1M vaccinations 
(#15-I-0044) ................................................................................................................................................ 52 
Figure 17: Pfs230 Functional Activity (SMFA) in the Presence or Absence of Complement ................... 53 
Figure 18. Antibody responses to Pfs25 and Pfs230 at low and high dosage and with single and co-
administration dosing obtained 8 weeks post vaccination #3. .................................................................... 62 
Figure 19: Functional activity of Pfs230 vaccinees by SMFA at 2 weeks and 12 weeks post Vaccination 
#2 in the open label safety cohort. .............................................................................................................. 63 
Figure 20 : Antibody responses to Pfs230 at 12 weeks post-vaccination #3 with either full or fractional 
dosing .......................................................................................................................................................... 66 
Figure 21: Functional activity of Pfs230 by SMFA at 12 weeks post-vaccination #3 with either full or 
fractional dosing .......................................................................................................................................... 67 
 


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Table 1: Phase 1 Study of Pfs25H-EPA Conjugates in US Healthy Adults ............................................... 31 
Table 2: Summary of Reported Adverse Events in Pfs25H Vaccine Arms, #13-I-N109, Mali Low Dose 
Cohort (2A) and Mali High Dose Cohort (2B) ........................................................................................... 37 
Table 3: Subject Enrollment and Vaccinations in US ................................................................................. 41 
Table 4: Summary of Reported Adverse Events in Pfs25M Vaccine Arms, #15-I-0044, US Cohort ........ 42 
Table 5: Subject Enrollment and Vaccination in Mali ................................................................................ 45 
Table 6: Summary of Reported Adverse Events in Pfs25M Safety Vaccine Arm versus Comparator, #15-
I-0044, Mali Cohort .................................................................................................................................... 46 
Table 7: Summary of BS and DSF completed for protocol 15-I-0044 during DSF evaluation period post 
Vaccination #3 and #4. ............................................................................................................................... 50 
Table 8: Summary of Reported Adverse Events in Pfs230D1M Vaccine Arms, #15-I-0044, US Cohort . 51 
Table 9: Summary of Reported Adverse Events in Pfs230D1M Safety Vaccine Arm versus Comparator, 
#15-I-0044, Mali Cohort ............................................................................................................................. 54 
Table 10: Summary of Reported Adverse Events in Pfs25 and Pfs230D1M Vaccine Arms, #15-I-0044, 
US Cohort ................................................................................................................................................... 56 
Table 11: Summary of Reported Adverse Events in Pfs25M and Pfs230D1M Safety Vaccine Arm versus 
Comparator, 15-I-0044, Mali Cohort .......................................................................................................... 58 
Table 12: TBV Formulation ........................................................................................................................ 80 
Table 13: Transmission Blocking Assays ................................................................................................... 92 
Table 14: Solicited Adverse Events .......................................................................................................... 101 
Table 15: Definitions for Severity of AE Grading .................................................................................... 102 
Table 16: Vaccination Dose Escalation Schedule and DSMB Meeting ................................................... 114 
Table 17: Estimated Compensation Schedule1 ......................................................................................... 129 
 
 
 


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List of Abbreviations 
AE 
adverse event/adverse experience 
AGC 
absolute granulocyte count 
ALT 
alanine transaminase 
ANC 
absolute neutrophil count 
AR 
adverse reaction 
β-hCG 
beta human choriogonadotropin 
CBC 
complete blood count 
CFR 
Code of Federal Regulations 
cGMP 
current Good Manufacturing Practices 
CHMI 
controlled human malaria infection  
CIR 
Center for Immunization Research (Johns Hopkins University) 
CRF 
case report form 
CRIMSON 
Clinical Research Information Management System of the NIAID 
CRP 
C-reactive protein 
CSO 
Clinical Safety Office 
CSP 
Circumsporozoite protein  
DHHS 
Department of Health and Human Services 
DNA 
deoxyribonucleic acid 
DSF 
direct skin feeds 
DSMB 
Data Safety Monitoring Board 
EC 
Ethics committee  
ELISA 
enzyme-linked immunosorbent assay 
ELISPOT 
enzyme-linked immunosorbent spot assay 
EMA 
European Medicines Agency  
EPA 
ExoProtein A 
EPI 
Expanded Program of Immunization  
FDA 
Food and Drug Administration 
FMPOS 
Faculte de Medecine Pharmacie d’OdontoStomatologie 
FWA 
Federal Wide Assurance 
GCP 
Good Clinical Practice 
GSK 
GlaxoSmithKline 
HBsAg 
hepatitis B surface antigen 
HCV 
hepatitis C virus 
HIV 
human immunodeficiency virus 
HPV 
human papillomavirus  
HRPP 
Human Research Protection Program 
IB 
Investigator’s Brochure 
IM 
intramuscular 
IND 
Investigational New Drug 


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List of Abbreviations 
IRB 
Institutional Review Board 
ISM 
independent safety monitor 
IV 
Intravenous therapy 
LMIV 
Laboratory of Malaria Immunology and Vaccinology (of NIAID) 
µg 
micrograms 
MPL 
monophosphoryl lipid  
MRTC 
Malaria Research and Training Center (Mali) 
MVI 
Malaria Vaccine Initiative  
n 
number (typically refers to subjects or participants) 
NIAID 
National Institute of Allergy and Infectious Diseases, NIH 
NIH 
National Institutes of Health 
NOCI 
New Onset of Chronic Illness  
OCRPRO 
Office of Clinical Research Policy and Regulatory Operations 
OHRP 
Office for Human Research Protections 
OHSR 
Office of Human Subjects Research 
PBS 
peripheral blood mononuclear cell 
PCR 
Polymerase Chain Reaction 
PE 
Pre-erythrocytic 
Pfs25/Pfs230 
Surface antigen of zygotes and ookinetes in the mosquito stage of 
Plasmodium falciparum 
PI 
Principal Investigator 
RNA 
ribonucleic acid 
RVF 
rift valley fever  
SAE 
serious adverse event/serious adverse experience 
SD 
standard deviation 
SERF 
Safety Expedited Report Form 
SMFA 
standard membrane feeding assay 
SOP 
Standard Operating Procedure 
SRCP 
Safety Review and Communication Plan 
SUSAR 
Serious and Unexpected Suspected Adverse Reaction  
TB 
tuberculosis 
TBA 
transmission blocking assay 
TBS 
TRIS-buffered saline  
TBV 
transmission blocking vaccine 
UP 
unanticipated problem 
USPHS 
U.S. Public Health Service 
USTTB 
University of Sciences, Techniques & Technologies of Bamako 
VIMT 
vaccine to interrupt malaria transmission  
WBC 
white blood cell 


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List of Abbreviations 
WHO 
World Health Organization 
WMW 
Wilcoxon Mann Whitney 
WRAIR 
Walter Reed Army Institute of Research 
 
 
 
 
 
 
 


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PROTOCOL SUMMARY 
Full Title: 
Phase 1 Dose Escalating, Double-Blind, Randomized Comparator 
Controlled Trial of the Safety and Immunogenicity of Pfs25M-
EPA/AS01 and Pfs230D1M-EPA/AS01 Vaccines, Transmission 
Blocking Vaccines against Plasmodium falciparum, Full and 
Fractional Dosing in Adults in Mali 
Short Title: 
Phase 1 Study of Pfs25M-Pfs230D1M/AS01  
Clinical Phase: 
1 
IND Sponsor: 
OCRPRO  
Conducted by: 
Laboratory of Malaria Immunology and Vaccinology 
(LMIV)/National Institute of Allergy and Infectious Diseases 
(NIAID)/National Institutes of Health (NIH) 
Clinical Sites: 
Malaria Research and Training Center (MRTC), University of 
Sciences, Techniques & Technologies of Bamako (USTTB), Mali, 
West Africa:  
 
Sotuba, Bamako, Mali 
 
Bancoumana and surrounding villages, Mali 
 
Doneguebougou and surrounding villages, Mali 
Principal Investigators: 
Patrick Duffy, MD (LMIV/NIAID/NIH) 
 
Issaka Sagara MD, MSPH, PhD (MRTC/DEAP/FMPOS) 
Sample Size: 
N = 305 Vaccinated 
Accrual Ceiling: 
N = 900;  
 
Reconsent: 301 previously enrolled and vaccinated 
 
   Pilot Study: 65 
 
   Main Study: 236 
Study Population: 
Healthy Malian adults 
Accrual Period: 
Approximately December 2016 to April 2017 
 
Approximately February 2018 to July 2018 (for re-consent) 
Study Design: 
 
In Sotuba, Bamako, Mali (N=65) 
 
 
Dose-escalating, open label, randomized, pilot study  
 
Group 1: Pfs25M-EPA/AS01 (n=15) 


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• Arm 1a (n=5), to receive 16 μg Pfs25M-EPA/AS01 on 
D0, D28, D168 
 
• Arm 1b (n=10), to receive 47 μg Pfs25M-EPA/AS01 on 
D0, D28, D168 
 
Group 2: Pfs230D1M-EPA/AS01 (n=15) 
 
• Arm 2a (n=5), to receive 13 μg Pfs230D1M-EPA/AS01 
on D0, D28, D168 
 
• Arm 2b (n=10), to receive 40 μg Pfs230D1M-EPA/AS01 
on D0, D28, D168 
 
Group 3: Pfs25M-EPA/AS01 and Pfs230D1M-EPA/AS01 (n=15) 
 
• Arm 3a (n=5), to receive 16 μg Pfs25M-EPA/AS01 and 
13 μg Pfs230D1M-EPA/AS01 on D0, D28, D168  
 
• Arm 3b (n=10), to receive 47 μg Pfs25M-EPA/AS01 and 
40 μg Pfs230D1M-EPA/AS01 on D0, D28, D168 
 
Group 4: Comparator (n=20) 
 
• Arm 4a (n=10), to receive ENGERIX-B on D0, D28, and 
D168  
 
• Arm 4b (n=10), to receive ENGERIX-B on D0, D28, and 
D168 
 
 
In Bancoumana and Doneguebougou, Mali (N=240) 
 
Double-blind, randomized, phase 1 clinical trial 
 
Group 2: Pfs230D1M-EPA/AS01 (n=120) 
 
• Arm 2c (n=60), to receive 40 μg Pfs230D1M-EPA/AS01 on D0, 
D28, D168; all subjects will undergo antimalarial drug treatment 
with Coartem® on D-7 (prior to vaccination #1); 4th vaccination on 
D476 
 
• Arm 2d (n=60), to receive 40 μg Pfs230D1M-EPA/AS01 (500 
µL TBV + AS01) on D0, D28, then 8 μg Pfs230D1M-EPA/AS01 
(100 µL TBV + AS01; fractional dose) on D168; all subjects will 
undergo antimalarial drug treatment with Coartem® on D-7 (prior 
to vaccination #1); 4th vaccination on D476 
 
Group 4: Comparators (n=120) 
 
• Arm 4c (n=120), to receive ENGERIX-B on D0, D28, and D168 
(start study with Arm 2c and 2d); all subjects will undergo 


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antimalarial drug treatment with Coartem® on D-7 (prior to 
vaccination #1); vaccination with Menactra® on D476 
 
Enrollment will be staggered for additional safety 
Study Duration: 
Start Date: Approximately December 2016 
End Date: Approximately March 2018 
 
Mali study participants will be enrolled for a total of 12 to 16 
months (at least 6 month follow-up after the last vaccination) 
depending on vaccination schedule and arm assignment and timing 
of screening 
 
Re-enrollment (long term immunogenicity follow-up; 4th 
vaccination) 
 
Start Date: Approximately February 2018 
 
End Date: Approximately August 2019 
 
Participants who had previously been enrolled in the study (up to 
301) will be re-enrolled for an additional 6 to 18 months depending 
on arm assignment and timing of reconsent. 
 
Study Agent/ 
Intervention Description: 
Pfs25M-EPA/AS01: Pfs25, a surface antigen of zygotes and 
ookinetes in the mosquito stages of P. falciparum, is a lead 
candidate for developing a malaria transmission blocking vaccine. 
Recombinant Pfs25M was expressed in Pichia pastoris (P. 
pastoris). Recombinant ExoProtein A (EPA), a mutant, non-toxic 
protein corresponding to sequence of EPA of Pseudomonas 
aeruginosa (P. aeruginosa), was expressed in Escherichia coli (E. 
coli). Pfs25M and EPA were chemically crosslinked to form 
Pfs25M-EPA conjugate. AS01B will be mixed directly with the 
vialed conjugated Pfs25M for a final formulation of either 16µg 
conjugated Pfs25M, 15 µg conjugated EPA in AS01 (25µg MPL+ 
25µg QS21 liposomal formulation) for Arms 1a, 3a or 47µg 
conjugated Pfs25M, 45µg conjugated EPA in AS01 for Arms 1b, 
3b.  All manufacturing processes including recombinant protein 
production and the chemical conjugation were performed in 
compliance with cGMP.  
 
 
 
Pfs230D1M-EPA/AS01: Pfs230 is a parasite antigen expressed in 
gametocytes and on the surface of emerging gametes in the 
mosquito host and is also a TBV target based on observations in 


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animal studies that the antigen-specific polyclonal and monoclonal 
antibodies conferred transmission-blocking activities in SMFA 
(Kaushal, Carter et al. 1983, Williamson, Keister et al. 1995). 
Recombinant Pfs230D1M was expressed in P. pastoris as an 
approximately 20 kDa fragment containing domain 1. 
Recombinant EPA, a mutant, non-toxic protein corresponding to 
sequence of EPA of P. aeruginosa, was expressed in E. coli. 
Pfs230D1M and EPA were chemically crosslinked to form 
Pfs230D1M-EPA conjugate. AS01B will be mixed directly with 
the vialed conjugated Pfs230D1M for a final formulation of either 
13µg conjugated Pfs230D1M, 10µg conjugated EPA in AS01 
(25µg MPL+ 25µg QS21 liposomal formulation) for Arms 2a, 3a 
or 40µg conjugated Pfs230D1M, 31µg conjugated EPA in AS01  
for Arms 2b, 3b, 2c, 2d.  The fractional dose (Dose #3) in Arm 2d 
will be 1/5th of the Pfs230D1M dose in AS01 (final formulation of 
8µg conjugated Pfs230D1M, 2µg conjugated EPA in AS01 [5µg 
MPL+ 5µg QS21 liposomal formulation]).   All manufacturing 
processes including recombinant proteins production and the 
chemical conjugation were performed in compliance with cGMP.  
 
 
 
ENGERIX-B (hepatitis B vaccine; recombinant): is a sterile 
suspension of noninfectious hepatitis B virus surface antigen 
(HBsAg) for intramuscular administration. It contains purified 
HBsAg obtained by culturing genetically engineered 
Saccharomyces cerevisiae cells, which carry the surface antigen 
gene of the hepatitis B virus. Each 1-mL adult dose contains 20 µg 
of HBsAg adsorbed on 0.5 mg aluminum as aluminum hydroxide. 
FDA approved for persons 20 years of age and older for a series of 
3 doses on a 0-, 1-, 6-month schedule. 
 
 
 
Menactra® (meningococcal serogroup A, C, Y, and W-135): is a 
sterile, intramuscularly administered vaccine that contains 
Neisseria meningitidis serogroup A, C, Y, and W-135 capsular 
polysaccharide antigens individually conjugated to diphtheria 
toxoid protein.  A single dose (0.5 mL) is recommended for those 
individuals 18 to 50 years of age and otherwise healthy who are at 
increased risk for meningococcal disease (e.g., individuals in an 
epidemic or highly endemic country such as Mali).  
 
Primary Objective: 
 


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• To assess safety and reactogenicity of Pfs25M-EPA/AS01, 
Pfs230D1M-EPA/AS01, and simultaneous administration of 
Pfs25M-EPA/AS01 and Pfs230D1M-EPA/AS01, including 
Pfs230D1M-EPA/AS01 at fractional dosing in Malian adults. 
(All arms) 
• To assess safety and reactogenicity of a booster dose of 
Pfs230D1M-EPA/AS01 at full dosing in Malian adults. (Arms 
2c, 2d, 4c) 
 
Secondary Objectives: 
 
• To assess the antibody response to the Pfs25 and Pfs230 
protein as measured by ELISA. (All arms) 
• To assess the functional antibody response to the Pfs25 and 
Pfs230 protein as measured by standard membrane feeding 
assay (SMFA).  (All arms except 1a, 2a, 3a)  
• To assess the functional antibody response to Pfs230 proteins 
as measured by direct skin feeds (DSF).  (Arms 2c, 2d, 4c 
only) 
Exploratory Objectives: 
 
  
• To explore cellular and transcriptomic responses to Pfs25 and 
Pfs230 vaccines when administered alone and in combination.  
(All arms except 1a, 2a, 3a) 
• To explore the impact of co-infections on malaria vaccine 
responses. (Arms 2c, 2d, 4c only) 
• To explore the antibody repertoire of functional antibody 
responses. (All arms except 1a, 2a, 3a) 
Primary Endpoint: 
 
• Incidence of local and systemic adverse events (AEs) and 
serious adverse events (SAEs) in Malian adults. (All arms) 
• Incidence of local and systemic adverse events (AEs) and 
serious adverse events (SAEs) in Malian adults. (Arms 2c, 2d, 
4c) 
Secondary Endpoints: 
 
• Anti-Pfs25 IgG levels as measured by ELISA. (All arms)  
• Anti-Pfs230 IgG levels as measured by ELISA. (All arms)  


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• TRA/TBA of induced antibody in SMFA. (All arms except 1a, 
2a, 3a) 
• Transmission interruption activity measured by DSF. (Arms 
2c, 2d, 4c only)  
Exploratory Endpoints: 
 
• Cellular immune responses to vaccination. (All arms except 1a, 
2a, 3a) 
• Whole genome transcriptional profiling. (All arms except 1a, 
2a, 3a) 
• Antibody levels against recombinant EPA, and other malaria 
antigens, such as Pfs48/45, expressed during the gametocyte 
development. (All arms except 1a, 2a, 3a)  
• Schistosomiasis detection (in urine). (Arms 2c, 2d, 4c only)  
• qPCR for helminth detection (from stool). (Arms 2c, 2d, 4c 
only)  
• Sequence B cell receptor/antibody genes. (All arms except 1a, 
2a, 3a) 
 
 
 
 


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Précis 
A vaccine to interrupt malaria transmission (VIMT) would be a valuable tool for local 
elimination or eradication of this disease, and may contain components that block transmission to 
mosquitoes (such as Pfs25 or Pfs230) or that prevent human infection (such as the vaccine 
RTS,S). Pfs25 and Pfs230, surface antigens of zygotes and ookinetes (and gametocytes for 
Pfs230) in the mosquito stage of P. falciparum, are the lead candidates for a malaria transmission 
blocking vaccine (TBV). Recombinant Pfs25M and recombinant Pfs230D1M have each been 
conjugated to P. aeruginosa ExoProtein A (EPA) and adjuvanted with AS01. Our ongoing 
experience with Pfs25M-EPA and Pfs230D1M-EPA in Malian adult trial participants, and the 
extensive experience with the AS01 adjuvants in African children and adults, justify conducting 
the first-in-human trial of Pfs25M-EPA and Pfs230D1M-EPA with AS01 in Malian adults. This 
dose-escalating phase 1 study will determine safety, immunogenicity, and functional activity of 
these vaccines in Malian adults. Pfs230D1M-EPA in AS01  will be assessed by mosquito feeding 
assays in Malian adults for evidence that they may reduce the number of malaria transmission 
events in study subjects. 
  
A total of 305 subjects will be enrolled at multiple sites in Mali, West Africa to receive 
escalating doses of a malaria transmission blocking vaccine (s): Pfs25M-EPA/AS01, 
Pfs230D1M-EPA/AS01, or simultaneous administration of Pfs25M-EPA/AS01 and 
Pfs230D1M-EPA/AS01; or a comparator vaccine (ENGERIX-B). Enrollment within each group 
will be staggered for additional safety and subjects will only be enrolled into the co-
administration group once each individual dose has been administered. Subjects will be followed 
for at least 6 months after the last vaccination. Safety outcomes will be local and systemic 
adverse events and serious adverse events. Immunogenicity outcomes will be antibody responses 
as measured by ELISA against recombinant Pfs25, Pfs230, EPA, CSP, and B cell and T cell 
responses. Functional activity of the induced antibodies will be assessed in TBV arms by 
standard membrane feeding assays conducted at the National Institute of Allergy and Infectious 
Diseases, and activity that interrupts malaria transmission will be measured in the double-blind 
portion of the study by direct skin feeding assays in Mali. 
 
Subjects in the open label safety cohorts (Arms 1a, 1b, 2a, 2b, 3a, 3b, 4a, 4b) will be offered re-
enrollment for follow-up laboratory assessment to explore the duration of immunogenicity and 
functional activity at approximately 9, 12 months post vaccination.  Following scheduled, 
intentional unblinding, subjects enrolled in Arms 2c, 2d, and 4c will be provided the opportunity 
to re-enroll for a fourth vaccination (Arm 2c and 2d with 40 µg dose of Pfs230D1M-EPA/AS01; 
Arm 4c with Menactra®) approximately 1 year post vaccination #3.  Subjects in these arms will 
also be eligible to re-enroll for follow up of duration of immunogenicity and functional activity 
at approximately 9, 12 months post vaccination if they choose not to enroll to receive the booster 
vaccination.  Subjects will be followed similarly to the previous year for safety, immunogenicity, 
and functional activity. 
  
 


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1 INTRODUCTION AND RATIONALE 
1.1 
Background  
According to the World Health Organization (WHO), global malaria control efforts have resulted 
in a reduction in the number of deaths since 2000; however, 438,000 people are estimated to 
have died due to malaria in 2015, a decline of 48% since 2000.1 Morbidity and mortality caused 
by malaria also has significant direct and indirect costs to the economic development of countries 
in which the disease is endemic.2 These factors, as well as growing drug resistance of the 
parasite, widespread resistance of mosquitoes to insecticide, and increased human travel 
necessitates new approaches to malaria control and eradication. While licensure of the partially 
protective malaria vaccine (RTS,S 25μg/AS01E; Mosquirix™) seems likely, both the level and 
duration of protection provided by the vaccine are suboptimal, and RTS,S appears to act by 
reducing clinical disease rather than preventing infection.3,4 A vaccine that interrupts malaria  
transmission (VIMT) would be a valuable resource to add in the fight to eliminate or eradicate 
this disease.5,6   
 
 
Malaria transmission blocking vaccines (TBVs) aim to induce anti-sporogonic antibodies in the 
human host. The antibody is subsequently taken up with the blood meal and blocks parasite 
development in the mosquito, thereby halting transmission to another human host. Pfs25, a 
surface antigen of zygotes and ookinetes in the mosquito stage, is expressed only in the mosquito 
host of P. falciparum.7 Pfs25 has long been a lead candidate for a malaria TBV;8 however, 
soluble recombinant Pfs25 is poorly immunogenic. LMIV investigators and collaborators have 
chemically conjugated Pfs25 to ExoProtein A (EPA), a mutant and detoxified protein from 
Pseudomonas aeruginosa (P. aeruginosa). These conjugates have induced sustained, 
significantly higher antibody responses in mice, rabbits, and rhesus monkeys than unconjugated 
Pfs25.9-11 The conjugate protein, recombinant EPA, is not a component of any licensed vaccines 
but has been extensively studied as a component of conjugated typhoid and shigellosis 
vaccines12-14 and LMIV/MRTC’s previous phase 1 TBV studies involving Pfs25H, Pfs25M, and 
Pfs230D1M formulated with Alhydrogel®.  
 
Pfs25H-EPA, which forms a conjugated nanoparticle to Alhydrogel®, was evaluated in a phase 1 
study of US adults (2011 to 2014; NIAID Protocol #11-I-N237) and Malian adults (2013 to 
2014; NIAID Protocol #13-I-N109).15,16 Additionally, Pfs25M-EPA, which no longer contains 
heterologous amino acids which were present in Pfs25H to facilitate early-stage production, has 
been recently evaluated in a phase 1 study in US adults (2015) and Malian adults (2015-2016) 
under NIAID Protocol #15-I-0044. Both have been demonstrated to be a safe, immunogenic 
vaccines with transmission-blocking activity as assessed by a SMFA. In the Pfs25H-EPA 
studies, specific anti-Pfs25 antibodies were detected by ELISA in sera from subjects receiving 4 
doses of Pfs25H-EPA/Alhydrogel® in US and Mali. With each vaccine dose, antibody responses 
and associated functional activity increased; however, it was seen in both studies that the 


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antibody responses, and along with it the functional activity, diminished quickly following 
vaccination.  
 
Pfs230 is also a TBV target based on observations in animal studies that the antigen-specific 
polyclonal and monoclonal antibodies conferred transmission-blocking activities in SMFA.17,18 
The full-length Pfs230 precursor of 360 kDa is expressed in gametocytes within erythrocytes, 
and is processed to become a ~300 kDa mature protein upon translocation to the surface of 
freshly emerged gametes from erythrocytes.19  Presence of Pfs230 protein in the human host is 
consistent with the detection of anti-Pfs230 immunity in malaria-exposed populations, which 
raised anticipation that a Pfs230-based vaccine may be boosted by natural malaria infection. To 
facilitate the recombinant protein production, several N-terminal sub-domains within this 300 
kDa protein were evaluated and found to be capable of inducing functional antibodies to block 
transmission in animal studies.20,21 Based on these findings, using a quality by design strategy, 
LMIV developed and manufactured a recombinant Pfs230D1M corresponding to amino acid 
sequence positions #542-#736 of the full-length Pfs230 using P. pastoris as a production system. 
The 20-kDa recombinant Pfs230D1M conjugated to EPA elicited strong transmission-blocking 
activities in mice, rabbits, and Aotus monkeys. Pfs230D1M-EPA conjugated to Alhydrogel® has 
been evaluated in a phase 1 study in US adults (2015) and Malian adults (2015-2016) under 
NIAID Protocol #15-I-0044 and has demonstrated to be safe and immunogenic both in malaria 
naïve and malaria exposed adults.   
 
Both antigens used in the TBV candidates proposed for this study, Pfs25M and Pfs230D1M, 
were developed at LMIV and selected for clinical development given their relative conserved 
sequences against circulating parasites in our targeted study population. Pfs25, which as stated 
prior is exclusively expressed in the mosquito, is not under immune pressure and consequently 
the deduced amino acid sequence for 11 Malian P. falciparum isolates are identical to the 
recombinant form of Pfs25 (identified as Pfs25M) with the exception that Pfs25M has had the 
insertion sequence and membrane anchor deleted, and the putative N-linked glycosylation sites 
removed (Figure 1A).  Pfs230, a parasite protein that is expressed during sexual development in 
the human host as a large protein of approximately 230 kDa, is known to contain various amino 
acid substitutions throughout the protein; however, the function of these changes is unknown.  
The recombinant protein Pfs230 domain 1 (also identified as Pfs230D1M) which comprises 
about 10% of the whole Pfs230 protein is also known to contain minor allelic variants in 
comparison to NF54 or the 3D7 clone of NF54.22  A comparative analysis of the deduced amino 
acid sequence of native Pfs230 domain 1 with the same 11 Malian isolates evaluated above for 
Pfs25M shows at least a 99% homology including the known N to Q at amino acid position 44 to 
remove the unique putative N-linked glycosylation site (Figure 1B).  Two other point mutations 
were observed within Pfs230D1: G to S at position 64 and K to N at position 120.  These same 
mutations were also observed in an analysis of over two-thousand parasite isolates.22  In 
particular, in West Africa, the minor allelic frequency was reported to be 0.111 and 0.339 for 
G605S and K661N which corresponds to the positions 64 and 120 shown in Figure 1B, 


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respectively.   Of note, rabbit antisera raised against Pfs230D1M blocked parasite transmission 
of a Thailand isolate with the G605S mutation, indicating the presence of this minor allele should 
not impact efficacy the Pfs230D1M-EPA conjugated vaccine.  The impact of the K661N 
mutation on the biological impact of the vaccine remains to be determined. 
 
Figure 1: Protein alignments of recombinant Pfs25M and Pfs230D1M to their respective 
native protein or protein fragment 
 
 
Figure 1A. Deduced amino acid sequence of full-length Pfs25 3D7 allele and the boundaries for 
recombinant Pfs25M.  Eleven additional Malian isolates, identified below, were identical to the 3D7 allele 
(not shown).  Figure 1B.  Deduced amino acid sequence of Pfs230D1 3D7 and eleven other Malian isolates 
in addition to the amino acid sequence of recombinant Pfs230D1M (abbreviated as 230D1M).  The amino 
acids highlighted in yellow denote point mutations relative to the 3D7 allele including those mutated N:Q 
to remove the putative N-linked glycosylation sites i.e., NXS/T. 
1.2 
Rationale for Study 
Our initial rationale for testing the combination of Pfs25- and Pfs230-based vaccines is that we 
believe this multi-component strategy may improve overall transmission-blocking efficacy. 
Firstly, Pfs230 is expressed early and Pfs25 is expressed late in sexual development of the 
parasite. The differential temporal expression may allow dual actions. Secondly, the combination 
may decrease the proportion of poor responders compared to vaccination with a single agent. 
Increased transmission-blocking activity was observed when antibodies raised separately against 


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recombinant Pfs25M and Pfs230D1M were mixed and fed together with a parasite culture to 
mosquitoes in SMFA. In non human primates, antibodies induced by co-administration of 
Pfs25M-EPA and Pfs230D1M-EPA also displayed stronger transmission-blocking activities in 
SMFA than those induced by the individual vaccine candidates.   
 
Given this information, our initial plans for this study were to proceed with a combination 
vaccine for the double-blind, comparator controlled portion of the study if the Pilot Safety Phase 
results were reassuring the vaccine at the targeted doses were safe. Following a planned interim 
group analysis post Vaccination #3 and #4 for protocol #15-I-0044, which looked at antibody 
responses and functional activity (via SMFA and DSF) of Pfs25M-EPA/Alhydrogel® alone, 
Pfs230D1M-EPA/ Alhydrogel® alone, Pfs25M-EPA/ Alhydrogel® and Pfs230D1M-EPA/ 
Alhydrogel® versus a comparator vaccine, the plan for this study has changed given these results. 
In summary, Pfs25M alone was seen to be inferior to Pfs230 alone or Pfs230 in combination 
with Pfs25 in regards to functional activity (SMFA and DSF). Thus the conclusion that Pfs25M 
alone was not recommended for the Main Phase of this study.  Pfs230 alone compared to Pfs25-
230 in combination, produced similar results in regards to immunogenicity (peak ELISA 
responses), functional activity (DSF, SMFA), and percentage of responders (detectable antibody 
responses). Overall there was no statistically significant difference between Pfs230 alone versus 
Pfs25 and Pfs230 given in combination, though Pfs230 did have a trend to higher overall peak 
ELISA responses and consistently higher SMFA responses.  Given that similar results were 
obtained in subjects who only received Pfs230 versus those who received Pfs25 and Pfs230 in all 
identified endpoint assays, the plan is to move forward with a single antigen, Pfs230D1M-
EPA/Alhdyrogel®.  
 
In addition to assessing various antigens and combination of antigens, LMIV has been interested 
in exploring additional vaccine strategies, including alterations in conjugation and change in 
adjuvant selection in attempt to achieve a higher, prolonged, quality antibody response to 
provide a long-lived, functional response that can last an entire malaria transmission season.  As 
noted above, the first step in this process was combining LMIV’s two leading TBV candidates, 
Pfs25M and Pfs230D1M, to assess that the combination was safe and did not interfere with each 
other when administered simultaneously.  Having achieved this objective with our current 
combination study (#15-I-0044), as determined by preclinical studies and an interim analysis 
completed in the still blinded #15-I-0044 study, the next step was to target improving our 
antibody responses, and associated functional activity, and durability of responses by changing 
the adjuvant fromAlhydrogel® to a safe, but more potent adjuvant AS01.   
 
Though AS01 adjuvants are not yet in licensed vaccines, one of the immunostimulant 
components, monophosphoryl lipid (MPL), is a component of a licensed GSK human 
papillomavirus vaccine, and the AS01 adjuvant system has been a key component of the 
Mosquirix™ (RTS,S 25μg/AS01E) and RTS,S/AS01B (RTS,S 50μg/AS01B) that has been widely 
tested in children and adults across Africa. Thus, the most logical and safe path forward in our 


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TBV clinical development plan was to evaluate Pfs25M-EPA and Pfs230D1M-EPA adjuvanted 
with AS01. Given availability of the adjuvant from our partners GSK and a strong safety profile, 
AS01E (25µg MPL+ 25µg QS21 liposomal formulation) rather than AS01B will be used for the 
final formulation. 
 
Utilizing GSK’s adjuvant AS01 (for this study, AS01 is AS01B diluted with conjugate/antigen in 
the formulation to have a dose equivalent to to 25µg of MPL and 2 µg of QS21 in a 0.5mL dose), 
which has shown to induce improved immune responses with AS01B and AS01E when compared 
to Alhydrogel®, while maintaining a strong safety profile, in conjunction  with LMIV’s leading 
transmission blocking vaccine candidates, Pfs25M-EPA and Pfs230D1M-EPA, this proposed 
study attempts to explore the use of Pfs230D1M-EPA/AS01 at full and fractional dosing to 
determine the safety, immunogenicity, functional activity, and transmission dynamics.   
2 Previous Preclinical Experience with Study Agents 
In preclinical studies evaluating Pfs25M-EPA and Pfs230D1M-EPA formulated with AS01 
adjuvant in rabbits at two-week intervals over a 43-day study interval, both vaccines when 
administered either alone or in combination were safe, with expected mild, transient injection 
site reactogenicity and mild, transient alterations in clinical laboratory parameters consistent with 
immune stimulation following vaccination.   
 
In animal studies conducted at LMIV, AS01 and AS01B adjuvanted products when compared to 
Alhydrogel® consistently yielded the highest and longest sustained antibody responses with 
associated significant functional activity by SMFA (Figure 2 and Figure 3).   


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Figure 2: Comparison of Transmission Blocking Activity of Pfs25M-EPA/AS01 and 
Pfs230D1M-EPA/AS01 to Pfs25M-EPA/Alhydrogel® and Pfs230D1M-EPA/Alhydrogel®  in 
CD1 Mice.   
 
 
Figure 2A shows antibody responses 14 days following Vaccination #2 to anti-Pfs25 at increasing antigen 
doses adjuvanted with AS01E, Alhydrogel®, and AS01B in CD1 mice that either received Pfs25 alone or 
Pfs25 and Pfs230 vaccinations.  Figure 2B shows antibody responses 14 days following Vaccination #2 to 
anti-Pfs230 at increasing antigen doses adjuvanted with AS01E, Alhydrogel®, and AS01B in CD1 mice that 
either received Pfs25 alone or Pfs25 and Pfs230 vaccinations.  Figure 2C and 2D reports SMFA results 
without and with complement from sera samples 14 days following Vaccination #2 at increasing antigen 
doses adjuvanted with AS01E, Alhydrogel®, and AS01B in CD1 mice that either received Pfs25 alone, 
Pfs230 alone, or Pfs25 and Pfs230 vaccinations.  In Figure 2A and 2B, circles represent antibody level per 
mouse and black bars indicate geometric mean of antibody levels.  In Figure 2C and 2D circles represent 
individual mosquitoes and black bars indicate geometric mean of oocyst/mosquito. AS01B and AS01E in 
this figure are designations that mean AS01B was diluted with conjugate/antigen in the formulation to have 
a dose equivalent to 50µg MPL and 50µg QS21 in a 0.5 mL dose (AS01B); and AS01E is the adjuvant 
AS01B diluted to a dose equivalent to 25µg of MPL and 25µg of QS21 in a 0.5 mL dose. 
 


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Figure 3: Measurement of Pfs25-specific Antibody Decay from Mouse AS01 study 
Comparing Alhydrogel® versus AS01. 
 
Mean half life for Pfs25M-EPA/Alhydrogel® is 59 days compared to mean half life for Pfs25M-EPA/AS01 
is 178 days. 
3 Previous Human Experience With Study Agents 
3.1 
Pfs25-EPA 
3.1.1 Pfs25H-EPA/Alhydrogel®  in Healthy US Adults  
A phase 1 dose escalation study of Pfs25H-EPA/Alhydrogel® was conducted in malaria-naïve 
adults at the Center for Immunization Research at the Johns Hopkins School of Public Health in 
Baltimore, Maryland, USA (NIAID Protocol #11-I-N237).23 The study enrolled 30 subjects as 
shown in Table 1, with Groups 1a and 1b sized for safety and Group 2 sized for safety and 
immunogenicity.  
Table 1: Phase 1 Study of Pfs25H-EPA Conjugates in US Healthy Adults 
Group 
n 
Vaccine 
Schedule (month) 
1a 
5 
8 µg Pfs25H-EPA/Alhydrogel® 
0, 2, optional 10 
1b 
5 
16 µg Pfs25H-EPA/Alhydrogel® 
0, 2, optional 10 
2 
20 
47 µg Pfs25H-EPA/Alhydrogel® 
0, 2, 4, 10 
 
Eligible subjects (n=15) in the high dose group initially received 3 vaccinations to explore the 
effect of the third dose on boosting and longevity of antibody responses; however, given the 
increasing antibody response to each subsequent dose of vaccine, an additional fourth boost was 
administered to all eligible subjects (n=11) in the high dose group and for 1 high responder in 
Group 1a. 


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3.1.1.1 Safety of Pfs25H-EPA/Alhydrogel® in Healthy US Adults 
Vaccinations were well-tolerated, with minimal local and systemic reactogenicity.15,23 There was 
an increase in the percentage of subjects reporting solicited adverse events (AEs) with each 
escalating dose, with AEs diminishing with each subsequent dose of vaccine given. No subjects 
were withdrawn due to related adverse events. A single serious adverse event (SAE) occurred 
during the course of the study that was unrelated to the vaccine. These findings are summarized 
in Figure 4. 
 
Figure 4: Related Local Injection Site Reactogenicity and Systemic Adverse Events.   
 
 
All injection site reactogenicity was deemed related to Pfs25H-EPA/Alhydrogel®. Clinical solicited (A-C) 
adverse events after each vaccination with each dose group are reported in individual graphs: Group 1a (A), 
1b (B) and Group 2 (C). All adverse events were mild except when indicated as moderate.  
 
All laboratory adverse events (related and unrelated) are shown in Figure 5.  While several 
subjects experienced anemia after vaccination, the majority of these events were judged to be 
unrelated to vaccination, either because these subjects had low, but normal, hemoglobin levels at 
baseline (day of first vaccination) or because of an alternate explanation (e.g., blood donation or 
previously undisclosed history of anemia).  An examination of trends in hematologic parameters 
does not show any pattern related to timing of vaccinations.15  


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Figure 5: Laboratory Adverse Events 
 
Laboratory (A-C) adverse events after each vaccination with each dose group are reported in individual 
graphs: Group 1a (A), 1b (B) and Group 2 (C). All adverse events were mild except when indicated as 
moderate. All neutropenia events occurred in the same subject. All hematuria occurred during menses. All 
leukopenia events occurred in the same subject. All laboratory adverse events except for a portion of the 
decreased hemoglobin (n=10) were found by the PI to be unrelated to Pfs25H-EPA/Alhydrogel® 
3.1.1.2 Immunogenicity of Pfs25H-EPA/Alhydrogel® in Healthy US Adults  
Specific anti-Pfs25 antibodies were detected by ELISA in sera from subjects receiving 2 or 3 
doses, and the titers increased further after the booster dose (Figure 6). 


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Figure 6: Anti-Pfs25 Antibodies in Volunteers Immunized with Pfs25-EPA/Alhydrogel® 
 
Groups 1a and 1b participants, receiving conjugated proteins comprising of 8µg and 16µg Pfs25H, 
respectively.  Group 2 participants, receiving 47µg Pfs25H. Arrows indicate the day of vaccination. 
Closed circles represent antibody level and individual participants, and black bars indicate geometric 
mean of antibody levels.   Anti-Pfs25 ELISA units were determined by using a reference standard 
generated from a previous study in which volunteers were immunized with recombinant Pfs25 formulated 
Montanide® ISA51. Anti-Pfs25 ELISA units were converted to Pfs25-specific antibodies µg/mL by 
multiplying a conversion factor (1 EU = 0.104 µg anti-Pfs25-specific IgG) obtained using the same 
methods described in Cheru et al.24   
The immunized antisera displayed various levels of transmission reducing activities, measured as 
reduction of oocyst development in mosquitoes in a standard membrane feeding assay (SMFA).  
The activity correlated with the antibody titers (Figure 7).  Following the fourth dose, a large 
proportion of subjects (9 of 11 vaccinated from the 47µg Group) developed functional antibodies 
with significant transmission reduction activity above 50% and up to 95%. 


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Figure 7: Transmission Reducing Activities Induced by Pfs25-EPA/Alhydrogel® 
 
Transmission reducing activity of sera collected 2 weeks after each initial vaccination and 8 weeks after 
the booster vaccination.  Each data point represents the average of two results in the SMFA, and lines 
indicate estimate EC50 and point-wise confidence interval by GEE model.  Thick red line indicates the 
threshold representing significant biological activity (50% TRA). The green, red, blue, and black dots 
correspond to the data points on D70: 14 days post Vaccination #2; D134: 14 days post Vaccination #3; 
D314: 14 days post Vaccination #4; D356: 2 months post Vaccination #4. 
3.1.2 Pfs25H-EPA/Alhydrogel® in Healthy Malian Adults 
A double-blind dose-escalating randomized controlled phase 1 study in malaria exposed adults of 
the safety and immunogenicity of Pfs25H-EPA/Alhydrogel® was conducted in Bancoumana, 
Mali. One hundred twenty subjects were enrolled and randomized to receive the low dose (16μg 
of conjugated Pfs25, n=10), the high dose (47μg of conjugated Pfs25, n=50), or the comparator 
vaccine (Euvax B for first 3 vaccinations, then Menactra® for the fourth vaccination, n=10 for 2 
vaccinations, n=50 for 4 vaccinations) (Figure 8). Enrollment within the high dose group was 
staggered for additional safety. The low dose and the matching comparator cohort (Group 1) 
received 2 vaccine doses given at 0 and 2 months. For the rest of the subjects, 3 doses were given 
at a 0, 2, 4 month dosing intervals in 2013, and a fourth (booster) dose at 12 months after the 
third dose at the start of the subsequent transmission season in 2014. Subjects were followed for 
at least 6 months following the last vaccination. In total, 82 subjects (40 comparator cohort and 
42 Pfs25 high dose cohort) received their fourth vaccination. 
 


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Figure 8: Phase 1 Study of Pfs25H-EPA Conjugates in Malian Healthy Adults 
 
Arrows indicate timing of vaccinations.  Direct skin feeds (DSF) in 2013 completed only on gametocyte or 
parasite carriers.  DSF in 2014, following receipt of vaccination #4, completed weekly on all eligible 
subjects starting 2 weeks post dose #4 and continuing for 6 consecutive weeks.   
3.1.2.1 Safety of Pfs25H-EPA/Alhydrogel® in Healthy Malian Adults 
Overall, vaccinations were well tolerated in both the Pfs25H-EPA/Alhydrogel® and comparator 
group, with the majority of AEs being mild or moderate, and the most commonly reported 
adverse events were local site injection pain, clinical malaria, cold, headache, rhinitis, and 
neutropenia (Table 2). Twenty-six (26) Grade 3 AEs were reported during the study, of which 
all were deemed by the investigator to be not or unlikely related to the vaccine except for a single 
episode of injection site pain, which was deemed definitely related and occurred in a comparator 
subject. In total three SAEs (spontaneous abortion, snake bite, post traumatic coma) were 
reported and all had been determined to be not related to vaccination.  No subject was withdrawn 
or deferred from vaccination secondary to an AE related to the vaccine.   
 
In comparison to the comparator group, those subjects who receive Pfs25H-EPA/Alhydrogel® 
reported no difference in the number of AEs, systemic or local AEs, laboratory abnormalities, 
clinical malaria episodes, or unsolicited AEs.  Though, overall Pfs25H-EPA/Alhydrogel® 
vaccinees reported statistically significant more solicited AEs (p=0.01, Fishers Exact test) and 
related AEs (p=0.019; Fishers Exact test) which increased number of solicited AEs held true 
when looking at Pfs25H-EPA/Alhydrogel® at the targeted antigen dose of 47µg versus 
comparator.   
 
 


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Table 2: Summary of Reported Adverse Events in Pfs25H Vaccine Arms, #13-I-N109, Mali Low Dose Cohort (2A) and Mali High 
Dose Cohort (2B) 
 
 
 
 
Each field has the format # (X) %, where # is the number of adverse events, X is the number of subjects with one or more episodes of the given event, 
and % is the number of participants with one or more episodes of the given event divided by the total number of participants who received the study 
agent (N) multiplied by 100. 
Vaccine 1
Vaccine 2
Total
Vaccine 1
Vaccine 2
Total
N=10
N=9
N=10
N=10
N=10
N=10
Total # AEs
23 (9) 90%
49 (9) 100%
72 (10) 100%
16 (8) 80%
73 (10) 100%
89(10) 100%
Local Reactogenicity
3 (2) 20%
2 (2) 22%
5 (3) 30%
3 (2) 20%
4 (3) 30%
7 (4) 40%
Systemic Reactogenicity
1(1)10%
0 (0) 0%
1(1)10%
0 (0) 0%
1 (1) 10%
1 (1) 10%
Laboratory Abnormalities
7 (4) 40%
7 (5) 56%
14 (7) 70%
0 (0) 0%
3 (2) 20%
3 (2) 20%
Unsolicited AEs
12 (7)70%
40(9) 100%
52 (10) 100%
13 (8) 80%
65 (10) 100%
78 (10) 100%
Grade 1      
13 (7) 70%
14 (6) 67%
27 (9) 90%
9 (6) 60%
13 (3) 30%
22 (7) 70%
Related to Pfs25
10 (5) 50%
6 (4) 44%
16 (8) 80%
3 (2) 20%
5 (4) 40%
8 (4) 40%
Grade 2     
9 (5) 50%
32 (9) 100%
41 (9) 90%
7 (6) 60%
59 (9) 90%
66 (10) 100%
Related to Pfs25
1 (1)10%
2 (2) 22%
3 (2) 20%
0 (0) 0%
2 (2)20%
2 (2) 20%
Grade 3     
1 (1) 10%
3 (2) 22%
4 (3) 30%
0 (0) 0%
1 (1) 10%
1 (1) 10%
Related to Pfs25
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Grade 4     
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
SAE     
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
16 µg Pfs25-EPA/Alhydrogel
Comparator/ Euvax B
Classification
Severity and Relationship
2A
Vaccine 1
Vaccine 2
Vaccine 3
Vaccine 4
Total
Vaccine 1
Vaccine 2
Vaccine 3
Vaccine 4
Total
N=50
N=50
N=45
N=42
N=50
N=50
N=46
N=42
N=40
N=50
Total # AEs
135 (45) 90%
152(36) 72%
368(45)100%
202(42)100%
857 (50) 100%
110 (45) 90%
121 (39) 85%
288(42)100%
161(40)100%
680 (49)98% 
Local Reactogenicity
22 (21) 42%
28 (26) 52%
17(17)38%
18(18)43%
82 (39) 78%
15 (15) 30%
14(13) 28%
20(20)48%
6(6)15%
55 (31) 62%
Systemic Reactogenicity
11(9)18%
9 (8) 16%
6(5)11%
4(4)10%
29(20)40%
9 (8) 16%
5 (4) 9%
1(1)2%
3(3)8%
18 (12) 24%
Laboratory Abnormalities
22 (18) 36%
30 (19) 38%
31(20)44%
31(18)43%
114 (33) 66%
19(13) 26%
23 (17) 37%
17(13)31%
23(16)40%
82 (31) 62%
Unsolicited AEs
80 (41) 82%
85(40) 80%
314(45)100%
149(40)95%
628 (50) 100%
67 (38) 76%
79 (35) 76%
250(42)100%
129(39)98%
525 (49) 98%
Grade 1      
84 (43) 86%
82 (37) 74%
74(34)76%
43(26)62%
283 (50) 100%
75 (39) 78%
65 (34) 74%
61(30)71%
34(22)55%
235(49) 98%
Related to Pfs25
42 (31) 62%
45(29) 58%
35(25)56%
20(18)43%
142 (47) 94%
39 (27) 54%
25 (20) 43%
31(22)52%
11(9)23%
106 (42) 84%
Grade 2     
51 (32) 64%
70(34) 68%
285(45)100%
159(42)100%
565 (49) 98%
34(23) 46%
55 (31)67%
219(42)100%
123(38)95%
431 (45) 90%
Related to Pfs25
7 (6)12%
7 (7) 14%
5(5)11%
11(10)24%
30 (20) 40%
2 (2) 4%
2 (2)4%
2(2)5%
2(2)5%
8 (5) 10%
Grade 3     
0 (0) 0%
0 (0) 0%
8 (7) 16%
0 (0) 0%
8 (7) 14%
1 (1) 2%
1(1)2%
8 (6) 14%
3(3)8%
13(10)20%
Related to Pfs25
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
1(1)3%
1(1)2%
Grade 4     
0 (0) 0%
0 (0) 0%
1 (1) 2%
0 (0) 0%
1 (1) 2%
0 (0) 0%
0 (0) 0%
0 (0) 0%
1(1)3%
1(1)2%
SAE     
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
47 µg Pfs25-EPA/Alhydrogel16 ug Pfs25-EPA/Alhydrogel
Comparator/ Euvax B + Menactra
Classification
Severity and Relationship
2B


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3.1.2.2 Immunogenicity of Pfs25H-EPA/Alhydrogel® in Healthy Malian Adults 
Immunogenicity outcomes were measured by IgG ELISA against recombinant Pfs25 and EPA 
(Figure 9) and functional activity by SMFA (Figure 10) conducted at the NIAID in the U.S.  
Following each vaccination (0, 2, 4, 16 months), anti-Pfs25 IgG increased with each dose and all 
but 1 subject responded after receipt of the fourth and final dose (Figure 9). Anti-Pfs25 titers 
correlated with anti-EPA but decayed more rapidly than did anti-EPA after 4th dose (Figure 9).   
 
Figure 9: Anti-Pfs25 and Anti-EPA IgG ELISA Titers in Phase 1 Trial of Pfs25H-
EPA/Alhydrogel® in Mali 
 
 
Anti-Pfs25 
Anti-EPA 
After dose: 
2 
3 
4 
2 
3 
4 
Geo mean titer 
93.05 
169.6 
422.3 
237.4 
468.1 
1406.0 
95% CI 
70-122 
127-225 
290-615 
171-329 
383-572 
1124-1759 
Non-
responders 
18/45 
(40%) 
5/44 
(11.3%) 
1/41 
(1.4%) 
3/45 
(6.7%) 
1/44 
(2.3%) 
0/41      
(0%) 
Geo mean titer 
of responders 
153.3 
201.6 
476.8 
267.8 
494.6 
1406.0 
 
Arrows indicate the day of vaccination.  Only data from Groups 2/3 subjects who received 47µg 
Pfs25H-EPA/Alhydrogel® vaccination presented.  Note all Group 2/3 comparator subjects were below 
the level of detection for anti-Pfs25 responses at all timepoints. Closed circles represent geometric (geo) 
mean antibody level and black bars 95% confidence intervals (CI).   
 
Statistically significant functional activity, as measure by SMFA, was not seen after the third 
dose but was observed after the fourth dose (Figure 10A). In general, anti-Pfs25 titers correlated 


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with functional activity (Figure 10B), which was assessed to be contained in the IgG fraction 
directed against Pfs25 (Figure 11).  
 
Figure 10: Serum Transmission Blocking Activity after Pfs25H-EPA/Alhydrogel® Dose 4 in 
Mali. 
 
SMFA of post-dose 4 sera (Day 494) from Pfs25-EPAH/ Alhydrogel® trial in Mali (Group 2/3) shows 
(Figure 10A) increase in serum functional activity in vaccinees over controls and (Figure 10B) 
correlation of antibody titers with serum activity.  For Figure 10A, each data point represents the 
average of two results in the SMFA in an individual subject with the thick black line indicating the 
median.  Thick red line indicates the threshold representing significant biological activity (50% TRA). 
The blue and green dots correspond to the data points on Day 126: 14 days post Vaccination #3; Day 
494: 14 days post Vaccination #4.  Comparator = Euvax B (for vaccinations #1, 2, 3) and Menactra 
(for vaccination #4). 


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Figure 11: Confirmation that Human Transmission Blocking Activity is Mediated by Anti-
Pfs25 IgG 
 
SMFA of post-dose 4 sera and IgG from two Pfs25-EPA/Alhydrogel® vaccinees in Mali with 
relatively high antibody titer (table above) shows high functional activity for serum as well as purified 
IgG fraction which can be ablated by depletion of Pfs25-specific IgG on an antigen-affinity column. 
3.1.3 Pfs25M-EPA/Alhydrogel® in Healthy US Adults 
The phase 1 study of the safety and immunogenicity of Pfs230D1M-EPA/Alhydrogel® and 
Pfs25M-EPA/Alhydrogel® in adults in the US (NIH Clinical Center) and Mali (Bancoumana, 
Mali) started in December 2014 in the US (NIAID Protocol #15-I-0044; clinicaltrials.gov: 
NCT02334462).  Study enrollment, as seen in Table 3, for the US portion of the study (N=35) 
was to evaluate safety and tolerability of increasing doses and Pfs25M and Pfs230D1M given 
alone or in combination prior to moving into Mali, West Africa for further safety, 
immunogenicity, and functional activity evaluation.    
 
 


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Table 3: Subject Enrollment and Vaccinations in US 
 
Arms 
n 
Vaccine 
Schedule 
(month) 
Pfs25 
1a 
5 
16µg Pfs25M-EPA/Alhydrogel® 
0, 1 
month 
1b 
5 
47µg Pfs25M-EPA/Alhydrogel® 
Pfs230 
2a 
5 
5µg Pfs230D1M-EPA/Alhydrogel® 
2b 
5 
15µg Pfs230D1M-EPA/Alhydrogel® 
2c 
5 
40µg Pfs230D1M-EPA/Alhydrogel® 
Pfs25 
+ 
Pfs230 
3a 
5 
16µg Pfs25M-EPA/Alhydrogel® + 15µg Pfs230D1M-
EPA/Alhydrogel® 
3b 
5 
47µg Pfs25M-EPA/Alhydrogel® + 40µg Pfs230D1M-
EPA/Alhydrogel® 
 
3.1.3.1 Safety of Pfs25M-EPA/Alhydrogel® in Healthy US Adults 
Overall vaccinations with Pfs25M-EPA/Alhydrogel® in protocol #15-I-0044 in US adults were 
well-tolerated with minimal local and systemic reactogenicity (Table 4).  The majority of the 
reported AEs were mild (Grade 1; 16µg: 23/27, 85%; 47µg: 21/24, 88%) and solicited. Local 
reactogenicity was by far the most frequently reported AEs in both the 16µg and 47µg Pfs25M-
EPA/Alhydrogel® arms, occurring with an increased frequency of symptoms with increasing 
dose (16 µg: 60% and 47 µg: 100%).  No significant differences were seen between doses with 
subsequent vaccinations nor with the duration of local symptoms.  Solicited systemic symptoms 
were only reported in two subjects in the low dose arm, 16µg Pfs25M, and only after the first 
dose.  As seen in previous Pfs25 studies, the most commonly noted laboratory abnormality was 
hemoglobin decreased, which the majority of these events being determined to be unlikely 
related to vaccination. There were no related Grade 3 AEs reported, and only two Grade 3 AEs 
(bronchitis, upper respiratory tract infection) were reported during the course of the study. There 
were no SAEs reported.   
 
 


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Table 4: Summary of Reported Adverse Events in Pfs25M Vaccine Arms, #15-I-0044, US 
Cohort 
 
Each field has the format # (X) %, where # is the number of adverse events, X is the number of subjects 
with one or more episodes of the given event, and % is the number of participants with one or more episodes 
of the given event divided by the total number of participants who received the study agent (N) multiplied 
by 100. 
 
3.1.3.2 Immunogenicity of Pfs25M-EPA/Alhydrogel® in Healthy US Adults  
As expected, no pre-existing immunity was seen prior to vaccination in US adults for Pfs25 in 
NIAID protocol #15-I-0044.  No subjects responded to the vaccine in Arm 1a (16µg of Pfs25M). 
The majority of subjects in Arms 1b (47µg of Pfs25M), 3a (16µg of Pfs25M + 15µg of 
Pfs230D1M), and 3b (47µg of Pfs25M + 40µg of Pfs230D1M) responded to the vaccine 
following Vaccination #2 (Figure 12).   
 
Vaccine 1
Vaccine 2
Total
Vaccine 1
Vaccine 2
Total
N=5
N=4
N=5
N=5
N=5
N=5
Total # AEs
17 (3) 60%
10 (2) 50%
27 (4) 80%
11 (5) 100%
13 (5) 100%
24 (5) 100%
Local Reactogenicity
4 (2) 40%
2 (2) 50%
6 (3) 60%
6 (5) 100%
7 (4) 80%
13 (5) 100%
Systemic Reactogenicity
8 (2) 40%
0 (0) 0%
8 (2) 40%
1 (1) 20%
0 (0) 0%
1 (1) 20%
Laboratory Abnormalities
3 (2) 40%
1 (1) 25%
4 (3) 60%
2 (2) 40%
2 (2) 40%
4 (3) 60%
Unsolicited AEs
2 (2) 40%
7 (1) 25%
9 (3) 60%
2 (2) 40%
4(3)60%
6 (5) 100%
Grade 1      
13 (2) 40%
10 (2) 50%
23 (4) 80%
11 (5) 100%
10 (4) 80%
21 (5) 100%
Pfs25 Related
9 (2) 40%
2 (2) 50%
11 (3) 60%
8 (5) 100%
 6(4)80%
14 (5) 100%
Grade 2     
4 (2) 40%
0 (0) 0%
4 (2) 40%
0 (0) 0%
1 (1) 20%
1 (1) 20%
Pfs25 Related
2 (2) 40%
0 (0) 0%
2 (2) 40%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Grade 3     
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
2 (2) 40%
2 (2) 40%
Grade 4     
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
SAE     
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Pfs25M, 16µg
Pfs25M, 47µg
Classification
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Figure 12: Pfs25–specific antibody responses in US naïve subjects receiving Pfs25M 
vaccinations (#15-I-0044)  
 
Results presented in ELISA units for each Arm.  Vaccinations occurred on Day 0, 28.  Day 0 was 
drawn pre-vaccination, D42 is 14 days post vaccination #2.  Each individual data point represents 
an individual subject anti-Pfs25 ELISA response. 
 
Anti-Pfs25 responses following two doses of Pfs25M-EPA/Alhydrogel® was similar to those 
seen with Pfs25H-EPA/Alhydrogel® in LMIV’s previous study in naïve U.S. adults described in 
Section 3.1.1.2 (p=0.43).   
 
Functional activity (as measured by SMFA) related to antibody titers following Vaccination #2 
have been assessed in only the highest antigen dose arms at this time.  Anti-Pfs25 responses 
following receipt of Pfs25M, 47µg alone for two doses (0,1 month) were similar to those seen in 
previous studies with Pfs25H (Figure 13).     
 
 
 


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Figure 13: Pfs25 and Pfs230 Functional Activity (SMFA) 
 
Samples obtained 14 days following receipt of vaccine #2 in the highest antigen dose arms (Pfs25M, 
47µg alone, Pfs230D1M, 40µg alone, and Pfs25M, 47µg + Pfs230D1M, 40µg co-administered). 
Each individual column represents an individual subject.  Each individual data point represents a 
single mosquito dissected. 
 
3.1.4 Pfs25M-EPA/Alhydrogel® in Healthy Malian Adults 
The phase 1 study of the safety and immunogenicity of Pfs230D1M-EPA/Alhydrogel® and 
Pfs25M-EPA/Alhydrogel® (NIAID protocol #15-I-0044) started in April 2015 in Mali and has 
completed enrollment (Table 5).  
 
Cohort 1, which included the single antigen safety Arms A1 (Pfs25M, 16µg), B1 (Pfs230D1M, 
15µg), and D1 (Twinrix®), and Cohort 2, which included the co-administration safety Arm C1 
(Pfs25M, 16µg + Pfs230D1M, 15µg) and D2 (Twinrix® + normal saline) have completed 
enrollment, all vaccinations (2 of 2 scheduled at 0, 1 month intervals), and undergone scheduled 
unblinding per protocol in October 2015.  
 


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Cohort 3, which includes increased sample size for immunogenicity and functional activity 
assessment, included Arms A2 (Pfs25M, 47µg + normal saline), B2 (Pfs230D1M, 40µg + 
normal saline), C2 (Pfs25M, 47µg + Pfs230D1M, 40µg) and D3 (Twinrix® + normal saline) 
have completed enrollment, 3 of the 4 scheduled vaccinations (0, 1, 6 months), and undergone 6 
weeks of intensive twice a week DSF assessment.  Subjects in cohort 3 who are currently active 
on protocol (171 of 200) are in long term safety follow-up and will receive Vaccination #4 in 
September 2016, undergo evaluation via DSF following receipt of Vaccination #4, and be 
unblinded in March/April 2017. 
 
Table 5: Subject Enrollment and Vaccination in Mali  
Arm 
Dose 
Enrolled 
Received 
Vaccination 
#1 (D0) 
Received 
Vaccination 
#2 (D28) 
Received 
Vaccination 
#3 (D168) 
Completed 
Final Study 
Visit/ 
On Protocol 
Cohort 1 - unblinded 
A1 
Pfs25M, 16µg 
5 
5 
5 
N/A 
5 
B1 
Pfs230D1M, 15µg 
5 
5 
4 
N/A 
4 
D1 
Twinrix® 
5 
5 
5 
N/A 
4 
Cohort 2 - unblinded 
C1 
Pfs25M, 16µg + 
Pfs230D1M, 15µg 
5 
5 
5 
N/A 
5 
D2 
Twinrix® + NS 
5 
5 
5 
N/A 
4 
Cohort 3 - blinded 
A2 
Pfs25M, 47µg + NS 
200 
200 
190 
177 
171 
B2 
Pfs230D1M 40µg + 
NS 
C2 
Pfs25M, 47µg + 
Pfs230D1M, 40µg 
D3 
Twinrix®  + NS, 
then Menactra + NS 
NS = normal saline.  N/A = not applicable. 
3.1.4.1 Safety of Pfs25M-EPA/Alhydrogel® in Healthy Malian Adults 
From the unblinded safety data provided by Arm A1 (low dose of Pfs25M, 16µg), overall 
vaccinations have been well tolerated with all of the reported AEs in the Pfs25M arm being 
either mild or moderate (Grade 1 and Grade 2; 19/19, 100%) and with the majority of AEs 
(14/19; 74%) reported being unsolicited AEs (Table 6). The only reported related AEs were 
injection site pain, which were all Grade 1 and did not increase in frequency of reporting with 
subsequent vaccination.  In comparison to the comparator group, who received Twinrix®, the 
Pfs25M vaccinees reported overall more AEs (19 versus 11), including increased frequency of 


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reporting of injection site pain (4 versus 1).  However, neither group reported any systemic 
reactogenicity and only a single laboratory abnormality (Grade 1) was noted following 
Vaccination #2 in a Pfs25M subject.  There were no Grade 3, Grade 4, or SAEs reported in the 
Pfs25M arm. 
 
Table 6: Summary of Reported Adverse Events in Pfs25M Safety Vaccine Arm versus 
Comparator, #15-I-0044, Mali Cohort 
 
Each field has the format # (X) %, where # is the number of adverse events, X is the number of subjects 
with one or more episodes of the given event, and % is the number of participants with one or more episodes 
of the given event divided by the total number of participants who received the study agent (N) multiplied 
by 100. 
 
Safety analysis of the high dose of Pfs25M-EPA/Alhydrogel®, 47µg of Pfs25M (Arm A2), is in 
process following unblinding in April 2017.  Overall, in the blinded cohort, the majority of 
reported AEs have been mild (Grade 1; 708/1431; 49%) with the most commonly report AEs 
being injection site pain, headache, malaria, neutropenia, nasopharyngitis, and rhinitis.  The most 
commonly reported related AEs have been injection site reactogenicity (pain, induration, 
pruritus, and edema), leukopenia, neutropenia, and headache, which have been all Grade 1 or 2.   
3.1.4.2 Immunogenicity of Pfs25M-EPA/Alhydrogel® in Healthy Malian Adults  
Evaluation of immunogenicity and functional activity for the low dose, unblinded cohorts 
(Cohort 1, 2) have been completed (Figure 14). As expected, no pre-existing immunity was seen 
prior to vaccination in Malian adults to Pfs25. The majority of subjects in all Arms (A1: 16µg of 
Pfs25M; C1: 16µg of Pfs25M + 15µg of Pfs230D1M) responded to the vaccine following 
Vaccination #2.  However, antibody responses to Pfs25 decayed rapidly post vaccination #2, 
similar to what has been seen previously with Pfs25H. 
 
Vaccine 1
Vaccine 2
Total
Vaccine 1
Vaccine 2
Total
N=5
N=5
N=5
N=5
N=5
N=5
Total # AEs
5 (4) 80%
14 (5) 100%
19 (5) 100%
3 (2) 40%
8 (4) 80%
11 (4) 80%
Local Reactogenicity
2 (2) 40%
2 (2) 40%
4 (3) 60%
0 (0) 0%
1 (1) 20%
1 (1) 20%
Systemic Reactogenicity
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Laboratory Abnormalities
0 (0) 0%
1 (1) 20%
1 (1) 20%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Unsolicited AEs
3 (2) 40%
11 (5) 100%
14 (5) 100%
3 (2) 40%
7 (4) 80%
10 (4) 80%
Grade 1      
2 (2) 40%
5 (4) 80%
7 (4) 80%
1 (1) 20%
3 (2) 40%
4 (2) 40%
Related to Pfs25
2 (2) 40%
2 (2) 40%
4 (3) 60%
0 (0) 0%
1 (1) 20%
1 (1) 20%
Grade 2     
3 (2) 40%
9 (5) 100%
12 (5) 100%
2 (1) 20%
4 (3) 60%
6 (3) 60%
Related to Pfs25
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Grade 3     
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
1 (1) 20%
1 (1) 20%
Related to Pfs25
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Grade 4     
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
SAE     
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Pfs25M, 16µg 
Comparator
Classification
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Figure 14: Pfs25–specific antibody responses in Malian subjects receiving Low Dose 
Pfs25M, Pfs230D1M, and Pfs25M + Pfs230D1M (#15-I-0044) 
 
Results presented in ELISA units for each Arm.  Vaccinations occurred on Day 0, 28.  Day 0 was 
drawn pre-vaccination, D42 is 14 days post vaccination #2.  Each individual data point represents 
an individual subject anti-Pfs25 (Arms A1, C1) or anti-Pfs230 (Arm B1, C1) ELISA responses. 
 
Evaluation of immunogenicity and functional activity in the targeted dose are in process 
following unblinding of the study in 2017, though an interim analysis was completed following 
Vaccination #3 and #4 in the targeted doses (Arm A2: Pfs25M, 47µg; Arm B2: Pfs230D1M, 
40µg; Arm C2: Pfs25M, 47µg AND Pfs230D1M, 40µg;  Arm D3: comparator, Twinrix®) that 
assisted in determining the final study plan for this proposed study.  
 
The two planned interim analyses (Jun 2016, Feb 2017) of immunogenicity (ELISA) and 
functional activity (DSF, SMFA) by Arm were completed after Vaccination #3 and #4 in the 
larger Main Cohort during the study to inform about proceeding to a booster 4th dose in 2016 and 
to inform the study design for protocol 17-I-N006 (TBV with AS01). In conclusion, by ELISA 
and SMFA, Pfs230 induced superior activity to Pfs25, and Pfs230 in combination with Pfs25, 
was not superior to Pfs230 alone (Figure 15).  


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Figure 15 : Pfs25 anf Pfs230 immunogenicity (ELISA) and functional activity (SMFA) in 
malaria-exposed Malian adults for 3 and 4 vaccine doses 
 
Samples obtained 14 days following receipt of vaccine #3 (study day 182) and #4 (study day 554) in the highest 
antigen dose arms (Pfs25M, 47µg alone; Pfs230D1M, 40µg alone; Pfs25M, 47µg + Pfs23D1M, 40µg; TWINRIX 
[received TWINRIX for first 3 doses, then Menactra® for fourth dose]). Vaccinations received at 0, 1, 6 months 
(2015) and 18 months (2016). Each individual data points represents an individual ELISA or SMFA results. Black 
bars represent 95% confidence intervals. Red asterix represent statistical significance when compared to the control 
(TWINRIX). Tables report the mean TRA (95% confidence intervals) following the third and fourth vaccination.  
 
In 2015, starting 1 week post vaccination #3 and using 60 mosquitoes total, 2005 DSF were 
performed in 175 individuals, of which 73% participated in all 12 feeds. Fifty-eight (2.9%) of the 
DSFs resulted in a positive DSF in 18 (10.3%) unique individuals. In 2016, starting 1 week post 
vaccination #4 and using 30 mosquitoes total, 1843 DSFs were performed in 160 individuals, of 
which 87% participated in all 12 feeds. Thirty (1.6%) of the DSFs resulted in a positive DSF in 
18 (11.3%) unique individuals. Analysis of the DSFs (accounting for all positive DSFs) post 
vaccination #4 using two models (GEE, GLMM) showed no significant difference in DSF results 
between Pfs230 alone, Pfs25M + Pfs230 in combination, and the control group. Pfs25M alone, 
when compared to the control group, in the GEE analyses did show a significant odds ratio of 


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infection (OR 6.75; 95% CI 1.16-39.35; p=0.034) but this significance was not seen in the 
GLMM analyses (OR 3.05; 95% CI 0.14-65.89; p=0.633). Further analyses are ongoing to 
account for possible transmission by other Plasmodium species (P. ovale, P. malariae), vaccine 
variants, and the impact of time post vaccination on DSF transmission events.  
As noted above, a change was made in the plan for DSF between 2015 and 2016, with a decrease 
of the number of mosquitoes per each feeding pint (30 to 15). This change was based on 
modeling with available DSF results from 2011 through 2015. However, in comparing the DSF 
transmission rates between 2015 and 2016 in the comparator group, there was no change in the 
percent of unique individuals transmitting overall, but there were substantial differences in the 
transmission dynamics in the comparator group from 2015 to 2016 as summarized below in 
Table 7. Given this result and the low transmission rate seen in the control arm in 2016 (post 
Vax #4), as described in Section 14.5, DSFs will completed twice a week starting one week post 
vaccination with a total of approximately 60 mosquitoes (30 mosquitoes/cup; 2 cups) for 12 
weeks total.  
 


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Table 7: Summary of BS and DSF completed for protocol 15-I-0044 during DSF evaluation period post Vaccination #3 and #4. 
No. unique 
subjects with ≥1 
DSF completedA 
No. unique 
subjects with ≥1 
positive DSFB 
No. DSFs 
completed over 
the 6 week periodC  
No. DSFs positiveD 
Median (range) of 
no. positive DSF per 
person 
Median (range) of 
no. positive 
mosquitoes per 
positive DSF 
Percent of 
positive 
mosquitoes in the 
positive DSFs E 
Vax #3 
Vax #4 
Vax #3 
Vax #4 
Vax #3 
Vax #4 
Vax #3 
Vax #4 
Vax #3 
Vax #4 
Vax #3 
Vax #4 
Vax #3 
Vax #4 
Comparator 
43 
(24.6%) 
39 
(24.5%) 
6  
(9.3%) 
4 
(10.3%) 
497 
(24.8%) 
452 
(24.5%) 
18 
(3.6%) 
4  
(0.9%) 
2  
(1-6) 
1  
(1) 
7  
(1-18) 
1  
(1-7) 
34.3% 
8.8% 
Pfs25 alone 
44 
(25.1%) 
41 
(25.9%) 
4  
(9.1%) 
9 
(21.4%) 
502 
(25.0%) 
480 
(26.0%) 
15 
(3.0%) 
18 
(3.7%) 
3.5  
(2-6) 
2  
(1-3) 
4  
(1-16) 
2.5  
(1-18) 
18.6% 
63.7% 
Pfs230 alone 
44 
(25.1%) 
41 
(25.9%) 
6 
(13.6%) 
2  
(4.9%) 
502 
(25.0%) 
475 
(23.7%) 
19 
(3.8%) 
3  
(0.6%) 
1.5  
(1-7) 
1.5  
(1-2) 
3  
(1-32) 
6  
(2-10) 
38.4% 
15.9% 
Pfs25+230 
44 
(25.1%) 
37 
(23.3%) 
2  
(4.6%) 
3  
(8.1%) 
504 
(25.1%) 
436 
(23.5%) 
6  
(1.2%) 
5  
(1.1%) 
 3  
(1-5) 
2  
(1-2) 
4  
(1-20) 
2  
(1-6) 
8.7% 
11.5% 
TOTAL 
175 
158 
18 
(10.3%) 
18 
(11.3%) 
2005 
1843 
58 
(2.9%) 
30 
(1.6%) 
2  
(1-7) 
1.5  
(1-3) 
5.5  
(1-32) 
2  
(1-18) 
100.0% 
100.0% 
A: Number of unique subjects who received vaccination #3 and then #4 and completed at least 1 DSF during the DSF evaluation period.  
B: Number of unique subjects who has ≥1 mosquito with ≥1 oocyst seen on dissection. % calculated as # unique subjects with +DSF / # unique subjects in that arm completing a DSF. 
C: Total number of DSF and blood smears completed by that arm over the six week period post Vaccination #3 and #4. % calculated as #DSFs completed per arm / total # DSFs 
completed by the entire cohort (total)  
D: % calculated as #positive DSFs by arm / #DSFs completed by that arm 
E: % calculated as #positive mosquitoes per arm / # total positive mosquitoes 
 
No. unique subjects gametocytemicA 
Total count gametocytemicB 
No. unique subjects parasitemicA 
Total count parasitemicB 
Vax #3 
Vax #4 
Vax #3 
Vax #4 
Vax #3 
Vax #4 
Vax #3 
Vax #4 
Comparator 
19 (44.2%) 
14 (35.9%) 
75 (15.1%) 
40 (8.7%) 
35 (81.4%) 
28 (71.8%) 
117 (23.5%) 
83 (18.0%) 
Pfs25 alone 
12 (27.3%) 
15 (36.6%) 
56 (11.2%) 
65 (13.5%) 
40 (90.9%) 
31 (75.6%) 
134 (26.7%) 
98 (20.4%) 
Pfs230 alone 
12 (27.3%) 
13 (31.7%) 
62 (12.6%) 
40 (8.4%) 
41 (93.2%) 
31 (75.6%) 
125 (24.9%) 
99 (20.8%) 
Pfs25+230 
7 (15.9%) 
13 (35.1%) 
28 (5.6%) 
31 (7.1%) 
34 (77.3%) 
27 (70.3%) 
99 (19.6%) 
76 (17.4%) 
TOTAL 
50 (28.6%) 
55 (34.8%) 
221 (11.0%) 
176 (9.5%) 
150 (85.7%) 
117 (74.1%) 
475 (23.7%) 
351 (19.3%) 
A: Number of unique subjects who had ≥ 1 bloodsmear with ≥ 1 gametocyte or ≥ 1 Pf parasite seen on it over the DSF evaluation period. % calculated as # unique subjects per arm / # 
unique subjects who received vaccination #3 or #4 and completed at least 1 DSF.  
B: Total count is the number of BS with ≥ 1 gametocyte or ≥ 1 Pf parasite seen on it over the DSF evaluation period. % calculated as # positive BS per arm / # BS completed per arm.


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3.2 
Pfs230-EPA 
3.2.1  Pfs230D1M-EPA/Alhydrogel® in Healthy US Adults 
Study enrollment, as seen in Table 3, for the US portion of study #15-I-0044 (N=35) was to 
evaluate safety and tolerability of increasing doses and Pfs25M and Pfs230D1M given in 
combination prior to moving into Mali, West Africa for further safety, immunogenicity, and 
functional activity evaluation.    
3.2.1.1 Safety of Pfs230D1M-EPA/Alhydrogel® in Healthy US Adults 
In the US portion of NIAID protocol #15-I-0044, Pfs230D1M vaccinations were well-tolerated, 
with minimal local and systemic reactogenicity as noted in Table 8.  The majority of the 
reported AEs were Grade 1 (5µg: 19/22, 86%; 15µg: 29/34, 85%; 40µg: 24/31, 77%).  All 
related AEs reported were Grade 1, except for a single Grade 2 episode of injection site pain 
following the first vaccination in the high dose group of 40µg Pfs230D1M.   Overall the reported 
local reactogenicity by vaccination and dose appeared to increase in the frequency of reporting 
and duration of symptoms with increasing antigen dose of Pfs230, but not with subsequent 
vaccinations. Solicited systemic symptoms were reported in all dosing arms and did not increase 
in frequency, severity, nor duration with increasing antigen dose.  Various laboratory 
abnormalities, all Grade 1, were noted across the dosing arms, seven in total in five subjects, but 
only three were deemed related to vaccination.  There were no Grade 3 AEs and no SAEs 
reported.   
 
Table 8: Summary of Reported Adverse Events in Pfs230D1M Vaccine Arms, #15-I-0044, 
US Cohort 
 
Each field has the format # (X) %, where # is the number of adverse events, X is the number of subjects 
with one or more episodes of the given event, and % is the number of participants with one or more episodes 
of the given event divided by the total number of participants who received the study agent (N) multiplied 
by 100. 
Vaccine 1
Vaccine 2
Total
Vaccine 1
Vaccine 2
Total
Vaccine 1
Vaccine 2
Total
N=5
N=5
N=5
N=5
N=4
N=5
N=5
N=5
N=5
Total # AEs
8 (4) 80%
14 (5) 100%
22 (5) 100%
10 (5) 100%
24 (4) 100%
34 (5) 100%
19 (5) 100%
12 (5) 100%
31 (5) 100%
Local Reactogenicity
2 (2) 40%
4 (4) 80%
6 (4) 80%
5 (4) 80%
4 (3) 75%
9 (4) 80%
7 (5) 100%
4 (4) 80%
11 (5) 100%
Systemic Reactogenicity
1 (1) 20%
1 (1) 20%
2 (2) 40%
3 (2) 40%
6 (2) 50%
9 (3) 60%
6 (3) 60%
0 (0) 0%
6 (3) 60%
Laboratory Abnormalities
0 (0) 0%
2 (2) 40%
2 (2) 40%
1 (1) 20%
0 (0) 0%
1 (1) 20%
3 (1) 20%
1 (1) 20%
4 (2) 40%
Unsolicited AEs
5 (3) 60%
7 (2) 40%
12 (3) 60%
1 (1) 20%
14 (4) 100%
15 (4) 80%
3 (2) 40%
7 (5) 100%
10 (5) 100%
Grade 1      
6 (4) 80%
13 (5) 100%
19 (5) 100%
10 (5) 100%
19 (4) 100%
29 (5) 100%
15 (5) 100%
9 (5) 100%
24 (5) 100%
Pfs230 Related
3 (3) 60%
6 (4) 80%
9 (4) 80%
7 (4) 80%
9 (3) 75%
16 (4) 80%
9 (5) 100%
5 (4) 80%
14 (5) 100%
Grade 2     
2 (2) 40%
1 (1) 20%
3 (3) 60%
0 (0) 0%
5 (3) 75%
5 (3) 60%
4 (2) 60%
3 (3) 60%
7 (4) 80%
Pfs230 Related
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
1 (1) 20%
0(0) 0%
1 (1) 20%
Grade 3     
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Grade 4     
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
SAE     
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Severity and Relationship
Classification
Pfs230D1M, 5µg
Pfs230D1M, 15µg
Pfs230D1M, 40µg


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3.2.1.2 Immunogenicity of Pfs230D1M-EPA/Alhydrogel® in Healthy US Adults 
As expected and seen with Pfs25 antibody responses, no pre-existing immunity was seen prior to 
vaccination in US for Pfs230.  Unlike Pfs25 responses, at the lower doses, majority of subjects 
vaccinated did respond to vaccination with Pfs230 alone.  The majority of subjects in Arms 2c 
(40µg of Pfs230D1M), 3a (16µg of Pfs25M + 15µg of Pfs230D1M), and 3b (47µg of Pfs25M + 
40µg of Pfs230D1M) responded to the vaccine following Vaccination #2 (see Figure 16).  There 
was no obvious interference seen with co-administration of Pfs25M-EPA/Alhydrogel® and 
Pfs230D1M-EPA/Alhydrogel® versus Pfs230D1M-EPA/Alhydrogel® alone, but these safety 
arms were not powered to assess interference. 
 
Figure 16: Pfs230–specific antibody responses in US naïve subjects receiving Pfs230D1M 
vaccinations (#15-I-0044) 
 
Results presented in ELISA units for each Arm.  Vaccinations occurred on Day 0, 28.  Day 0 was 
drawn pre-vaccination, D42 is 14 days post vaccination #2.  Each individual data point represents an 
individual subject anti-Pfs230- ELISA response. 
 
Functional activity by SMFA, in US adults in protocol #15-I-0044, related to antibody titers 
following Vaccination #2 has only been assessed in the highest antigen dose arms at this time 
and will be further assessed in the larger Mali cohort once unblinded.  A few individuals, after 
two doses of either Pfs230D1M alone or in combination with Pfs25M, developed appreciable 
vaccine specific antibody responses which was associated with a reduction in transmission 
activity (decrease in the number of oocysts/mosquito versus day 0 pool) via SMFA (Figure 13) 


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and for Pfs230 activity, is complement dependent (Figure 17). However, the numbers in each 
arm are too small to be of statistical significance nor were these arms of the study powered for 
this endpoint. 
Figure 17: Pfs230 Functional Activity (SMFA) in the Presence or Absence of Complement  
 
Each individual column represents an individual subject with Day 0 (pre-vaccination #1), Day 42 (14 
days post vaccination #2).  Each individual data point represents a single mosquito dissected.  Top set 
of figures is the assay completed with complement present, the bottom set of figures is the assay 
completed without complement present in the assay. Only the highest antigen dose arms 
(Pfs230D1M, 40µg alone, n=5 and Pfs25M, 47µg + Pfs230D1M, 40µg co-administered, n=5) 
presented.  
3.2.2 Pfs230D1M-EPA/Alhydrogel® in Healthy Malian Adults 
Status of protocol #15-I-0044 is already summarized in Section 3.1.4.   
3.2.2.1 Pfs230D1M-EPA/Alhydrogel® in Healthy Malian Adults 
From the unblinded safety data provided by Arm B1 (low dose of Pfs230DM, 15µg), overall 
vaccinations have been well tolerated with the majority of the reported AEs being mild or 
moderate (Grade 1 and Grade 2; 14/17, 82%) with very few reported related AEs (headache, 
n=1; leukopenia, n=1), both of which were Grade 1. The most commonly reported related AEs 
were injection site pain, neutropenia, and headache, which were all Grade 1.  In a single subject, 
a Grade 3 gastroenteritis was reported with associated Grade 4 laboratory abnormalities 
(leukocytosis, blood creatinine increased), all deemed unlikely related to vaccination and 
resolved shortly after resolution of subject’s gastroenteritis symptoms.  Due to a continued Grade 
1 lab abnormality at time of this subject’s next scheduled vaccination, it was decided for the 


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subject’s safety to not proceed with Vaccination #2 and to follow them for safety.  As had been 
seen with low dose Pfs25M, in comparison to the comparator group, who received Twinrix®, the 
Pfs230D1M vaccinees reported overall more AEs (17 versus 11).   However, the Pfs230D1M 
group reported no local reactogenicity and only a single solicited symptom (Grade 1, headache).  
As noted in Table 9, there were more laboratory abnormalities reported in the Pfs230D1M arm, 
including the Grade 4 as noted above, but of these laboratory abnormalities, only one (Grade 1 
leukopenia) was determined possibly related to vaccination.  There were no SAEs reported in the 
Pfs230D1M arm. 
 
Table 9: Summary of Reported Adverse Events in Pfs230D1M Safety Vaccine Arm versus 
Comparator, #15-I-0044, Mali Cohort 
 
Each field has the format # (X) %, where # is the number of adverse events, X is the number of subjects 
with one or more episodes of the given event, and % is the number of participants with one or more 
episodes of the given event divided by the total number of participants who received the study agent (N) 
multiplied by 100. 
 
Safety analysis of the high dose of Pfs230D1M-EPA/Alhydrogel®, 40µg of Pfs230D1M, is still 
blinded.  Overall, in the blinded cohort, the majority of reported AEs have been mild (Grade 1; 
708/1431; 49%) with the most commonly report AEs being injection site pain, headache, 
malaria, neutropenia, nasopharyngitis, and rhinitis.  The most commonly reported related AEs 
have been injection site reactogenicity (pain, induration, pruritus, and edema), leukopenia, 
neutropenia, and headache, which have been all Grade 1 or 2.   
3.2.2.2 Immunogenicity of Pfs230D1M-EPA/Alhydrogel® in Healthy Malian Adults 
Evaluation of immunogenicity and functional activity for the low dose, unblinded cohorts 
(Cohort 1, 2) have been completed (Figure 14). Pre-existing immunity to Pfs230 was seen prior 
to vaccination. The majority of subjects in all Arms (B1: 15µg of Pfs230D1M; C1: 16µg of 
Vaccine 1
Vaccine 2
Total
Vaccine 1
Vaccine 2
Total
N=5
N=4
N=5
N=5
N=5
N=5
Total # AEs
7 (4) 80%
10 (4) 100%
17 (5) 100%
3 (2) 40%
8 (4) 80%
11 (4) 80%
Local Reactogenicity
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
1 (1) 20%
1 (1) 20%
Systemic Reactogenicity
1 (1) 20%
0 (0) 0%
1 (1) 20%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Laboratory Abnormalities
3 (1) 20%
2 (2) 50%
5 (3) 60%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Unsolicited AEs
3 (3) 60%
8 (2) 50%
11 (5) 100%
3 (2) 40%
7 (4) 80%
10 (4) 80%
Grade 1      
2 (2) 40%
4 (4) 100%
6 (5) 100%
1 (1) 20%
3 (2) 40%
4 (2) 40%
Related to Pfs230
1 (1) 20%
1 (1) 25%
2 (1) 20%
0 (0) 0%
1 (1) 20%
1 (1) 20%
Grade 2     
2 (2) 40%
6 (2) 50%
8 (3) 60%
2 (1) 20%
4 (3) 60%
6 (3) 60%
Related to Pfs230
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Grade 3     
1 (1) 20%
0 (0) 0%
1 (1) 20%
0 (0) 0%
1 (1) 20%
1 (1) 20%
Related to Pfs230
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Grade 4     
2 (1) 20%
0 (0) 0%
2 (1) 20%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Related to Pfs230
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
SAE     
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Pfs230D1M, 15µg
Comparator -- Twinrix®
Classification
Severity and Relationship


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Pfs25M + 15µg of Pfs230D1M) responded to the vaccine following Vaccination #2.  Antibody 
responses to Pfs230 appeared to persist longer than has been previously seen with Pfs25. 
 
3.3 
Pfs25-EPA and Pfs230-EPA 
3.3.1 Pfs25M-EPA/Alhydrogel® and Pfs230D1M-EPA/Alhydrogel® in Healthy US Adults 
Study enrollment, as seen in Table 3, for the US portion of the study (N=35) was to evaluate 
safety and tolerability of increasing doses and Pfs25M and Pfs230D1M given in combination 
prior to moving into Mali, West Africa for further safety, immunogenicity, and functional 
activity evaluation.    
3.3.1.1 Safety of Pfs25M-EPA/Alhydrogel® and Pfs230D1M-EPA/Alhydrogel® in Healthy 
US Adults 
Overall vaccinations were well-tolerated with the co-administration of Pfs25 and Pfs230 in US 
adults in protocol 15-I-0044, with minimal local and systemic reactogenicity as noted in Table 
10.  The majority of the reported AEs were Grade 1 (16µg + 15µg: 27/27, 100%; 16µg + 15µg: 
42/44, 95%).  All related AEs reported were Grade 1.  There were more AEs reported overall 
given the increased reporting of local reactogenicity per each vaccination received.   Overall the 
reported local reactogenicity by vaccination and dose appeared to increase in the frequency of 
reporting with increasing doses and was more likely to be attributed to Pfs230 than Pfs25, but 
not significantly so.  However, with increasing antigen dose, the duration of symptoms and 
severity of symptoms remained unchanged. Solicited systemic symptoms were reported in all 
dosing arms and did not increase in frequency, severity, nor duration with increasing antigen 
dose.  Various laboratory abnormalities, all Grade 1 and only 1 of 5 reported deemed related to 
vaccination, were noted across the dosing arms. Hemoglobin decrease was the most commonly 
reported laboratory abnormality. In the high dose arms, there was one Grade 2 AE (breast 
disorder) and one Grade 3 AE (systolic hypertension), both determined not related to 
vaccination, and no SAEs were reported.   
 


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Table 10: Summary of Reported Adverse Events in Pfs25 and Pfs230D1M Vaccine Arms, 
#15-I-0044, US Cohort 
 
Each field has the format # (X) %, where # is the number of adverse events, X is the number of 
subjects with one or more episodes of the given event, and % is the number of participants with one 
or more episodes of the given event divided by the total number of participants who received the 
study agent (N) multiplied by 100. 
 
Overall there has been no significant difference in reported local or systemic solicited 
reactogenicity or laboratory abnormalities between Pfs25M-EPA/Alhydrogel® alone, 
Pfs230D1M-EPA/Alhydrogel® alone, or co-administration of Pfs25M-EPA/Alhydrogel® and 
Pfs230D1M-EPA/Alhydrogel®. 
3.3.1.2 Immunogenicity of Pfs25M-EPA/Alhydrogel® and Pfs230D1M-EPA/Alhydrogel® 
in Healthy US Adults 
As already described in Sections 3.1.3.2 and 3.2.1.2, no pre-existing immunity was seen prior to 
vaccination in US for either Pfs25 or Pfs230.  The majority of subjects in the highest antigen co-
administration dose arm (Pfs25M, 47µg + Pfs230D1M, 40µg, n=5) responded to the vaccine 
following Vaccination #2 for both antigens (see Figure 12).  There was no significant 
interference seen with co-administration of Pfs25M-EPA/Alhydrogel® and Pfs230D1M-
EPA/Alhydrogel® compared to those who received Pfs25 or Pfs230 alone, though overall the 
antibody responses were slightly lower in the co-administration when compared to the single 
antigen arms, but as noted above, these safety arms were not powered to assess interference. 
Additionally, in a few individuals, appreciable functional activity via SMFA was achieved 
following Vaccination #2, as seen in Figure 13 and Figure 17, which for Pfs230 was 
complement dependent.   
 
In conclusion, for the US portion of  study #15-I-0044, in malaria naïve adults, Pfs25M-
EPA/Alhydrogel® given alone or in combination with Pfs230D1M-EPA/Alhydrogel® and 
Vaccine 1
Vaccine 2
Total
Vaccine 1
Vaccine 2
Total
N=5
N=5
N=5
N=5
N=5
N=5
Total # AEs
14 (5) 100%
13 (5) 100%
27 (5) 100%
23 (5) 100%
21 (5) 100%
44 (5) 100%
Local Reactogenicity
6 (4) 80%
8 (4) 80%
14 (4) 80%
9 (5) 100%
11 (4) 80%
20 (5) 100%
Systemic Reactogenicity
3 (2) 40%
1 (1) 20%
4 (2) 40%
5 (2) 40%
4 (1) 20%
9 (2) 40%
Laboratory Abnormalities
2 (2) 40%
2 (1) 20%
4 (2) 40%
1 (1) 20%
0 (0) 0%
1 (1) 20%
Unsolicited AEs
3 (3) 60%
2 (2) 40%
5 (4) 80%
8 (4) 80%
6 (2) 40%
14 (5) 100%
Grade 1      
14 (5) 100%
13 (5) 100%
27 (5) 100%
22 (5) 100%
20 (5) 100%
42 (5) 100%
Related to Pfs25
4 (2) 40%
4 (4) 80%
8 (4) 80%
9 (5) 100%
7 (3) 60%
16 (5) 100%
Related to Pfs230
6 (4) 80%
4 (3) 80%
10 (4) 80%
9 (5) 100%
8 (4) 80%
17 (5) 100%
Grade 2     
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
1 (1) 20%
1 (1) 20%
Related to Pfs25
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Related to Pfs230
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Grade 3     
0 (0) 0%
0 (0) 0%
0 (0) 0%
1 (1) 20%
0 (0) 0%
1 (1) 20%
Related to Pfs25
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Related to Pfs230
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Grade 4     
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
SAE     
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Pfs25M, 16µg AND Pfs230D1M, 15µg
Pfs25M, 47µg AND Pfs230D1M, 40µg
Classification
Severity and Relationship


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Pfs230D1M-EPA/Alhydrogel® given alone or in combination with Pfs25M-EPA/Alhydrogel® is 
safe and tolerable.  Detectable responses were seen in the majority of vaccinated subjects to 
Pfs25, 47µg and/or Pfs230, 40µg following receipt of two vaccinations at the targeted dose. 
Appreciable functional activity, though in a limited number of individuals, is achievable 
following two doses of Pfs230D1M-EPA/Alhydrogel® or Pfs230D1M-EPA/Alhydrogel® and 
Pfs25M-EPA/Alhydrogel® at the highest vaccine dose administered. 
 
3.3.2 Pfs25M-EPA/Alhydrogel® and Pfs230D1M-EPA/Alhydrogel® in Healthy Malian 
Adults 
Status of protocol #15-I-0044 is already summarized in Section 3.1.4.   
3.3.2.1 Safety of Pfs25M-EPA/Alhydrogel® and Pfs230D1M-EPA/Alhydrogel® in Healthy 
Malian Adults 
From the unblinded safety data provided by Arm C1 (low dose of Pfs25M, 16 µg AND  
Pfs230DM, 15µg), overall vaccinations have been well tolerated with the majority of the 
reported AEs being mild (Grade 1; 15/23, 65%; Table 11).  There have been few reported related 
AEs (majority of which have been injection site pain) and all reported related AEs have been 
Grade 1. There was a single Grade 3 AE (malaria) reported that occurred over 100 days post 
vaccination and was not related.  There were no Grade 4 AEs or SAEs reported in the Pfs25M 
and Pfs230D1M arm. In comparison to the comparator group, who received Twinrix® and 
normal saline, the Pfs25M and Pfs230D1M vaccinees reported overall more AEs (23 versus 17), 
including increased frequency of reporting injection site pain (6 versus 3) and related AEs (9 
versus 4).  However, both groups reported few to no systemic reactogenicity, and overall, the 
comparator arm reported more laboratory abnormalities in comparison to the vaccine group.   
 


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Table 11: Summary of Reported Adverse Events in Pfs25M and Pfs230D1M Safety Vaccine 
Arm versus Comparator, 15-I-0044, Mali Cohort 
 
Each field has the format # (X) %, where # is the number of adverse events, X is the number of subjects 
with one or more episodes of the given event, and % is the number of participants with one or more episodes 
of the given event divided by the total number of participants who received the study agent (N) multiplied 
by 100. 
 
Safety analysis of the high dose of Pfs25M-EPA/Alhydrogel®, 47µg of Pfs25M and high dose of 
Pfs230D1M-EPA/Alhydrogel, 40µg of Pfs230D1M, is still blinded.  Overall the in the blinded 
cohort, the majority of reported AEs have been mild (Grade 1; 708/1431; 49%) with the most 
commonly report AEs being injection site pain, headache, malaria, neutropenia, nasopharyngitis, 
and rhinitis.  The most commonly reported related AEs have been injection site reactogenicity 
(pain, induration, pruritus, and edema), leukopenia, neutropenia, and headache, which have been 
all Grade 1 or 2.   
3.3.2.2 Immunogenicity of Pfs25M-EPA/Alhydrogel® and Pfs230D1M-EPA/Alhydrogel® 
in Healthy Malian Adults 
Evaluation of immunogenicity and functional activity for the low dose, unblinded cohorts 
(Cohort 1, 2) have been completed (Figure 14) and were previously summarized in Section 
3.1.4.2 and 3.2.2.2.  
3.4 
Rationale for Fractional Dosing 
Dosing sparing by fractional-dose vaccination has been a concept explored in many vaccine 
fields, including yellow fever, polio, and malaria. In the first ever conducted controlled human 
malaria infection (CHMI) evaluating vaccines based on the RTS,S antigen, one study group 
(receiving RTS,S/AS02 at 0, 1, 7-month schedule; WRMAL-003) who received a reduced third 
immunization dose (fifth of the standard 0.5mL dose) showed high levels of vaccine efficacy 
Vaccine 1
Vaccine 2
Total
Vaccine 1
Vaccine 2
Total
N=5
N=5
N=5
N=5
N=5
N=5
Total # AEs
7 (4) 80%
16 (5) 100%
23 (5) 100%
5 (2) 40%
12 (5) 100%
17 (5) 100%
Local Reactogenicity
3 (3) 60%
3 (2) 40%
6 (4) 80%
1 (1) 20%
2 (2) 40%
3 (2) 40%
Systemic Reactogenicity
1 (1) 20%
0 (0) 0%
1 (1) 20%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Laboratory Abnormalities
1 (1) 20%
1 (1) 20%
2 (1) 20%
3 (2) 40%
1 (1) 20%
4 (2) 20%
Unsolicited AEs
2 (2) 40%
12 (5) 100%
15 (5) 100%
1 (1) 20%
9 (4) 80%
10 (4) 80%
Grade 1      
5 (4) 80%
10 (5) 100%
15 (5) 100%
4 (2) 40%
3 (3) 60%
7 (4) 80%
Related to Pfs25 or Pfs230
5 (4) 80%
4 (3) 60%
9 (4) 80%
2 (1) 20%
2 (2) 40%
4 (2) 40%
Grade 2     
2 (2) 40%
5 (3) 60%
7 (4) 80%
1 (1) 20%
9 (4) 80%
10 (4) 80%
Related to Pfs25 or Pfs230
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Grade 3     
0 (0) 0%
1 (1) 20%
1 (1) 20%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Related to Pfs25 or Pfs230
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Grade 4     
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
SAE     
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Pfs25M, 16µg AND Pfs230D1M, 15µg
Comparator -- Twinrix® + Normal Saline
Classification
Severity and Relationship


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against homologous sporozoite challenge, with six out of seven subjects protected.25 The 0, 1, 7-
month schedule, with a fractional dose delivered as the third immunization which had achieved 
this high level of protection had never been repeated again until recently (Malaria-071) when the 
study was reproduced with similar high levels of vaccine efficacy against homologous CHMI 
seen.25 
 
Building off results seen in WRMAL-003, the biological hypothesis going into Malaria-071 was 
that the delayed fractional dose had allowed a better qualitative immune maturation in the 
germinal center, with antigen selection of the B cells harboring surface immunoglobulin with 
highest antigen affinity. Preliminary analyses showed again high efficacy in the fractional dose 
group (4/30 subjects developed parasitemia; vaccine efficacy: 86.7% [95% CI: 66.8; 94.6; 
p<0.0001]) compared to standard doses given at 0, 1, 2-months (6/16 subjects developed 
parasitemia; vaccine efficacy: 62.5% [95% CI: 29.4; 80.1; p=0.0009]).  To note all 12 control 
subjects (no vaccine) developed parasitemia confirming the validity of the malaria challenge. 
Malaria-071 was not powered to detect superiority of the fractional dosing group over the 
standard dosing group, but the study did show that protection against P. falciparum parasitemia 
in the fractional dose group represented an increase in VE over the standard doses assesses as a 
reduction in the proportion of unprotected subjects, of 64.4% [95% CI: -7.9; 88.3], p=0.0741, 
Fisher’s exact; difference in time to parasitemia p=0.0455, logrank). Additionally, in Malaria-
071, anti-CS antibody GMCs at all timepoints from 3 weeks to 6 months post-Dose 3 were 
similar when RTS,S/AS01B was given at 0, 1, 7- months with a fractional 0.1 mL dose at month 
7 (range 75.7-32.8 EU/mL) and when given at 0, 1, 2-months (range 110.1-34.6 EU/mL). 
Preliminary immunological analyses of serum samples from subjects participating in Malaria-
071 suggest that the fractional delayed dose does lead to the production of antibodies with higher 
antigen avidity as compared to a classical immunization regimen with three full doses.  
 
In the follow-up phase of the Malaria-071 study, subjects who were protected in the initial 
challenge were randomized to receive a fractional fourth dose or no fourth dose, before being 
exposed to a second sporozoite challenge approximately six months after the initial challenge. In 
the absence of a booster dose, efficacy had waned at the time of the rechallenge (subjects from 
fractional dose group: 42.9% [95% CI: -8.5, 69.9], p=0.1923; subjects from standard dose group: 
20.0% [95% CI: -24.0, 48.4], p=0.4545). Boosting with a fractional dose was shown to maintain 
high protection in subjects in the fractional dose group (90.0% [95% CI: 35.8, 98.4]; p=0.0009). 
Reduced protection was observed following fractional dose boosting in subjects in the standard 
dosing group (25.0% [95% CI: -32.1, 57.4]; p=0.4000). The fractional dose boosting 
was shown to reverse susceptibility to infection in the first challenge to protection against re-
challenge in 4 out of 5 subjects who were infected in the primary/challenge phase. The results 
from the study follow-up phase and the second challenge suggest that there is waning immunity 
with the fractional schedule too but that the protection can be extended with an additional 
fractional dose.  
 


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These results from the Malaria-071 challenge study may indicate a simple way to improve the 
functional activity or protective efficacy of a malaria vaccine candidate and requires further 
assessment in the endemic population under conditions of natural exposure. Building off this 
observation and similar observations seen in yellow fever and polio vaccination, we plan to 
assess the impact of a fractional third dose in our TBVs in the field. 
3.5 
Rationale for Long Term Immunogenicity Follow-up (Pilot/Sotuba) and Evaluation 
of Fourth Vaccination (Main/Bancoumana-Doneguebougou) 
3.5.1 Long Term Immunogenicity Follow-up (Pilot/Sotuba) 
The pilot phase (n=65) of this trial (#17-I-N006) completed follow up per protocol in December 
2017/January 2018. The pilot group was designed to assess the safety and reactogenicity of 
increasing dosages of either Pfs25 alone, Pfs230 alone, or Pfs25 co-administered with Pfs230. 
Safety data, as summarized below, overall indicated that the TBV alone or in combination at low 
and high dosages were safe and tolerable which allowed the main study group to be enrolled.  
3.5.1.1 Safety Results 
3.5.1.1.1 Pfs25M-EPA/AS01 alone 
Overall vaccinations in Arm 1a (low dose of Pfs25M, 16µg) and Arm 1b (high dose of Pfs25M, 
47 µg) have been well tolerated with few adverse events (AEs) reported overall and the majority 
of which have been mild (Grade 1; Arm 1a: 21/23, 91%; Arm 1b: 24/30, 80%). One Grade 3 AE 
in Arm 1a (gastroenteritis) has been reported and determined not related to vaccination.  
3.5.1.1.2 Pfs230D1M-EPA/AS01 alone 
Overall vaccinations in Arm 2a (low dose of Pfs230D1M, 13µg) and Arm 2b (high dose of 
Pfs230D1M, 40 µg; dose used in the Activity Main Phase of the study) have also been well 
tolerated. Most of the related AEs reported have been mild (Grade 1; Arm 2a: 23/29, 79%; Arm 
2b: 42/51, 82%) with the majority being injection site pain. One subject in Arm 2a and three 
subjects in Arm 2b have experienced four Grade 3 events (neutropenia, gastritis, respiratory tract 
infection, malaria), all of which were determined not related to vaccination.  
3.5.1.1.3 Pfs25M-EPA/AS01 and Pfs230D1M-EPA/AS01 
Overall vaccinations in Arm 3a (low dose of Pfs25M, 16µg and low dose of Pfs230D1M, 13µg) 
and Arm 3b (high dose of Pfs25M, 47µg and high dose of Pfs230D1M, 40µg), have been well 
tolerated with majority of AEs reported being mild (Grade 1; Arm 3a: 25/26, 96%; Arm 3b: 57/64, 
89%) and with the most commonly reported AEs being local site injection abnormalities (47/90, 
52%). To note, the frequency of local reactogenicity reported is increased compared to other Arms, 
given in these Arms two vaccinations are given at each vaccination time point, one in each arm.  
Two subjects in Arm 3b reported moderate (Grade 2) injection site pain following receipt of 


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Vaccination #1, but all other reported local site injection reactions have been Grade 1. No Grade 
3 AEs have been reported. 
3.5.1.1.4 Engerix-B (comparator) 
Overall vaccinations for Arm 4a and Arm 4b (Engerix-B) have been well tolerated. The majority 
of AEs reported have been mild (Grade 1; 54/67, 81%). A single subject was diagnosed with 
pulmonary tuberculosis (previously reported SAE; Grade 3; not related to vaccination) and has 
since completed TB treatment and fully recovered. Two additional Grade 3 AEs have been 
reported, both malaria AEs, not related to vaccination.  
In summary, vaccinations in all Arms of the Safety Pilot Phase thus far have been well tolerated. 
The most common vaccine related AE seen in the TBV arms have been expected mild local 
reactogenicity post vaccination that appears on the day of vaccination or one-day post vaccination 
and resolves within 2-3 days. As expected solicited systemic reactogenicity (such as headache, 
arthralgia, fatigue) are reported and usually are Grade 1 (mild) and transient in presentation. 
Laboratory abnormalities appear, with the majority being transient, asymptomatic Grade 1 (mild) 
and Grade 2 (moderate) neutropenias. In the TBV Arms, a few Grade 3 AEs (5 out of 45 subjects, 
11%) have been reported overall (versus 3 out of 20 subjects in the comparator arm, 15%), all of 
which have been determined by the investigator as not related to study product/procedures. As 
expected, given the time of year, the number of malaria cases has increased overall post 
Vaccination #3. 
3.5.1.2 Immunogenicity and Functional Activity 
In the unblinded Safety Pilot Phase, immunogenicity results (ELISA) have been completed 
through study day 224 (Figure 18; 8 weeks post Vaccination #3) and functional activity 
(standard membrane feeding assays; SMFA) have been completed through study day 112 
(Figure 19; 12 weeks post Vaccination #2).   
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Figure 18. Antibody responses to Pfs25 and Pfs230 at low and high dosage and with single and co-
administration dosing obtained 8 weeks post vaccination #3. 
 
Figure 18. Antibody responses to Pfs25 and Pfs230 at low and high dosage and with single and co-
administration dosing. Samples obtained 14 days and then at least monthly following receipt of vaccination. 
Vaccinations received at 0, 1, 6 months.  Each individual data point represents the ELISA geometric mean value 
with black bars represent 95% confidence intervals. Comparison group (dashed lines) is the same dosage used with 
the Alhydrogel® adjuvant from NIAID protocol 15-I-0044.  
 
As expected, no pre-existing immunity was seen prior to vaccination in Malian adults to Pfs25. 
Most subjects in all Arms receiving Pfs25M (Arm 1a: low dose of Pfs25M, 16µg and Arm 1b: 
high dose of Pfs25M, 47µg and Arm 3a: low dose of Pfs25M, 16µg and low dose of 
Pfs230D1M, 13µg and Arm 3b: high dose of Pfs25M, 47µg and high dose of Pfs230D1M, 40µg) 
responded to the vaccine following Vaccination #1, which is significantly different from the lack 
of antibody responses seen with the same dosage of Pfs25M but with the Alhydrogel® adjuvant. 
Antibody responses continued to increase with the second vaccination, but were the same or 
slightly less after the third vaccination. There was no difference in the antibody responses to 
Pfs25 between Arm 1a versus Arm 1b, nor between those who received the combination of Arm 
3a versus Arm 1a and Arm 3b versus Arm 1b, but the overall trend was lower antibody responses 
to Pfs25 were seen in those individuals that received the combination dose (Arms 3a, 3b) than 
those who received Pfs25M alone (Arms 1a, 1b).   
 
Consistent with previous findings, a small percentage of subjects who received Pfs230D1M 
vaccinations had pre-existing antibodies to the Pfs230D1M vaccine antigen. Similar to Pfs25, 
most subjects in all Arms receiving Pfs230D1M (Arm 2a: low dose of Pfs230D1M, 13µg and 
Arm 2b: high dose of Pfs230D1M, 40µg and Arm 3a: low dose of Pfs25M, 16µg and low dose 
of Pfs230D1M, 13µg and Arm 3b: high dose of Pfs25M, 47µg and high dose of Pfs230D1M, 


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40µg) responded after one vaccination. Antibody responses were similar between those who 
received the low dose (Arm 2a) and the high dose (Arm 2b) of Pfs230D1M alone.  And as seen 
with Pfs25M, overall lower antibody responses to Pfs230 were seen in those individuals that 
received the combination dose (Arms 3a, 3b) than those who received Pfs230D1M alone (Arms 
2a, 2b).   
 
Standard membrane feeding assays (SMFA) have been completed in Arms 2a and 2b and 
associated comparator Arms (Arm 4a, 4b) at two timepoints post Vaccination #2 (2 weeks and 
12 weeks) to date. As seen in Figure 19, significant functional activity, including transmission 
blocking activity, can be seen in the Pfs230D1M alone Arms at 2 weeks and persisting even at 
12 weeks post Vaccination #2. This is a significant improvement from the lack of activity seen 
with the same TBV product with the Alhydrogel® adjuvant (NIAID protocol #15-I-0044).  
 
Figure 19: Functional activity of Pfs230 vaccinees by SMFA at 2 weeks and 12 weeks post Vaccination #2 in 
the open label safety cohort. 
 
Figure 19. Functional activity of Pfs230 vaccinees by SMFA at 2 weeks and 12 weeks post Vaccination #2 in 
the open label safety cohort. Samples obtained 14 days and then at least monthly following receipt of vaccination, 
including at 12 weeks post vaccination #2. Vaccinations received at 0, 1, 6 months. Each individual data point 
represents an individual mosquito dissection in the SMFAs with the black bars representing the geometric mean 
oocyst count per mosquito.   
 
Looking at the kinetics of anti-Pfs25 and anti-Pfs230 antibodies, although there is increased 
durability with the addition of AS01, the rate of decay is unknown. To answer this question, the 
vaccinated subjects in the pilot arm and some from the main arms will be invited for additional 
blood draws at 9 and 12 months to evaluate the kinetics of these antibodies.  


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3.5.2 Evaluation of Fourth Vaccination (Main/Bancoumana-Doneguebougou) 
3.5.2.1 Safety 
In the blinded Activity Main Phase, most AEs reported have been mild or moderate with most AEs 
being unrelated to vaccination (1830/2336, 78%). Of the related AEs (506/2336,22%), the majority 
have been mild (Grade 1; 423/506, 84%), with the most commonly report related AEs being local 
site reactogenicity (369/506, 73%; Grade 1: 324/369, 88%; Grade 2: 45/369, 12%) and mild (Grade 
1) solicited systemic reactogenicity (e.g. headaches, fever, myalgias, arthralgias). Local injection 
site pain did not appear to increase in severity or frequency with subsequent vaccinations, but 
following Vaccination #2 the occurrence of other local site reactogenicity (edema, pruritus, 
impairment of movement, redness) appeared to be reported more frequently, but not post 
Vaccination #3.  
Various laboratory abnormalities have been noted, though most of them have been mild (Grade 1) 
and transient. A few Grade 2 (moderate) neutropenias and leukopenias have been reported.  But 
no laboratory abnormalities have been reported in the frequency or intensity meeting further 
evaluation or study halt. As expected, given the time of year, the number of malaria cases has 
increased overall post Vaccination #3. 
A total of thirty-seven Grade 3 AEs have been reported (malaria, snake bite, pyrexia, 
bronchitis,systolic hypertension,), all determined as not related to vaccination.  A single SAE 
(snake bite) was previously reported and has resolved completely.  
In summary, vaccinations have been well tolerated with no identified safety concerns at this time, 
but safety will be further assessed upon scheduled, intentional unblinding. 
3.5.2.2 Immunogenicity and Functional Activity 
ELISA and SMFA results are currently pending for the Activity Main Phase, which remains 
blinded at this time; however, the Pfs230D1M-EPA/AS01 dosing regimen (0, 1, 6 months) and 
dosage (40µg) assessed in the main study was also evaluated on a smaller scale during the pilot 
study. As presented in Section 3.5.1.2, immunological data from the pilot group have been 
encouraging. Results to date have aligned with what was seen in preclinical studies, that 
adjuvanting with AS01 does lead to significantly higher and more durable antibody responses, as 
determined by ELISA against Pfs25 and Pfs230 (Figure 18), as well as antibody responses 
developing with fewer vaccine doses.  
 
For a vaccine to play a vital role in blocking malaria transmission, the vaccine will have to be 
active for more than one transmission season. While increasing the durability of the vaccine by 
adjuvant is one method, another is boosting at the beginning of a transmission season. In order to 
explore the effect of boosting, vaccinated participants from the main functional activity arms 
(Arm 2c and 2d: Pfs230D1M; Arm 4c: comparator) will be invited to receive a 4th vaccination 
(Arm 2c and 2d: full dose Arm 4c: Menactra®) prior to the next transmission season.  


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The dose of the fourth vaccination (full versus fractional dosing of Pfs230D1M-EPA/AS01) has 
been determined after scheduled, intentional unblinding and associated safety review and 
analysis of anti-Pfs230 ELISA and SMFA results completed in May 2018. There was no 
significant difference in safety between the Arms. The rates of clinical malaria (Full vs Control 
p= 0.75; Fractional vs Control p=0.87 and Full vs Fractional p=0.71) and blood smear positivity 
(Full vs Control p= 0.82; Fractional vs Control p=0.82 and Full vs Fractional p=0.60) were also 
not clinically significant between the Arms.  
A summary of immune responses and functionality results are provided below in Figure 20 and 
Figure 21.  In brief, while there was a significant difference between the full and fractional arms 
detected by antibody responses to Pfs230 (as seen in Figure 20 with p=0.00014), functional 
activity by SMFA showed no significant difference between the two groups (p=0.0597, Figure 
21).  The similarity in SMFA responses may be due to antibody titers remaining elevated above a 
possible threshold that may provide high levels of TRA at 12 weeks post-vaccination.  These 
levels would be expected to decrease over time allowing for analysis of the difference of the 
effectiveness of the two doses at later timepoints.  With no clearly superior regimen evident after 
a third vaccination with either a full or a fractional dose of Pfs230, we will plan to assess a full 
boosting dose in both Arms 2c and 2d.  This approach was also recommended in recent 
discussion with the DSMB. 
 


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Figure 20 : Antibody responses to Pfs230 at 12 weeks post-vaccination #3 with either full or fractional 
dosing 
 
Figure 20: Antibody responses to Pfs230 at 12 weeks post-vaccination #3 with either full of fractional dosing. 
Participants in the fractional dosing group (Arm 2d) received 8µg Pfs230 for their third vaccination, while full 
dosing (Arm 2c) group received 40µg Pfs230 for their third vaccination.  Both groups received 40µg Pfs230 for 
their first and second vaccinations.  Wilcoxon p values for comparisons between arms are as follows: Full vs. 
Fractional:  p=0.00014; Full vs. Control: p<0.0001; Fractional vs. Control: p<0.0001. 
 
 


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Figure 21: Functional activity of Pfs230 by SMFA at 12 weeks post-vaccination #3 with either full or 
fractional dosing 
 
 
Figure 21: Functional activity of Pfs230 by SMFA at 12 weeks post-vaccination #3 with either full or 
fractional dosing. Participants in the fractional dosing group (Arm 2d) received 8µg Pfs230 for their third 
vaccination, while full dosing group (Arm 2d) received 40µg Pfs230 for their third vaccination.  Both groups 
received 40µg Pfs230 for their first and second vaccinations.  Wilcoxon p values for comparisons between arms are 
as follows: Full vs. Fractional:  p=0.0597; Full vs. Control: p<0.0001; Fractional vs. Control: p<0.0001. 
 
3.6 
Clinical Development Plan 
LMIV has a mandate to develop vaccines that will interrupt malaria transmission, and therefore 
will be useful for malaria elimination campaigns. Vaccines can target either preerythrocytic (PE) 
malaria parasites to prevent human infection, or can target sexual stage malaria parasites to 
prevent transmission to mosquitoes (TBV), in order interrupt malaria transmission. 
 
LMIV has the most advanced program for clinical development of TBV. TBV are intended to 
interrupt malaria transmission by producing antibodies in humans that block parasite 
development in mosquitoes. The immunological data from a Phase 1 Pfs25H-EPA/Alhydrogel® 
study in the US and in Mali have shown a consistent increase in Pfs25 antibody response by 


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ELISA following each subsequent vaccination, but these responses appear short lived.  Data 
from these studies also have shown that increasing antibody titers correlated with increased 
functional transmission reducing activity in SMFA. Ongoing studies in the US and Mali have 
thus far determined that adding another transmission blocking target, Pfs230D1M, either alone or 
in combination with Pfs25M is safe and immunogenic.  The immunological data from a Phase 1 
Pfs25M-EPA/ Alhydrogel®  and Pfs230D1M-EPA/ Alhydrogel®  study in the US and Mali has 
likewise shown a consistent increase in Pfs25 and Pfs230D1M antibody response by ELISA 
following each subsequent vaccination that correlate with functional activity with SMFA.  
Specifically, vaccine regimens containing Pfs230 can achieve high functional activity by SMFA, 
however the duration of this activity is still to be determined. Additionally, preclinical studies of 
both Pfs25M and Pfs230D1M have shown that formulation with AS01 adjuvant induces 
significantly higher and longer lived antibody by ELISA titers, and significantly greater 
transmission blocking activity by SMFA, when compared to the existing formulations in 
Alhydrogel® (Figure 2). 
 
In parallel, we will evaluate the vaccine antigen Pfs230D1M, at full dosing and at fractional 
dosing, when formulated with AS01 for its ability to induce antibodies that prevent parasite 
transmission to mosquitoes. Vaccine activity will be assessed by testing sera in SMFA and by 
direct skin feeds to measure the reduction of parasite transmission in vaccinated individuals 
versus controls. 
 
If evidence from these initial trials indicate that Pfs230D1M has measurable efficacy/activity, we 
envision subsequent trial(s) of a pre-erythrocytic vaccine (such as RTS,S/AS01B; adult 
formulation of Mosquirix™) in combination with Pfs230D1M formulated in AS01 to assess their 
combined activity for interrupting malaria transmission in naturally exposed populations.  
 
A step forward in this clinical plan is to assess the durability of TBV vaccine responses with the 
adjuvant AS01. As described in Version 7.0 of the protocol, given the promising 
immunogenicity and functional activity results from the unblinded pilot study (including the 
same vaccination regimen and dose utilized in the main study) and the reassuring safety data 
from the currently blinded main study, the plan is to 1) continue to monitor and evaluated the 
durability of vaccine specific antibody responses and associated functional activity in both the 
pilot and main study; 2) assess the safety, immunogenicity, and functional activity over 
subsequent malaria transmission season following receipt of an additional vaccination.  
4 Study Objectives 
4.1 
Primary Objective 
• To assess safety and reactogenicity of Pfs25M-EPA/AS01, Pfs230D1M-EPA/AS01, and 
simultaneous administration of Pfs25M-EPA/AS01 and Pfs230D1M-EPA/AS01, 
including Pfs230D1M-EPA/AS01 at fractional dosing in Malian adults. (All arms) 


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• To assess safety and reactogenicity of a booster dose of Pfs230D1M-EPA/AS01 at full 
dosing in Malian adults. (Arms 2c, 2d, 4c)  
4.2 
Secondary Objective 
• To assess the antibody response to the Pfs25 and Pfs230 protein as measured by ELISA. 
(All arms) 
• To assess the functional antibody response to the Pfs25 and Pfs230 protein as measured 
by standard membrane feeding assay (SMFA). (All arms except Arms 1a, 2a, 3a) 
• To assess the functional antibody response to Pfs230 protein as measured by direct skin 
feeds (DSF).  (Arms 2c, 2d, 4c only) 
4.3 
Exploratory Objectives 
• 
To explore cellular and transcriptomic responses to Pfs25 and Pfs230 vaccines when 
administered alone or in combination. (All arms except 1a, 2a, 3a) 
• 
To explore the impact of co-infections on malaria vaccine responses. (Arms 2c, 2d, 4c 
only)  
• 
To explore the antibody repertoire of functional antibody responses. (All arms except 1a, 
2a, 3a) 
5 Study Design 
5.1 
Overall Design  
This is a phase 1 study designed to evaluate safety, reactogenicity, immunogenicity, and 
transmission-blocking activity (via SMFA)) of Pfs25M-EPA and Pfs230D1M-EPA conjugates 
formulated on AS01 and transmission-blocking activity (via DSF) of Pfs230D1M-EPA 
conjugates formulated on AS01 at full and fractional dosing.  We will enroll a total of 305 
vaccinated subjects to be divided between multiple sites in Mali as shown in Appendix A and 
described below.  
 
In Bamako/Sotuba, Mali (N=65) 
Dose-escalating, open label, randomized, pilot study  
 
1. Group 1: Pfs25M-EPA/AS01 (n=15) 
• Arm 1a (n=5), to receive 16 μg Pfs25M-EPA/AS01 on D0, D28, D168  
• Arm 1b (n=10), to receive 47 μg Pfs25M-EPA/AS01 on D0, D28, D168 
 
 
2. Group 2: Pfs230D1M-EPA/AS01 (n=15) 
• Arm 2a (n=5), to receive 13 μg Pfs230D1M-EPA/AS01 on D0, D28, 
D168  
• Arm 2b (n=10), to receive 40 μg Pfs230D1M-EPA/AS01 on D0, D28, 
D168 


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3. Group 3: Pfs25M-EPA/AS01 and Pfs230D1M-EPA/AS01 (n=15) 
• Arm 3a (n=5), to receive 16 μg Pfs25M-EPA/AS01 and 13 μg 
Pfs230D1M-EPA/AS01 on D0, D28, D168  
• Arm 3b (n=10), to receive 47 μg Pfs25M-EPA/AS01 and 40 μg 
Pfs230D1M-EPA/AS01 on D0, D28, D168 
 
 
4. Group 4: Comparator (n=20) 
• Arm 4a (n=10), to receive ENGERIX-B on D0, D28, and D168  
• Arm 4b (n=10), to receive ENGERIX-B on D0, D28, and D168 
 
In Bancoumana and Doneguebougou, Mali (N=240) 
Double-blind, randomized, phase 1 clinical trial 
 
1. Group 2: Pfs230D1M-EPA/AS01 (n=120) 
• Arm 2c (n=60), to receive 40 μg Pfs230D1M-EPA/AS01 on D0, D28, 
D168, and a fourth dose of 40 μg on D476  
• Arm 2d (n=60), to receive 40 μg Pfs230D1M-EPA/AS01 on D0, D28, 
then 8 μg Pfs230D1M-EPA/AS01 (fractional dose) on D168, and a fourth 
dose of 40 μg on D476 
 
 
2. Group 4: Comparators (n=120) 
• Arm 4c (n=120), to receive ENGERIX-B on D0, D28, and D168 (start 
study with Arm 2c and 2d), and Menactra® on D476.  
 
The multisite study in Mali is designed in a stepwise manner such that each dose of the TBVs is 
administered in a dose-escalating fashion, and each vaccine (either Pfs25M-EPA/AS01 or 
Pfs230D1M-EPA/AS01) is administered first as a single vaccination at the target dose before it 
is administered simultaneously. Additionally, two doses of each vaccine will be administered and 
assessed for safety as outlined in Section 12.14.4 and Appendix A prior to implementation of 
the first vaccination in the double blind portion of the study.  
5.1.1 Sotuba/Bamako Pilot Study 
The open-label, dose-escalating pilot study is designed to evaluate vaccine safety and 
reactogenicity in healthy Mali adults living in a low malaria transmission setting (Sotuba, a 
suburb of Bamako, Mali). Subjects enrolled in the initial safety cohort will receive a single dose 
of a single antigen (either Pfs25M or Pfs230D1M) prior to vaccinating new enrolled subjects 
who will receive the co-administration of Pfs25M and Pfs230D1M.  Additional subjects (n=10 
per arm; Arms 1b, 2b, 3b, 4a, 4b) will be enrolled to receive the targeted vaccine dose for further 
safety and immunogenicity evaluation as outlined in Appendix A. Data from subjects enrolled in 
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and will be used for exploratory objectives examining the antibody repertoire of functional 
antibody responses to vaccination. No blinding will be implemented, but comparators will be 
enrolled to control for variable natural blocking activity that may present during the rainy season 
and as safety controls.  Randomization will occur in Arms 1b, 2b, 3b, 4a, 4b.  Arms 1a, 2a, 3a 
will enroll in the study on an available basis.  
5.1.2 Bancoumana and Doneguebougou Main Study 
The double-blind, randomized, comparator controlled portion of the study is designed to also 
evaluate study vaccine safety and reactogenicity in healthy malaria-exposed individuals, but is 
powered as well to assess for immunogenicity and functional activity via SMFA and DSF.  
 
Following scheduled, intentional unblinding at study day 336, subjects from the main study will 
be offered re-enrollment into the open label fourth vaccination. 
5.2 
Study Endpoints 
5.2.1 Primary Endpoint 
• Incidence of local and systemic AEs and SAEs in Malian adults. (All arms) 
• Incidence of local and systemic AEs and SAEs in Malian adults. (Arms 2c, 2d, 4c) 
5.2.2 Secondary Endpoints 
• Anti-Pfs25 IgG levels as measured by ELISA. (All arms) 
• Anti-Pfs230 IgG levels as measured by ELISA. (All arms) 
• TRA/TBA of induced antibody in SMFA. (All arms except 1a, 2a, 3a) 
• Transmission interruption activity measured by DSF. (Arms 2c, 2d, 4c only) 
5.2.3 Exploratory Endpoints 
• Cellular immune responses to vaccination. (All arms except 1a, 2a, 3a) 
• Whole genome transcriptional profiling. (All arms except 1a, 2a, 3a) 
• Antibody levels against recombinant EPA, and other malaria antigens, such as Pfs48/45, 
expressed during the gametocyte development. (All arms except 1a, 2a, 3a) 
• Schistosomiasis detection (in urine). (Arms 2c, 2d, 4c only)  
• qPCR for helminth detection (from stool). (Arms 2c, 2d, 4c only)  
• Sequence B cell receptor/antibody genes. (All arms except 1a, 2a, 3a) 
5.3 
Sample Size and Estimated Duration of Study 
A total of 305 subjects will be vaccinated in this trial. Note: more subjects may be initially 
enrolled, receive drug treatment (Coartem®) prior to vaccination, but no more than 305 subjects 


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will receive at least one vaccine.  Each subject will be monitored actively for at least 6 months 
after the last vaccination, for a total of 12 to 16 months, depending on the vaccination schedule 
and timing of screening for the study.  Up to 900 subjects will be screened to accommodate 
possible screening failures. 
Participants who had previously been enrolled in the study (up to 301) will be re-enrolled for an 
additional 6 to 18 months depending on arm assignment and timing of reconsent. 
 
Definitions for the purpose of this study: 
• Screened – subjects will receive a study identification number when the informed consent 
is signed and will either be determined as “enrolled” or “screen failures” as noted below. 
o Screening may be completed over the course of multiple visits. 
o Screening, in most cases, will occur within 56 days prior to enrollment into the 
study 
 If the screening visit is >56 days prior to enrollment, then an updated 
medical review, physical exam/vital signs, repeat Malaria Comprehension 
Exam, and repeat laboratory evaluation (inclusive of safety labs, HIV, 
Hepatitis B, C) will be completed to determine eligibility for enrollment. 
• Enrolled – subject will be considered enrolled beginning with the receipt of the first 
vaccination or at time completion of randomization. 
• Randomized – the subjects are considered randomized when they meet the following 
criteria: 
o Confirmation that the inclusion/exclusion criteria are met. 
o Randomization number assigned.  Note, randomization number may be the same 
as the study identification number.  
• Screen Failures – subjects are considered screen failures when they meet 1 of the 
following criteria after signing consent: 
o Screening results reveal that the subject is ineligible per Section 6.4 and/or 6.5. 
o Subject withdraws consent before being vaccinated and/or randomized  
• Discontinued – subject are considered discontinued when they meet 1 or more of the 
following criteria: 
o Subject withdraws consent after being vaccinated and/or randomized. 
o Subject is withdrawn by the PI/Sponsor after being vaccinated and /or 
randomized. 


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• Completed  -- subjects are considered completed when they complete the final study visit 
for their arm. 
• Re-enrolled – subject will be considered re-enrolled beginning with signing of the re-
enrollment consent form. 
6 Study Population 
6.1 
Description of Population and Sites 
6.1.1 Rationale for Research Subject Selection 
Healthy volunteers will be recruited for this study. 
6.2 
Clinical Sites 
The study will be carried out in collaboration between the LMIV/NIAID/NIH and the MRTC in 
Bamako, Mali. The study will be conducted by the MRTC at three locations in Mali, West Africa  
(see Map 1). The MRTC staffing at the Mali site is employed by NIAID/USTTB programs and 
assigned to the Sotuba/Bamako, Bancoumana, and Doneguebougou sites for the duration of the 
study to provide clinical care and execute the protocol.    
 
Map 1: Map Showing the Three Study Sites 
 


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6.2.1.1 Sotuba/Bamako Site 
Bamako is the capital of Mali, located on the Niger river and has a population of about 1.7 
million people.  The district of Bamako is divided into urban (hypoendemic) and periurban 
(mesoendemic) areas and peripheral villages (hyperendemic). Sotuba is a village located on the 
outskirts of Bamako on the bank of the Niger River, consisting of ∼6,500 inhabitants. Malaria 
transmission follows the same seasonality as in Bancoumana and Donéguébougou, although 
entomological inoculation rates are historically much lower. The annual rainfall varies between 
800 mm and 1000 mm and occurs from June to October. Many clinical trials (malaria vaccine 
and drug trials), as well as epidemiological and entomologic malaria studies, have been done in 
Sotuba. As in Donéguébougou and Bancoumana, the MRTC maintains a medical clinic and 
laboratory in Sotuba and has been working at this site since 1993. 
 
To note, if any issues arise prohibiting starting the study within Bamako and/or Sotuba, this 
phase of the study (as outlined in Appendix A) can be completed in either Bancoumana or 
Doneguebougou. 
6.2.1.2 Bancoumana and Doneguebougou Site 
Bancoumana is located 60 kilometers southwest of Bamako and has a population of about 9,000 
people. The site is situated in the south-Sudanian area of Mali. The climate is hot, with daily 
temperatures ranging from 19°C to 40°C. The annual rainfall varies between 600 mm and 1200 
mm and occurs from June to October. Many clinical trials, as well as epidemiological and 
entomologic malaria studies, have been done in Bancoumana.26-28   
 
Doneguebougou is a community located 30 km north of Bamako and has a population of about 
2,000, with another 2,000 inhabitants in the surrounding villages.  For the purpose of vaccine 
trials, adequate facilities for conducting an interventional trial have been put in place at 
Doneguebougou within walking distance to the residents’ homes. At Doneguebougou, the 
malaria transmission is highly seasonal, with the transmission season taking place from June 
until December. Doneguebougou is situated in a high transmission area, with entomological 
inoculation rates (determined by human landing catch) as high as 137 to 167 infectious bites per 
person over 1 transmission season. 
 
Healthy adult subjects will be recruited from the surrounding community and villages, and the 
demographics of the study population will therefore be representative of that community.  
6.3 
Recruitment 
Community permission will be obtained from village elders and other community members in 
Sotuba/Bamako, Bancoumana, and Doneguebougou after explanation and discussion of the study 
at a community meeting (see Section 15.2.1). A general announcement inviting household and 


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family members to the participating clinic to learn about the study will be made at the time of 
community permission, using local radio or any traditional channel of communication.   
6.4 
Inclusion Criteria (Initial enrollment and Re-enrollment) 
All of the following criteria must be fulfilled for a volunteer to participate in this trial: 
1. Age ≥ 18 and ≤ 50 years. (Note for re-enrollment: ≤ 52 years) 
2. Available for the duration of the trial.  
3. Able to provide proof of identity to the satisfaction of the study clinician completing the 
enrollment process. 
4. In good general health and without clinically significant medical history in the opinion of the 
investigator. 
5. Females of childbearing potential must be willing to use reliable contraception (as defined 
below) from 21 days prior to Study Day 0 (Study Day 476 for re-enrollment) and then until 3 
months after  last vaccination.  (Arms 2c, 2d, 4c only for re-enrollment) 
• Reliable methods of birth control include one of the following: confirmed pharmacologic 
contraceptives (parenteral) delivery; intrauterine or implantable device.  
• Reliable methods of birth control include concurrent use of a pharmacologic and a barrier 
method, i.e.  two of the following: confirmed pharmacologic contraceptives (oral, 
transdermal) delivery or vaginal ring AND condoms with spermicide or diaphragm with 
spermicide.  
• Non-childbearing women will also be required to report date of last menstrual period, 
history of surgical sterility (i.e. tubal ligation, hysterectomy) or premature ovarian 
insufficiency (POI), and will have a baseline urine or serum pregnancy test performed. 
6. Willingness to have blood samples stored for future research. 
7. Willingness to undergo DSFs (Arms 2c, 2d, 4c only). 
8. Known resident of Bancoumana or Doneguebougou or surrounding area or known student or 
long term resident (more than 1 year) of Bamako/Sotuba, Mali  
6.5 
Exclusion Criteria (Initial enrollment and Re-enrollment) 
A subject will be excluded from participating in this trial if any one of the following criteria is 
fulfilled:  
1. Pregnancy as determined by a positive urine or serum human choriogonadotropin (β-
hCG) test (if female). (Arms 2c, 2d, 4c only for re-enrollment) 
NOTE: Pregnancy is also a criteria for discontinuation of any further dosing or non-
safety related interventions for that subject. 
2. Currently breast-feeding (if female). (Arms 2c, 2d, 4c only for re-enrollment) 


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3. Behavioral, cognitive, or psychiatric disease that in the opinion of the investigator affects 
the ability of the participant to understand and comply with the study protocol. 
4. Hemoglobin, WBC, absolute neutrophils, and platelets outside the local laboratory-
defined limits of normal (subjects may be included at the investigator’s discretion for ‘not 
clinically significant’ values outside of normal range and ≤ Grade 1). (Arms 2c, 2d, 4c 
only for re-enrollment) 
5. Alanine transaminase (ALT) or creatinine (Cr) level above the local laboratory-defined 
upper limit of normal (subjects may be included at the investigator’s discretion for ‘not 
clinically significant’ values outside of normal range and ≤ Grade 1). (Arms 2c, 2d, 4c 
only for re-enrollment) 
6. Infected with human immunodeficiency virus (HIV), hepatitis C virus (HCV), or 
hepatitis B (HBV). (Arms 2c, 2d, 4c only for re-enrollment) 
7. Evidence of clinically significant neurologic, cardiac, pulmonary, hepatic, endocrine, 
rheumatologic, autoimmune, hematological, oncologic, or renal disease by history, 
physical examination, and/or laboratory studies including urinalysis. 
8. History of receiving any investigational product within the past 30 days. 
9. Participation or planned participation in a clinical trial with an investigational product 
prior to completion of the follow up visit 28 days following last vaccination OR planned 
participation in an investigational vaccine study until the last required protocol visit 
10. Subject has had medical, occupational, or family problems as a result of alcohol or illicit 
drug use during the past 12 months. 
11. History of a severe allergic reaction or anaphylaxis. (Arms 2c, 2d, 4c only for re-
enrollment) 
12. Severe asthma, defined as asthma that is unstable or required emergent care, urgent care, 
hospitalization, or intubation during the past 2 years, or that has required the use of oral 
or parenteral corticosteroids at any time during the past 2 years. (Arms 2c, 2d, 4c only for 
re-enrollment) 
13. Pre-existing autoimmune or antibody-mediated diseases including but not limited to: 
systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, Sjögren’s 
syndrome, or autoimmune thrombocytopenia. (Arms 2c, 2d, 4c only for re-enrollment) 
14. Known immunodeficiency syndrome. (Arms 2c, 2d, 4c only for re-enrollment) 
15. Known asplenia or functional asplenia. (Arms 2c, 2d, 4c only for re-enrollment) 
16. Use of chronic (≥14 days) oral or intravenous corticosteroids (excluding topical or nasal) 
at immunosuppressive doses (i.e., prednisone >10 mg/day) or immunosuppressive drugs 
within 30 days of Study Day 0. (Arms 2c, 2d, 4c only for re-enrollment) 
17. Prior to Study Day 0 and every subsequent vaccination day, receipt of a live vaccine 
within the past 4 weeks or a killed vaccine within the past 2 weeks. (Arms 2c, 2d, 4c only 
for re-enrollment) 


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18. Receipt of immunoglobulins and/or blood products within the past 6 months. (Arms 2c, 
2d, 4c only for re-enrollment) 
19. Previous receipt of an investigational malaria vaccine in the last 5 years. (For re-
enrollment – does NOT include receipt of Pfs230D1M-EPA/AS01) (Arms 2c, 2d, 4c only 
for re-enrollment) 
20. History of severe reaction to mosquito bites (Arms 2c, 2d, 4c only) 
21. History of allergy to the comparator vaccine (Initial enrollment: such as latex, yeast, or 
previous Hepatitis B vaccine) (Re-enrollment: Arm 4c only; previous meningococcal 
vaccine components, diptheria toxoid or CRM197 containing vaccine) 
22. History of Guillain-Barre syndrome (Re-enrollment: Arm 4c only) 
23. Known allergies or contraindications (such as significant cardiac disease; prolonged QTc 
>450 ms; currently taking medications that may prolong your QTc; serious side effects 
from Coartem® in the past) to study treatment (Coartem® [artemether/lumefantrine]) 
(Arms 2c, 2d, 4c only) (Initial enrollment only) 
24. Other condition 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. 
6.6 
Justification for Exclusion of Pregnant Women 
This study will not enroll pregnant and/or breastfeeding women since the effects of Pfs25M-
EPA/AS01 and Pfs230D1M-EPA/AS01 vaccines on the developing human fetus are unknown 
with the potential for teratogenic or abortifacient effects.  Because this is an unknown but 
potential risk for adverse events in nursing infants secondary to treatment of the mother with 
Pfs25M-EPA/AS01 and Pfs230D1M-EPA/AS01 women that are breastfeeding will also be 
excluded from the study.  
6.7 
Justification for Exclusion of Children 
This study will not enroll children, since appropriate dosing or safety have not yet been 
established in adults.   
7 Study Agents 
7.1 
Transmission Blocking Vaccines (Pfs25M-EPA/AS01, Pfs230D1M-EPA/AS01) 
7.1.1 Pfs25M-EPA/AS01 
The PpPfs25M and EcEPA lots, both manufactured at Walter Reed Bioproduction facility (Silver 
Spring, Maryland) in cGMP compliance, were used to manufacture the conjugate. PpPfs25M is a 
Pichia-expressed recombinant Pfs25 with a molecular mass of 18,713 Daltons.  EcEPA is an E. 
coli-expressed recombinant protein with molecular mass of 66,975 Daltons. The Pfs25M-EPA 
conjugate was produced by reaction between thiolated PpPfs25M and maleimide-activated 


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EcEPA, followed by purification using size-exclusion chromatography. The Pfs25M-EPA 
conjugate was manufactured at Walter Reed Bioproduction facility in cGMP compliance in 
January 2015.   
 
The Pfs25M-EPA was formulated as conjugated Pfs25M in 4 mM PBS to a 2X dilution of the 
high dose (188µg/mL in 0.5mL volume) in cGMP compliance at Walter Reed Bioproduction 
facility in April 2016 and will be provided as a single use vial. AS01B adjuvant will also be 
provided as a single use vial by GSK, with a concentration of 100µg/mL MPL and 100 µg/mL 
QS21 in a liposomal formulation in a volume of 0.625mL. For each dose of vaccine, the two 
single-use vials, one of conjugated Pfs25M in 4mM PBS, and one of AS01B, will be mixed 1:1 at 
bedside. A dilution of the conjugated Pfs25M in 4mM PBS with normal saline will be performed 
prior to admixing with AS01B for the 16 µg dose. For the 47µg dose, the AS01B will be mixed 
directly with the vialed conjugated Pfs25M in 4mM PBS.  
 
Dosing volumes of 0.5 mL will be prepared for delivery of either 16µg conjugated Pfs25M, 
15µg conjugated EPA in diluted AS01B (25µg MPL+25µg QS21 liposomal formulation); or 
47µg conjugated Pfs25M, 4 µg conjugated EPA in diluted AS01B (25µg MPL+25µg QS21 
liposomal formulation). The vaccine will be mixed and drawn up into the syringes directly prior 
to injection. 
7.1.2 Pfs230D1M-EPA/AS01 
The PpPfs230D1M and EcEPA lots, were also both manufactured at Walter Reed Bioproduction 
facility (Silver Spring, Maryland) in cGMP compliance, were used to manufacture the conjugate. 
PpPfs230D1M is a Pichia-expressed recombinant a sub-segment (S542-G736) of Pfs230 with a 
molecular mass of 21,854 Daltons. EcEPA is an E. coli-expressed recombinant protein with 
molecular mass of 66,975 Daltons. The Pfs230D1M-EPA conjugate was produced by reaction 
between thiolated PpPfs230D1M and maleimide-activated EcEPA, followed by purification 
using size-exclusion chromatography. The Pfs230D1M-EPA conjugate was manufactured at 
Walter Reed Bioproduction facility in cGMP compliance in May 2015.   
 
The Pfs230D1M-EPA was formulated as conjugated Pfs230D1M in 4 mM PBS to a 2X dilution 
of the high dose (160µg/mL in 0.5 mL volume) in cGMP compliance at Walter Reed 
Bioproduction facility in April 2016 and will be provided as a single use vial. AS01B adjuvant 
will also be provided as a single use vial by GSK, 100µg/mL MPL and 100µg/mL QS21 in a 
liposomal formulation in a volume of 0.625 mL. For each dose of vaccine, the two single-use 
vials, one of Conjugated Pfs230D1M in 4mM PBS, and one of AS01B, will be mixed 1:1 at 
bedside. A dilution of the conjugated Pfs230D1M in 4 mM PBS with normal saline will be 
performed prior to admixing with AS01B for the 13µg. For the 40µg dose, the AS01B will be 
mixed directly with the vialed conjugated Pfs230D1M in 4 mM PBS. For the 8µg fractional 
dose, the AS01B will be mixed with Pfs230D1M directly as noted above for the 40µg dose, but 
only 1/5th (0.1 mL) of the volume will be administered. 


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Dosing volumes of 0.5mL will be prepared for delivery of either 13µg conjugated Pfs230D1M, 
10µg conjugated EPA in diluted AS01B (25µg MPL+ 25µg QS21 liposomal formulation); or 
40µg conjugated Pfs230D1M, 31µg conjugated EPA in diluted AS01B (25µg MPL+ 25µg QS21 
liposomal formulation). Dosing volume of 0.1mL (fractional dose) will be prepared for delivery 
of 8µg conjugated Pfs25M, 6µg conjugated EPA in AS01 (5µg MPL+ 5µg QS21 liposomal 
formulation). The vaccine will be mixed and drawn up into the syringes directly prior to 
injection. 
7.1.3 Disposition and Dispensation 
The Pfs25M-EPA and Pfs230D1M-EPA vials will be supplied to the study site pharmacist by the 
Sponsor or Sponsor Representative. The Sponsor receives the product from Pilot Bioproduction 
Facility, WRAIR, Silver Spring, Maryland, where the materials were formulated and packaged. 
Vaccines will be labeled for investigational use only. The AS01B vials will be supplied by GSK. 
7.1.4 Formulation, Packaging, and Labeling 
Pfs25M-EPA was manufactured at Walter Reed Bioproduction facility in cGMP compliance in 
April 2016. Each single-use vial contains 188µg/mL conjugated Pfs25M, 180µg/mL conjugated 
EPA in 4mM PBS in a volume of 0.5 mL. The vial label reads: 188µg/mL Conjugated Pfs25M in 
4mM PBS. 
 
Pfs230D1M-EPA was manufactured at Walter Reed Bioproduction facility in cGMP compliance 
in April 2016. Each single-use vial contains 160µg/mL conjugated Pfs230D1M, 124µg/mL 
conjugated EPA in 4mM PBS in a volume of 0.5mL. The vial label reads: 160µg/mL Conjugated 
Pfs230D1M in 4mM PBS. 
 
AS01 is a liposome-based Adjuvant System containing the immunoenhancers MPL (3-Odesacyl- 
4’-monophosphoryl lipid A) and QS21 (a saponin molecule purified from the bark extract of 
Quillaja saponaria Molina tree). There are two formulations in the AS01 family: AS01B 
Adjuvant System and AS01E Adjuvant System. While AS01B contains 50µg of MPL and 50µg 
of QS21 per one human dose, AS01E contains half the quantity of each immunoenhancer, i.e. 25 
µg of MPL and 25 µg of QS-21.  For this study, the AS01B adjuvant will be used at a diluted 
concentration similar to the concentration of AS01E, but not same formulation as AS01E, thus the 
designation of AS01 or diluted AS01B throughout the protocol.  
7.1.5 Study Agent Storage and Shipping 
Pfs25M-EPA and Pfs230D1M-EPA must be stored at -80°C until time of transport to field site. 
At this time, vaccine can gradually thaw from -80°C to 9°C on day of use. Vaccine can not be 
refrozen for use after a thaw. AS01B must be stored at +2°C to +8°C. Vials will be transported 
and stored at temperature-controlled conditions, as per SOPs. Temperature data loggers will 


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accompany the vaccines at all times to ensure storage temperatures limits have not been violated. 
Adjuvant should NOT be frozen at any time. Refrigerator and freezer temperatures will be 
continuously monitored.   Access to study vaccine will be limited to authorized study personnel.  
Any temperature excursion outside the defined range must be reported to the Sponsor prior to 
use.  The impacted products must not be used and must be stored in quarantine at label 
temperature conditions until usage approval has been obtained from the Sponsor. 
7.1.6 Preparation, Administration, and Dosage of Study Agents 
The TBV conjugates are stored at -80C until just before transport to the field for use. Then stored 
2-8°C as described in Section 7.1.5. AS01 adjuvant are stable in the refrigerator at +2°C to +8°C. 
The final vaccine for administration is obtained by admixing Pfs25M-EPA or Pfs230D1M-EPA 
with AS01 and has to be injected intra-muscularly within 4 hours of reconstitution. The 
formulation of the study vaccine is presented in Table 12. 
 
 
 
Table 12: TBV Formulation   
Vaccine 
Product Name 
Formulation 
Volume to be 
administered 
Number of 
doses 
Number of 
booster dose 
 
 
TBV/AS01 
(Low dose): 
Arms 1a, 2a, 3a 
Pfs25M-EPA 
(Arm 1a, 3a) 
Pfs25M = 16µg 
EPA = 15µg  
0.5 mL 
 
 
 
 
3 doses 
0 doses 
Pfs230D1M-EPA 
(Arm 2a, 3a) 
Pfs230D1M = 13µg 
EPA = 10µg 
AS01B 
(Arms 1a, 2a, 3a) 
AS01 = 25µg 
MPL+ 25µg QS21 
liposomal 
formulation 
 
TBV/AS01 
(Targeted dose): 
Arms 1b, 2b, 
3b, 2c, 2d 
Pfs25M-EPA 
(Arms 1b, 3b) 
Pfs25M = 47µg 
EPA = 45µg  
0.5 mL 
 
Arm 1b, 3b: 
3 doses  
 
0 doses 
Pfs230D1M-EPA 
(Arms 2b, 3b, 2c, 
2d) 
Pfs230D1M = 40µg 
EPA = 31µg 
Arm 2b, 2c, 
3b: 3 doses  
 
Arm 2d: 2 
doses 
Arms 2c, 2d: 1 
dose of 40µg 
 
AS01B 
(Arms 1b, 2b, 3b, 
2c, 2d) 
AS01 = 25µg 
MPL+ 25µg QS21 
liposomal 
formulation 


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TBV/AS01 
(fractional 
dose): 
Arms 2d only 
Pfs230D1M-EPA 
Pfs230D1M = 8µg 
EPA = 6µg 
0.1 mL 
Arm 2d: 1 
dose 
AS01B 
AS01 = 5µg MPL+ 
5µg QS21 
liposomal 
formulation  
7.2 
Comparator Vaccines 
7.2.1 ENGERIX-B (Hepatitis B Vaccine) 
ENGERIX-B (Hepatitis B [Recombinant] Vaccine; GlaxoSmithKline) is a sterile suspension for 
intramuscular administrations that contains inactivated, noninfectious hepatitis B virus surface 
antigen (HBsAg).  The purified HBsAg is obtained by culturing genetically engineered 
Saccharomyces cerevisiae yeast cells, which carry the surface antigen gene of the hepatitis B 
virus.  HBsAg is then adsorbed separately onto aluminum salts. A 1 mL dose of vaccine contains 
20 µg of recombinant HBsAg protein, 0.5 mg of aluminum in the form of aluminum hydroxide.  
The vaccine is manufactured by GlaxoSmithKline.  ENGERIX-B is FDA approved for the active 
immunization against disease caused by all known subtypes of hepatitis B virus in neonates, 
infants, children, and nonpregnant adults given at 0-, 1-, and 6-month schedule.  
 
A 1 mL dose is recommended for adults aged 20 years or older at a 0-, 1-, and 6-month schedule. 
While 0.5 mL dose is the package insert recommended dosing for neonates, infants, children, 
adolescents, and young adults (neonates through 19 years of age), for consistency within the trial 
and given 1 mL at 0-, 1-, and 6-months is an approved alternate schedule for 11 through 19 years 
of age, all subjects enrolled in the study (18-50 years old) will receive 1mL ENGERIX-B at 0-, 
1-, and 6-months.   
  
Additional information is provided in the ENGERIX-B package insert provided.  
 
For subjects who received a TBV during the study, the comparator vaccine will be offered at no 
cost to the participants. For Hepatitis B, this may be ENGERIX-B or another FDA approved or 
WHO certified Hepatitis B containing vaccine, such as TWINRIX. 
7.2.2 Menactra® 
Menactra® (Sanofi Pasteur) is a sterile, intramuscularly administered vaccine that contains 
Neisseria meningitidis serogroup A, C, Y, and W-135 capsular polysaccharide antigens 
individually conjugated to diphtheria toxoid protein.  No preservative or adjuvant is added during 
the manufacturing process.  Menactra® is FDA approved for active immunization to prevent 
invasive meningococcal disease caused by Neisseria meningitidis serogroups A, C, Y, and W-
135 (but does not protect against serotype B) for use in individuals 9 months through 55 years of 


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age.  A single dose (0.5 mL) is recommended for those individuals 18 to 50 years of age and 
otherwise healthy who are at increased risk for meningococcal disease (e.g., individuals in an 
epidemic or highly endemic country such as Mali). 
 
Additional information is provided in the Menactra® package insert provided. 
 
Subjects who received a 4th dose of TBV during the study will be offered Menactra® or an 
equivalent appropriate FDA approved or WHO certified Neisseria meningitidis vaccine at no 
cost to the participants.  
 
7.3 
Normal Saline 
Sterile isotonic (0.9%) normal saline will be commercially procured in the US and shipped to 
Mali at ambient temperature. Normal saline will be used for diluting Pfs25M-EPA and 
Pfs230D1M-EPA prior to formulation with AS01B for the lower dose groups in Bamako, Mali. 
7.4 
Administration 
Vaccines will be administered as intramuscular injections into the deltoid muscle. Arms may be 
alternated with successive vaccinations if a single vaccination is given. If simultaneous 
vaccinations are to be administered (two individual vaccinations at the same time), each vaccine 
is drawn up and delivered separately.   When choosing an arm for the vaccine injection, 
clinicians should consider whether there is an arm injury, local skin problems such as scaring or 
rash, or significant tattoo that precludes administering the injection or will interfere with 
evaluating the arm after injection.  In keeping with the MRTC practices and procedures, and 
good medical practice, acute medical care will be provided to subjects for any immediate allergic 
reactions or other injury resulting from participation in this research study.   
 
Due to the variance in volume and appearance of the study product (Pfs230D1M-EPA/AS01) in 
comparison to the control vaccines (ENGERIX-B), opaque tape will be wrapped around the 
vaccine syringe(s) when being administered in the double blind study in Mali. 
7.4.1.1 Contraindications to Vaccination  
The following criteria should be checked prior to each immunization and are contraindications to 
further immunization:  
• Hypersensitivity reaction following administration of the study vaccine. 
• Positive urine or serum β-hCG test prior to vaccination. 
 
Subjects will be encouraged to remain in the study for safety evaluation of doses already 
received and completed research visits for immunogenicity, functional activity, and protective 
efficacy if deemed safe by the PI. 


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7.4.1.2 Indications for Deferral of Vaccination 
If any of the following AEs occurs at the time of the scheduled vaccination, the subject may be 
either vaccinated at a later date within the allowable visit window as specified in the protocol or 
withdrawn at the discretion of the Investigator: 
• Oral temperature >37.5˚C at the time of vaccination will warrant deferral of 
immunization until fever resolves (within protocol-defined vaccination window). 
• Any other condition that in the opinion of the Investigator poses a threat to the individual 
if immunized or that may complicate interpretation of the safety of vaccine following 
immunization. 
 
Such individual(s) will be followed in the clinic until the symptoms resolve or the window for 
immunization expires. No further vaccination will be performed if the subject does not recover 
(temperature ≤37.5˚C and/or lack of symptoms) within the vaccination window, though subject, 
if well may proceed to receive additional vaccines at future vaccination visits at the PI’s 
discretion.  The subject will be monitored for safety and immunogenicity for 6 months after their 
last vaccination, unless the subject has withdrawn consent. If the subject does not receive the 
second or third vaccination some scheduled safety blood draws may or may not be performed at 
the discretion of the PI. Blood draws scheduled to measure immune responses and protective 
efficacy will be obtained if possible.   
 
If the subject meets any of the above criteria for deferral on the day of first immunization, the 
Investigator may elect to exclude the subject from further participation in the study and that 
subject may be replaced. If the subject meets any of the above criteria for deferral on the day of 
second or third immunization, the Investigator may elect to include the subject in future 
vaccinations (if missed second) or continue to follow them per protocol without additional 
vaccinations for the remainder of the study. Subjects who miss vaccination #2 or #3 cannot be 
replaced.  
7.5 
Coartem® 
Artemether/lumefantrine (Coartem®) is a licensed antimalarial in the US and Mali for treatment 
of uncomplicated malaria.  It has an excellent safety profile and is widely used to treat malaria. 
Subjects who may have any contraindications to the use of these drugs will be excluded at 
screening.  Coartem® will be dosed with food and administered over 3 days for a total of 6 doses, 
as per package insert and standard adult dosing. 
 
All antimalarial medications used for the study, including Coartem® which is used for pre-
emptive treatment of all subjects in Arms 2c, 2d, and 4c prior to Vaccination #1 will be 
maintained at the study site. Coartem® tablets will be purchased from commercial sources and 
provided by the MRTC study team to subjects. Drug accountability will be managed by the site 
clinical team. 


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7.5.1 Preparation and Administration 
Coartem® will be provided as tablets for oral administration.  Administration will be by study 
staff according to dosing parameters.   
7.5.2 Storage and Handling 
Coartem® tablets will be maintained in the manufacturer’s original packaging and stored at the 
clinic under recommended storage conditions until prepared for dispensing. 
 
7.5.3 Return of Study Product 
Final accountability of drug supplies will be performed at the conclusion of the study.  Final 
disposition of any remaining Coartem® will be determined and documented. 
7.6 
Prohibited Medications and Procedures 
Treatment with any of the following medications/procedures could potentially interfere with 
vaccine-induced immunity and will not be permitted. Use of any of these during the study may 
exclude a subject from receiving further doses of the study vaccine.  However, the subject will be 
encouraged to remain in the study for safety evaluations. 
• Licensed killed vaccines in the two week period prior to and following each vaccination or 
licensed live vaccines in the four week period prior to and following each vaccination. 
• Receipt of immunoglobulins and/or any blood products up to 6 months prior to the first 
vaccination and for 30 days after administration of the last dose of vaccine. 
• Chronic oral or intravenous administration (≥30 days) of immunosuppressive doses of 
steroids, i.e., prednisone >10 mg per day, immunosuppressants, or other immune-modifying 
drugs from each day of vaccination to 2 weeks following each vaccination. 
• Any investigational drug or investigational vaccine other than the study vaccine during the 
study period. 
• Required surgical removal of the spleen or the development of a hematologic or other disease 
that would interfere with normal immunity. 
 
Over-the-counter medications, such as acetaminophen or ibuprofen, may be used to help relieve 
symptoms from vaccination and are not considered prohibited. 
 
Use of antimalarial medications or antibiotics that have antimalarial activity administered during 
the study period is not exclusionary, but will be documented by clinical staff and will be taken 
into consideration during data analysis. 


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7.7 
Vaccine Accountability 
After administration of a vaccine dose, the single-dose vials of antigen and adjuvant will be 
accounted for according to the site standard operating procedures and in agreement with the 
study IND Sponsor for appropriate monitoring.   
Accurate inventory and accountability record of vaccine supplies for this study will be 
maintained by the study site pharmacist (or designee).  Partially used vials may not be 
administered to other subjects. 
8 Study Schedule 
The study schedule and approximate amounts of blood drawn are summarized in Appendix B 
and detailed in Appendix C. 
8.1 
Screening Procedures 
8.1.1 For Initial Enrollment (2016/2017) 
The purpose of the screening visit is to determine subject eligibility for study participation. 
Screening procedures include the informed consent process, Malaria Comprehension Exam, 
laboratory assessments (completed within 56 days of first vaccination), and clinical assessments.  
Screening activities can occur over multiple visits if necessary, including the day of enrollment.  
 
In the event that a chronic illness and/or HIV, HBV, or HCV is discovered during the course of 
screening, long-term treatment and care will not be reimbursed by the study, but referral for 
continuing care can be provided to subjects.   
 
Per national requirements for reporting communicable diseases, confirmed positive test results 
for HIV, HBV, and HCV will be reported to the local health department according to applicable 
laws and appropriate medical referrals initiated. 
 
The following screening evaluations must be completed for all subjects within the 56 days prior 
to first vaccination:  
• Explain the study and informed consent document to the subject. 
• Ensure the subject has acknowledged consent by signing or fingerprinting the informed 
consent document. Ensure that the subject receives a signed copy of the informed 
consent. 
• Ensure the subject has correctly answered ≥80% of the questions (see Section 15.2.1.2) 
on the Malaria Comprehension Exam.  
• Elicit a complete medical history, including menstrual and contraceptive history and/or 
history of surgical sterility for females, and medication use. 


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• Females of childbearing potential must be willing to use reliable contraception from at 
least 21 days prior to first vaccination through three months after the last vaccination. 
• Administer a complete physical examination, including vital signs (height, weight, blood 
pressure, temperature, and heart rate). 
• HIV pre- and post-test counseling as indicated including follow-up contact with subject 
to report the results and referral for appropriate medical care if indicated. 
• Obtain approximately 10 mL of blood for complete blood count (CBC) with differential 
and platelet count, ALT, creatinine (Cr), hepatitis B surface antigen, hepatitis C antibody, 
and HIV antibody. 
• Obtain urine (or serum) for pregnancy testing (for females) and urinalysis/urine dipstick 
for protein and blood. 
• Obtain a 12-lead electrocardiogram (EKG) (for Arms 2c, 2d, and 4c subjects only) 
  
If screening laboratories are completed within ≤ 2 days prior to Study Day 0, these clinical 
laboratory values (CBC with differential, ALT, Cr) may be used for Study Day 0 (day of first 
vaccination) assessments and do not need to be repeated. 
8.1.2 Rescreening for Re-enrollment (2018) 
The purpose of the re-screening visit is to determine subject eligibility for continued study 
participation. Screening procedures include the informed consent process, laboratory 
assessments (for Arm 2c, 2d, 4c only on study day 364) and clinical assessments.  Rescreening 
activities can occur over multiple visits if necessary, including the day of re-enrollment.  
 
For Arms 2c, 2d, 4c, in the event that a chronic illness and/or HIV, HBV, or HCV is discovered 
during the course of rescreening, long-term treatment and care will not be reimbursed by the 
study, but referral for continuing care can be provided to subjects.   
 
Per national requirements for reporting communicable diseases, confirmed positive test results 
for HIV, HBV, and HCV will be reported to the local health department according to applicable 
laws and appropriate medical referrals initiated. 
 
The following screening evaluations must be completed for all subjects upon re-enrollment into 
the study:  
• Explain the study and informed consent document to the subject. 
• Ensure the subject has acknowledged consent by signing or fingerprinting the informed 
consent document. Ensure that the subject receives a signed copy of the informed 
consent. 
• Elicit a complete medical history, including menstrual and contraceptive history and/or 
history of surgical sterility for females, and medication use. 


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• Females of childbearing potential must be willing to use reliable contraception from at 
least 21 days prior to first vaccination through three months after the last vaccination. 
(not required for re-enrollment of the pilot study) 
• Administer a complete physical examination, including vital signs (height, weight, blood 
pressure, temperature, and heart rate).  
• HIV pre- and post-test counseling as indicated including follow-up contact with subject 
to report the results and referral for appropriate medical care if indicated. (not required 
for re-enrollment of the pilot study) 
• Obtain approximately 10 mL of blood for complete blood count (CBC) with differential 
and platelet count, ALT, creatinine (Cr), hepatitis B surface antigen, hepatitis C antibody, 
and HIV antibody. (not required for re-enrollment of the pilot study) 
• Obtain urine (or serum) for pregnancy testing (for females) and urinalysis/urine dipstick 
for protein and blood. (not required for re-enrollment of the pilot study) 
 
Note, for re-enrollment, rescreening procedures will be completed >56 days prior to vaccination 
#4 as subjects enrolled in this study in Arms 2c, 2d, 4c have already been screened for HIV, Hep 
B, Hep C prior as well as had clinical labs monitored on multiple occasions over the course of 
their study participation in the last year. Given the background rate of these diseases in the 
community, the likelihood of a new infection with HIV, Hep B, Hep C is low. In addition repeat 
clinical labs will be completed prior to vaccination #4 and those with clinically significant 
findings will not be vaccinated or will have their vaccination delayed. 
 
8.2 
Assignment to Groups 
For the dose-escalating, open label, randomized, pilot study in Sotuba/Bamako, the study will be 
randomized and then enrolled on a first available basis in the following manner: 
1. Arms 1a and 2a, n=10; then 
2. Arm 3a, n=5; then 
3. Arms 1b, 2b, 4a; n=30; then 
4. Arms 3b, 4b; n=20 
 
For the field site (Bancoumana and Doneguebougou), the study will be enrolled in the following 
manner in a double-blind, en-bloc randomization and stratification by village within each of the 
following groups: 
1. Arms 2c, 2d, 4c, n=240 
 
Enrollment and randomization of subjects into the TBV portion of the double blind, randomized 
study in Bancoumana and Doenguebougou, Mali, as specified above and in Section 14.6, will 


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not occur until at least two vaccinations (Pfs25M and Pfs230D1M) have been administered to the 
corresponding arms in the Bamako/Sotuba, Mali (see Appendix A).  
 
Enrollment occurs with receipt of the first vaccination.  Subjects who are screened but not 
enrolled and/or randomized may be replaced.   
8.3 
Study Subject Schedule 
Day to day study schedule will be completed as outlined in Appendix B and Appendix C. 
9 Study Procedures/Evaluations 
9.1 
Photographs of Rash or Injection Site Reactions 
If a subject develops a rash or injection site reaction, photographs may be taken by the 
investigators. These photographs will not include the subject’s face or any identifying scars, 
marks, or tattoos. 
9.2 
Clinical Laboratory Testing 
Using standard techniques, the clinical laboratory will perform the following tests: 
1. Complete blood count (CBC) plus white blood cell differential and platelet count. 
• The following CBC parameters will be assessed for safety throughout the trial: white 
blood cell count (WBC), absolute neutrophil count (ANC)/absolute granulocyte count 
(AGC), hemoglobin (Hgb), and platelet count (plt). 
2. Serum creatinine (Cr). 
3. Alanine aminotransferase (ALT). 
4. HBsAg test (can include rapid diagnostics, ELISA, PCR if indicated). 
5. HCV test (can include rapid diagnostics, ELISA, PCR if indicated). 
6. HIV test (can include rapid diagnostics, ELISA, Western Blot if indicated). 
7. Urine dipstick/Urinalysis (at screening only). 
8. Urine and/or serum pregnancy testing (β-hCG). 
 
Schistosomiasis testing (urine microscopic examination for S. haematobium and stool PCR for S. 
mansoni) and helminth testing (stool PCR for 8 common gastrointestinal parasite pathogens) will 
be completed in subjects enrolled in Arms 2c, 2d, 4c on Study Day 0. Test results will be 
available to the clinical trial sites, and if subjects did not receive the mass drug administration as 
expected since testing, individuals will be treated.   
 


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Urine microscopy for S. haematobium will be completed on site and results made available to the 
study team and subjects in real time.  Subjects who test positive for S. haematobium will be 
offered treatment with praziquantel upon diagnosis.  Stool results for helminth testing, which are 
being conducted on a research basis, will be provided retrospectively to the study site once 
completed, which may be after the completion of the study, and will be handled on a case by 
case basis for treatment indications of asymptomatic carriage. Positive results from either of 
these tests (stool or urine) will not be exclusionary for enrollment. 
9.3 
Malaria Diagnostics  
9.3.1 Blood Smears 
The gold standard for malaria diagnosis and evaluation of vaccine efficacy endpoints is the 
detection of malaria parasites on Giemsa-stained thick blood films. Giemsa stained thick and thin 
films will be examined for asexual and sexual parasites in the MRTC clinical laboratory, as 
outlined in Appendix B. Blood smears are prepared in duplicate according to standard 
procedures and evaluated by trained study microscopists.  
 
For detection of gametocytemia, each blood smear is read by two independent trained 
microscopists and is reported per 1,000 WBC.  A positive gametocyte read is defined as a single, 
confirmed gametocyte seen by one reader and confirmed by the other microscopist per 1,000 
WBC. 
 
Thick blood smears may be prepared from the blood remaining in the IV cannula, or (at time 
points when no IV blood collection is planned) from a finger prick or venous blood sample at the 
subject’s request.  
 
Thin/thick blood smears will be used for diagnosis throughout the study.  
9.3.1.1 Symptomatic Malaria 
Clinical or symptomatic malaria for this study is defined as the presence of asexual P. falciparum 
parasites at any parasitemia with either an axillary temperature of ≥37.5 °C or more of the 
following symptoms: headache, myalgia, arthralgia, malaise, nausea, dizziness, or abdominal 
pain and will be reported as an AE. 
9.3.2 Rapid Diagnostics 
For unscheduled visits and clinical diagnostics, rapid diagnostic tests may be utilized for 
determination of an acute malarial illness; however, they cannot be used in place of microscopy 
for determination of parasitemia status for DSF.  


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9.3.3 Malaria PCR 
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 
29,30.  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. 31-33 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 P. 
falciparum with a detection limit of at least 500 parasites/mL that will be used during the study 
for comparison to traditional thick blood smears.   
 
P. falciparum qPCR may be performed from all scheduled visits with a malaria blood smear 
noted (see Appendix B) to capture infections that remain below the detection limit for 
microscopy. For subject convenience, a finger prick sample can be used for both preparation of 
the microscopy slide and for DNA preservation, but venous blood draws can also be used. 
9.3.4 Malaria Clinical Management 
In accordance with the Malian Government treatment guidelines, subjects will be treated with 
approved anti-malarial treatment, such as artemether/ lumefantrine (Coartem®, 80mg/480 mg per 
dose) or artesunate/amodiaquine (ASAQ; fixed dose artemisinin based combination therapy) that 
is indicated and approved for the management of uncomplicated P. falciparum malaria.  Clinical 
or symptomatic malaria for this study is defined in Section 9.3.1.1.   
9.4 
Immunologic Laboratory Testing 
9.4.1 Antibody Assay 
Anti-Pfs25, anti-Pfs230 ELISAs, anti-CSP, and anti-HBsAg will be performed on sera or plasma 
obtained from immunized subjects at LMIV in Bethesda, Maryland and may also be performed 
at collaborating laboratories.  
 
For Pfs25 and Pfs230, briefly, microwell plates are coated with antigen solution. Plates are 
washed with TRIS-buffered saline (TBS) containing Tween-20 (T-TBS) and blocked with TBS 
containing skim milk powder. After washing with T-TBS, diluted serum samples are added in 
triplicate and incubated at room temperature for 2 hours. After incubation, unbound antibodies 
are removed by washing the plates with T-TBS, and alkaline phosphatase-conjugated goat anti-
human IgG solution is added to each well and incubated for 2 hours at room temperature. Plates 
are then washed with T-TBS, followed by adding phosphatase substrate solution to each well; 
the plates are then covered and incubated for 20 minutes at room temperature for color 
development. The plates are read immediately at 405 nm with a microplate reader. The optical 


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density values are used to determine antibody levels by comparing to a standard curve generated 
from a known positive-control plasma included on each ELISA plate.  
 
Additionally, the magnitude and kinetics of IgG responses may be explored over time post 
Vaccination #3.  Quality of antibody responses may be explored via antibody avidity as well as 
assessment of antibody subclasses to provide useful insight into the humoral immune response. 
9.4.2 B-Cell and T-Cell Assays 
Specimens collected for B-cell and T-cell studies will undergo initial processing and cell 
separation at the clinical site and will be transported to LMIV according to standard procedures. 
 
B-cell studies will be done at LMIV and associated immunology laboratory partners (MVI 
PATH, Adaptive Biotechnologies, Atreca). The analysis of the generation and maintenance of 
antigen-specific memory B cells will be carried out to determine if these cells can be elicited and 
maintained by vaccination. Peripheral blood lymphocytes will be obtained and assayed for the 
presence of antigen-specific memory B cells, total number of memory B cells, and plasmablast 
responses using flow cytometry and ELISPOT assays.   
 
T-cell studies will be performed at LMIV. Antigen-specific T-cell responses to vaccination will 
be determined by ELISPOT and/or intracellular cytokine staining flow cytometry.   
 
Ex vivo studies will be performed at MRTC using whole blood to enumerate various immune 
subsets (T, B, NK cells) prior to and after each vaccination.  
9.4.3 Transcriptional Profiling 
Whole genome transcriptional profiling will be performed to explore possible gene expression 
profiles or pathways that predict optimal responses to vaccination and to determine if innate 
immune responses are sensitively reflected in the PBMC transcriptome shortly after vaccination. 
Gene expression profiling following vaccination will allow the predictive capacity of eventual 
high and low responders, and thus will assist in defining the correlates of protection induced by 
vaccination. 
 
Transcriptional analyses will be performed on whole blood collected as outlined in Appendix B.    
Blood will be collected via venous puncture and placed in PAXGene tubes to preserve RNA 
integrity until the RNA is extracted. Specimens will be analyzed at the Research Technologies 
Branch, NIAID and/or the NIH Intramural Sequencing Center. 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.   


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9.4.4 Transmission-Blocking Assays/Evaluations 
The transmission-blocking assays which will be conducted are summarized in Table 13.   
Table 13: Transmission Blocking Assays 
Assay 
Mosquitoes 
Test Samples 
Site 
Standard 
Membrane-
Feeding Assay 
(SMFA) 
Lab strain  
(A. stephensi) 
Membrane feeds with lab cultured parasites 
mixed with test serum/plasma/IgG  
LMIV 
LMVR  
Direct Skin Feed 
(DSF) 
MRTC lab colonies  
(A. gambiae) 
Direct skin feeds on vaccinees 
MRTC 
 
Feeding assays demonstrate biologic activity of transmission-blocking antibodies, and are critical 
to selection of transmission-blocking vaccine candidates. Subjects will be screened periodically 
by blood smear (as seen in Appendix B) for the presence of asexual parasites and gametocytes. 
Priority for feeding assays (SMFA and DSF) will be conducted post Vaccination #3 when 
gametocyte carriage and parasite carriage rates are the highest and antibodies secondary to 
Pfs230D1M are expected to be peaking post vaccination.   
 
Multiple feeds might be conducted on a single subject at the multiple screening time points, but a 
single subject will not undergo more than 32 DSFs (inclusive of the twice a week feeds during 
the intense DSF period following Vaccination #3 and #4) following a vaccination. 
9.4.4.1 Standard Membrane-Feeding Assay  
Membrane-feeding assays demonstrate biologic activity of transmission-blocking antibody and 
are critical to selection of vaccine candidates. SMFAs will be performed on blood obtained at 
baseline and periodically after vaccination as outlined in Appendix B.   In a SMFA, test serum 
or plasma obtained from immunized subjects is mixed with parasites from a laboratory culture 
and the mixture is placed in a feeding cup covered with an artificial membrane. Pre-starved 
mosquitoes from a laboratory colony are allowed to feed through the membrane. A similar 
procedure is carried out on a malaria-naïve control serum at the same time using mosquitoes 
raised from the same laboratory colony. One week after the feed, mosquitoes are dissected and 
midguts are stained with mercurochrome for the oocyst form of the parasite. The reduction of the 
proportion of oocyst-laden mosquitoes or the reduction of average oocyst numbers per mosquito 
compared to mosquitoes fed on the control group demonstrate biologic function of the antibody, 
and may be predictive of efficacy in the field. SMFA results have been shown to correlate with 
ELISA antibody titers against Pfs25 in several species.24 The SMFAs will be conducted at LMIV 
and LMVR in Rockville, Maryland. 
 
At the time of the direct-feeding assays, venous blood may be collected from each subject as 
outlined in Appendix B. The blood aliquot will be processed immediately for SMFA.  The 


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process will be maintained at approximately 37°C to avoid temperature-induced gametogenesis. 
SMFA will be conducted at LMIV using laboratory-strain mosquitoes and parasites. Assays will 
compare feedings with: 
• Plasma/sera; 
• IgGs purified from the selected plasma/sera, mixed with a malaria-naïve human 
sera pool (to eliminate non-specific factors which may be present in plasma).  
 
To confirm anti-Pfs25-specific and anti-Pfs230-specific transmission-blocking activities, SMFAs 
may also be conducted  
• using Pfs25-specific and Pfs230-specific IgG purified using affinity 
chromatography;  
• using test plasma/sera that has been depleted of Pfs25-specific and Pfs230-
specific antibodies using recombinant Pfs25 and Pfs230 proteins.    
9.4.4.2 Direct Skin Feeds  
While the SMFA is standardized and reproducible and has been shown to correlate with ELISA 
antibody titers in several species, the parasite strain, gametocyte load in the feeder, and lab 
colony mosquitoes are all significantly different from those found under field conditions.24 DSFs, 
in which insectary-raised clean mosquitoes are directly fed on infected individuals, we believe 
may be more likely to be predictive of an intervention’s impact on transmission than membrane 
feeds. Thousands of subjects in Bancoumana or neighboring areas have previously been enrolled 
in direct-feeding tests without significant adverse effects reported. In 1994 and 1995, Toure and 
colleagues conducted direct feeds on 72 children aged 2 years or more without any risks 
identified.34 From 1996 to 1998, Diallo and colleagues conducted DSFs on 372 children aged 4 
to 18 years without any safety problems.26  In 2002 and 2003, DSFs were carried out in 
Bancoumana and in a neighboring village on a total of 44 gametocyte carriers aged 6 to 18 years 
old,35 also without any reported safety problems. 
 
More recently, under four previously conducted clinical trials (NIAID Protocol #13-I-N109, #11-
I-N143, #14-I-N159, #15-I-0044) the study team has performed over 2000 DSFs on subjects older 
than 5 years since June 2011. All DSF participants were actively followed 24 hours post feed and 
as many as 12 DSFs over a 2 week period have been conducted.  Except for 1 case of definitely 
related Grade 2 erythema that resolved within 48 hours and 2 episodes of definitely related Grade 
1 pruritus, there have been no other expected or unexpected AEs recorded as definitely, probably, 
or possibly related to the feeding procedures in either study.  Additionally, under this protocol 
(#17-I-N006), 3518 DSFs have been completed with only four pruritus AEs (4/3518, 0.1%) in 
three subjects have been reported related to the DSFs. All have been mild (Grade 1), all starting 
on the day of DSF, and all resolved within 2 days. None of the DSF related AEs have met criteria 
for withdrawal of the subject from participating in future DSFs.  


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Additionally, under protocol #11-I-N143, multiple DSFs during a 24 hour period have been 
completed in 18 subjects to date.  In these 18 subjects, there have been no reported AEs 
secondary to having multiple direct skin feeds completed in a short period of time and have been 
tolerated well by the involved subjects. 
 
For DSFs, 2 feeding pints with approximately 30 pre-starved female mosquitoes in each will be 
prepared. Each subject will be exposed to the feeding pints for 15-20 minutes. All subjects will 
be offered a topical antihistamine and/or topical antipruritic to use following the feeds. Systemic 
medications may also be prescribed for treatment of local or systemic symptoms following DSFs 
if clinically indicated.  
 
During the time periods outside of the intense twice a week DSF for approximately 12 weeks 
following Vaccinations #3 attempts to identify and optimize parameters that contribute to DSF 
variability may be implemented during the conduct of the feeding assays. Subjects will be 
provided the details of the variation on the DSFs prior to participation and are not required to 
deviate from the standard DSF (2 feeding pints; approximately 30 pre-starved female mosquitoes 
per pint; application to the bilateral calves or arms; exposure for 15-20 minutes; feed conducted 
at subject’s convenience, usually dusk or dawn).   
 
These variables include the following:  
• Body location (for example, ankle, leg, forearm),  
• Number of cups applied (for example, 2 cups or 3 cups),  
• Time of day feeds conducted (for example, dawn, dusk, late night),  
• Number of feeds per day (up to 2 feeds in a single calendar day),   
• Duration of feeds (for example, decreasing time to 10 minutes). 
 
The total number of mosquitoes used for each DSF will be maintained at appropriately 60 
mosquitoes total regardless of these variables, and no subject will undergo more than 32 DSFs 
within any 6 month period post vaccination.  Note, if subjects are not willing to undergo twice a 
week feeds at the time of the DSF, they will not be withdrawn but will be followed per protocol 
but with periodic DSF or without DSF being completed.  
 
Previously, DSFs started either 1 or 2 weeks post vaccination in order to time the DSFs with 
expected peak antibody responses. Although antibody responses have significantly improved, in 
peak and more importantly durability, the question of functional activity no longer is focused on 
peak time periods, but rather duration of activity, we will still plan to start DSFs 1 week after 
vaccination #4 in order to assess activity early in the transmission season. DSFs will  continue 


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twice a week for 16 weeks in order to increase timepoints available for analysis to evaluate 
vaccine efficacy.  
 
Some experiments have already been completed under protocols #13-I-N109 and #11-I-N143 to 
try to help determine which time of day and body part will maximize positive DSFs. Although 
there was not a significant difference found between location and time of feed in this small 
experiment, we did find that feeding at dawn and on the arm were estimated to have the highest 
counts. The data collected under this protocol will provide additional information about how to 
optimize/maximize the DSF procedure.  
 
After the feed, surviving mosquitoes will be assessed for infectiousness by microscopy or 
molecular assays.  
9.4.4.2.1 Withdrawal from DSFs 
The following criteria will be checked prior to each DSF and are contraindications to DSFs:   
• Severe local or systemic reaction to mosquito bites following a previous DSF. 
• Positive urine or serum β-hCG test (pregnancy testing results obtained within the last 7-10 
days before the DSF are acceptable; see Appendix B). 
• Acute illness with an oral temperature >37.5˚C at the time of the DSF. 
• Any other condition that in the opinion of the Investigator poses a threat to the individual if 
he/she undergoes DSF or that may complicate interpretation of the safety of DSF. 
 
Recent use of antimalarial medications is not a contraindication to participating in the DSFs. The 
medication used and time period the medication was taken should be recorded on the subject’s 
source documents and case report forms accordingly. 
9.5 
Entomological Procedures and Insectary Used in DSF  
9.5.1 Mosquito Collection 
Mosquitoes may be collected from residences or compounds or communities of study subjects on 
a periodic basis to assess mosquito infection rates and their relation to the transmission-blocking 
activity measured in blood samples as described above from consented subjects. 
9.5.2 Mosquito Rearing at the Insectary 
A laboratory colony of A gambiae established from a local catch in 2008 will be used for the 
DSFs. This colony has been used in the current assay development study, and was demonstrated 
to have similar susceptibility as the F1 progeny of the wild-caught mosquitoes. The colony has 
been maintained using blood meals collected under standard procedures of the blood transfusion 
center from local healthy donors. To ensure that the donor is free of potentially transovarial 
arbovirus in incubation, the donated blood is only used 5-7 days after the collection and after the 


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donor is confirmed to remain healthy during this period. Potentially transovarial arbovirus in 
mosquitoes or in blood meals may be detected by ELISA and PCR methods. However, it is not 
feasible to conduct these tests routinely on blood donor volunteers due to highly restricted access 
to positive controls required in these tests, but blood samples from donors and mosquitoes from 
the colony are tested for RVF on a regular basis in the US.   
 
The insectary in which mosquitoes are reared in Bamako has been in use for more than 15 years. 
Security is ensured by the use of double doors, which prevents the escape of reared mosquitoes 
as well as the entry of non-insectary mosquitoes. Mosquitoes are transported to Bancoumana or 
Doneguebougou in net-sealed feeding cups secured in wooden holders inside a cooler, with wet 
towels to maintain adequate humidity. After arrival at the assigned insectary in Bancoumana or 
Doneguebougou, mosquitoes are secured within the transporting containment. The insectary is 
adequately equipped with rooms with humidifiers which are regularly monitored according to 
standard insectary procedures, and access to the insectary is limited to study personnel only. 
After feeds, mosquitoes, still in net-sealed cups, are transported back to the insectary in Bamako. 
All subsequent handling of mosquitoes will take place in Bamako. 
9.5.3 Mosquito Dissection 
Mosquitoes are knocked down and then transferred into a Petri dish containing a slightly wet 
paper towel. Under a dissecting scope and on a slide, each individual specimen is placed in a 
drop mercurochrome solution. The midgut is pulled and covered with a coverslip. The oocysts 
are detected under a light microscope. The presence of oocyst and oocyst counts for each 
specimen will be recorded. A mosquito will be considered positive if ≥1 oocyst is present. 
 
If needed, the head and thorax of the mosquitoes may be processed for ELISA to detect 
sporozoites and for PCR to identify species and molecular form of the mosquito. 
 
Additional details on mosquito rearing, feeds, and dissection will be described in study SOPs. 
9.6 
Schistosomiasis and Helminthes Infection Testing  
Endemic helminthes infections in sub-Saharan Africa, such as helminth and schistosomiasis 
infections, are known to elicit a wide range of immunomodulating responses and may impact the 
efficacy of vaccinations.36-38 This potential impact of co-infections on vaccine immunogenicity 
will depend on the prevalence of local helminth and schistosomiasis infections. With the 
introduction of phase 1 malaria trials into Africa, consideration and initial exploration into the 
existence of co-infections in vaccinees and the impact on vaccine efficacy and immunogenicity 
needs to be explored. 
 
To assess the impact of co-infections on immune responses, schistosomiasis testing (may include 
urine microscopic examination for S. haematobium and stool PCR for S. mansoni; urine antigen 
detection) and helminthes testing39 (stool PCR for 8 common gastrointestinal pathogens) will be 


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completed on Study Day 0 in Arms 2c, 2d, 4c only.   Given these tests are run for research 
pruposes and retrospectively, if these results are available prior to the final study visit and 
parasites are detected, and recommended, subjects will be treated with standard treatment and 
continued in the study.  
9.7 
Other Laboratory Assays 
If there is adequate remaining blood sample available to fulfill the laboratory objectives, other 
laboratory assays may be performed as follows: 
• Malaria rapid diagnostic tests (RDTs) may be used for clinical diagnosis purposes. 
• qPCR may be used to detect gametocytes using whole blood collected on the day of DSF 
and/or positive blood smears.  
• Filter paper filled with whole blood, whole blood, or mosquitoes may be used to 
determine parasite genotype. 
• Antibodies against sporozoite, pre-erythrocytic, blood, and sexual stages may be 
determined by ELISA.  
• Mosquito species and molecular forms may be identified by qPCR..40   
• Hemoglobin variants (Hgb C, S, alpha thalassemia), glucose-6-phosphate dehydrogenase 
deficiency, and ABO blood group/Rhesus factor may be assessed using high-performance 
liquid chromatography and/or other standard methodology. 
• Human leukocyte antigen genotypes may be evaluated for genetic polymorphisms, allelic 
and antigenic diversity. 
• B cell receptor/antibody genes may be sequenced to explore the antibody repertoire of 
functional antibody responses. 
• Cytokine levels may be evaluated during vaccination and following vaccination. 
• Direct membrane feeding assays may be completed following Vaccination #3 to compare 
to SMFA and DSF results. 
 
Study physicians may ask for additional laboratory exams related to subject care. 
10 Research Use of Stored Human Samples, Specimens, or Data 
Intended Use: Samples and data collected under this protocol will be used to study malaria and 
related diseases and possible adverse reactions to vaccination. Genetic testing may be performed 
in accordance with the genetic testing information that is included in the study informed consent. 
 
Storage: Access to stored research samples will be limited using either a locked room or a 
locked freezer. Temporary storage of samples collected in Mali, prior to shipment to LMIV, may 
occur at the Core Immunology Laboratory or the MRTC CAP laboratory.  Samples will be stored 
at the LMIV in Rockville, MD or at LMIV’s designated repository, Thermo Scientific, 


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Rockville, MD, with the exception of retention specimens which may be kept at the MRTC in 
Mali for quality control. Samples and data will be stored using codes assigned by the 
investigators or their designees. Data will be kept in password-protected computers. Only 
investigators or their designees will have access to the samples and data.  
 
Tracking: Samples will be tracked using a sample-tracking software program, e.g., 
Freezerworks.  
 
Disposition at the Completion of the Protocol: In the future, other investigators (both at the 
NIH and outside) may wish to study these samples and/or data. In that case, IRB approval must 
be sought prior to any sharing of samples. Any clinical information shared about the sample with 
or without patient identifiers would similarly require prior IRB approval.   
 
At the completion of the protocol (termination), samples and data will either be destroyed or, 
after IRB and FMPOS EC approval, transferred to another existing protocol.   
 
Reporting the Loss or Destruction of Samples/Specimens/Data to the IRB: Any loss or 
unanticipated destruction of samples (for example, due to freezer malfunction) or data (for 
example, misplacing a printout of data with identifiers) that meets the definition of Protocol 
Deviation and/or compromises the scientific integrity of the data collected for the study, will be 
reported to the NIH IRB and FMPOS EC. 
 
Consent to allow long term storage of study samples is a part of the inclusion criteria for this 
study.  However, if a subject decides following enrollment not to have their samples stored the 
Principal Investigator or designee will destroy all known remaining samples and report this 
destruction to the subject and the NIH IRB and FMPOS EC. This decision will not affect the 
subject’s continued participation in this protocol or any other protocols supported by the NIH. 
11 Data Sharing Plan 
In NIH's and MRTC’s view, all data should be considered for data sharing. Data should be made 
as widely and freely available as possible while safeguarding the privacy of subjects, and 
protecting confidential and proprietary data. We recognize that the public dissemination of our 
scientific results can facilitate the creation of collaborative efforts with domestic and 
international collaborators. Furthermore, we recognize that the proposed project may result in 
novel ideas for new methods, technologies, and data that could benefit the entire research 
community. Therefore, final research data will be shared openly and timely in accordance with 
the most recent NIH guidelines (http://grants.nih.gov/grants/policy/data_sharing/) while being 
mindful that the confidentiality and privacy of participants in research must be protected at all 
times. Timelines for distribution of data will vary depending on any required restrictions in 
accordance with federal and/or institutional policies and guidelines. In general we expect de-
identified data will be available through NIH-funded or approved public repository, speaking 


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engagements and publications, presentations at scientific symposia and seminars. Effort will be 
made to publish our research findings in scientific journals. All final peer-reviewed manuscripts 
that arise from this proposal will be submitted to the digital archive PubMed Central. For tools, 
reagents, data and model organisms generated by the proposed study, pending third parties 
rights, LMIV/MRTC will transfer materials to outside researchers in both the private and public 
sectors under a Material Transfer Agreement or Research Collaboration Agreement.  Discussions 
of unpublished ELISA and SMFA data from this trial with collaborators will be conducted under 
a Confidential Disclosure Agreement. 
12 Assessment of Safety 
12.1 Documenting, Recording, and Reporting Adverse Events 
At each contact with the subject, information regarding adverse events will be elicited by 
appropriate questioning and examinations and will be: 
• immediately documented in the subject’s medical record/source document, 
• recorded on the Adverse Event Case Report Form (AE CRF) or electronic database, and 
• reported as outlined below (e.g., IND Sponsor, IRB, FDA). 
 
A study clinician will be available during the study period and will be available to the study 
subjects at all times. Should a subject call a study clinician to report an AE, it will be discussed 
with the PI and documented, recorded, and reported appropriately. 
12.2 Definitions 
Adverse Event (AE) 
An adverse event is any untoward or unfavorable medical occurrence in a human subject, 
including any abnormal sign (e.g., abnormal physical exam or laboratory finding), symptom, or 
disease, temporally associated with the subject’s participation in the research, whether or not 
considered related to the research. 
 
Adverse Reaction (AR) 
An adverse event that is caused by an investigational agent (drug or biologic). 
 
Suspected Adverse Reaction (SAR) 
An adverse event for which there is a reasonable possibility that the investigational agent caused 
the adverse event. ‘Reasonable possibility’ means that there is evidence to suggest a causal 
relationship between the drug and the adverse event. A suspected adverse reaction implies a 
lesser degree of certainty about causality than adverse reaction which implies a high degree of 
certainty. 
 
Serious Adverse Event (SAE) 


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A Serious Adverse Event (SAE) is defined as an AE that results in any of the following 
outcomes: 
• death 
• a life threatening (i.e., an immediate threat to life) event 
• an inpatient hospitalization or prolongation of an existing hospitalization 
• a persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions 
• a congenital anomaly/birth defect 
• a medically important event* 
 
* Medical and scientific judgment will be exercised in deciding whether expedited reporting is 
appropriate in other situations, such as important medical events that may not be immediately 
life threatening or result in death or result in hospitalization but may jeopardize the subject or 
may require intervention to prevent one of the other outcomes listed above.  
 
Unexpected Adverse Event 
An AE is considered unexpected if it is not listed in the Investigator’s Brochure or Package 
Insert (for marketed products) or is not listed at the specificity or severity that has been observed.  
It is the responsibility of the IND Sponsor to make this determination. 
 
Serious and Unexpected Suspected Adverse Reaction (SUSAR) 
A SUSAR is a Suspected Adverse Reaction that is both serious and unexpected. 
 
Unanticipated Problem (UP) 
An Unanticipated Problem (UP) is any event, incident, experience, or outcome that is 
1. unexpected in terms of nature, severity, or frequency in relation to 
a. the research risks that are described in the IRB-approved research protocol 
and informed consent document, Investigator’s Brochure, or other study 
documents; and 
b. the characteristics of the subject population being studied; and 
2. possibly, probably, or definitely related to participation in the research; and 
3. places subjects or others at a greater risk of harm (including physical, psychological, 
economic, or social harm) than was previously known or recognized. (Per the IND 
Sponsor, an AE with a serious outcome will be considered increased risk.) 
 
Serious Unanticipated Problem (UP) 
A UP that meets the definition of a Serious Adverse Event or compromises the safety, welfare or 
rights of subjects or others. 


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Unanticipated Problem that is not an Adverse Event (UPnonAE) 
An unanticipated problem that does not fit the definition of an adverse event, but which may, in 
the opinion of the investigator, involve risk to the subject, affect others in the research study, or 
significantly impact the integrity of research data.  Such events would be considered a non-
serious UP. For example, we will report occurrences of breaches of confidentiality, accidental 
destruction of study records, or unaccounted-for study drug. 
 
New Onset of Chronic Illness (NOCI) 
The new onset of chronic illness is defined as a diagnosis of a new medical condition that is 
chronic in nature, including those potentially controllable by medication (e.g., diabetes, asthma). 
Any NOCI will be recorded in the same manner as unsolicited AEs. 
 
12.3 Investigator Assessment of Adverse Events 
If a diagnosis is clinically evident (or subsequently determined), the diagnosis rather than the 
individual signs and symptoms or lab abnormalities will be recorded as the AE. 
 
All solicited (see Table 14 below) and unsolicited AEs will be recorded through Day 7 after each 
vaccination, including injection site reactions, or until resolved. Adverse reactions related to 
direct skin feeds will be recorded until Day 7 after feeds or until resolved. After that period only 
unsolicited AEs (including symptomatic malaria), SAEs, UPs, and NOCIs will be recorded.  
Note that a positive blood smear without associated clinical symptoms will not be reported as an 
AE. 
 
Table 14: Solicited Adverse Events 
Systemic adverse events 
Fever (temperature ≥38.0 °C) 
Headache  
Nausea/Vomiting  
Diarrhea 
Abdominal pain 
Fatigue 
Myalgia  
Arthralgia  
Urticaria  
Laboratory adverse events 
Hemoglobin – decreased hemoglobin 
WBC – leukopenia, leukocytosis 
ANC/AGC – decreased neutrophil/granulocyte count 


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Platelet count – thrombocytopenia  
ALT – increased ALT  
Creatinine (Cr) – increased Cr 
Local reactogenicity 
Injection pain/tenderness 
Injection erythema/redness 
Injection swelling 
Injection induration 
Injection pruritus 
Limitation of arm movement 
 
Additional laboratory abnormalities other than those specified as safety labs in the protocol 
should be reported as adverse events if they require intervention. Interventions include, but are 
not limited to, discontinuation of treatment, dose reduction/delay, additional assessments, or 
concomitant treatment. In addition, any medically important laboratory abnormality may be 
reported as an adverse event at the discretion of the investigator. This could include a laboratory 
result for which there is no intervention but the abnormal value suggests a disease or organ 
toxicity. In addition, similar to solicited AEs, all laboratory AEs as defined in Appendix D, will 
be collected and graded through 7 days after each vaccination or until resolved. 
 
The investigator will evaluate all AEs with respect to Seriousness (criteria listed above), Severity 
(intensity or grade), and Causality (relationship to study agent and relationship to research) 
according to the following guidelines. 
12.3.1 Severity 
Severity of AEs will be assessed by the investigator as described in Appendix D.   AEs not 
included in the Appendixes will be graded for severity using the followings definitions as seen in 
Table 15. 
 
Table 15: Definitions for Severity of AE Grading 
Severity 
Definition 
Grade 1 (Mild) 
No interference with activity, may use 1 dose 
of an over the counter medication 
Grade 2 (Moderate) 
Repeated use of non-narcotic pain reliever > 
24 hours or some interference with activity 
Grade 3 (Severe) 
Activities of daily living limited to <50% of 
baseline, medical evaluation/therapy required 
Grade 4 (Potentially Life-Threatening) 
Extreme limitation in activity, significant 
assistance required; immediate medical 


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intervention or therapy required to prevent 
death 
Grade 5 
Death 
 
12.3.2 Causality 
Causality (likelihood that the event is related to the study agent) will be assessed considering the 
factors listed under the following categories: 
 
Definitely Related 
• reasonable temporal relationship 
• follows a known response pattern 
• clear evidence to suggest a causal relationship 
• there is no alternative etiology 
 
Probably Related 
• reasonable temporal relationship 
• follows a suspected response pattern (based on similar agents) 
• no evidence of a more likely alternative etiology 
 
Possibly Related 
• reasonable temporal relationship 
• little evidence for a more likely alternative etiology 
 
Unlikely Related 
• does not have a reasonable temporal relationship  
 
OR 
• good evidence for a more likely alternative etiology 
 
Not Related 
• does not have a temporal relationship 
 
OR 
• definitely due to an alternative etiology 
 
Note: Other factors will also be considered for each causality category when appropriate. 
Causality assessment is based on available information at the time of the assessment of the AE. 


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The investigator may revise the causality assessment as additional information becomes 
available. 
 
The degree of certainty with which an AE can be attributed to administration of the study 
vaccine will be determined by how well the event can be understood in terms of one or more of 
the following: 
 
• The event being temporally related with vaccination or reproduced on re-vaccination. 
• A reaction of similar nature having previously been observed with this type of vaccine and/or 
formulation. 
• The event having been reported in the literature for similar types of vaccines. 
• Whether or not there is another identifiable cause. 
 
All local (injection site) reactions will be considered causally related to vaccination.  All malaria 
cases will be reported as not related to vaccination.  Asymptomatic parasitemia (positive blood 
smears without related malaria clinical symptoms) will not be reported as an AE.  Clinical 
malaria will be reported as an AE. 
 
Reports will further classify AEs as follows:   
• Related - all AEs that are assessed as definitely, probably, or possibly related. 
• Unrelated - all AEs assessed as unlikely or definitely not related. 
 
When reporting to regulatory authorities and IRBs is needed, AE relationship will be determined  
as noted above. 
12.4 Investigator Reporting Responsibilities to the Sponsor 
12.4.1 Adverse Events 
Line listings, frequency tables and other summary AE data will be submitted to the IND Sponsor 
when needed for periodic safety reviews, review of IND annual reports, review of IND safety  
reports, and preparation of final study reports. 
12.4.2 Serious Adverse Events 
All SAEs (regardless of relationship and whether or not they are also UPs) will be reported on 
the Safety Expedited Report Form (SERF) and sent to the Sponsor Clinical Safety Office (CSO) 
by fax or email attachment. Deaths and immediately life-threatening SAEs will be reported 
within 1 business day after the clinical site becomes aware of the event. All other SAEs will be 
reported to the CSO within 3 business days after the site becomes aware of the event. 
 


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Sponsor Clinical Safety Office Contact Information: 
 
 
OCRPRO Clinical Safety Office 
 
 
5705 Industry Lane 
 
 
Frederick, MD 21704 
 
 
Tel: (301) 846-5301 
 
 
Fax: (301) 846-6224 
 
 
Email: rchspsafety@mail.nih.gov 
 
The clinical site Investigator in Mali will also notify LMIV PI and the Site Medical Monitor in 
Mali by email, fax, or telephone within 1 working day of notification of an SAE occurrence. 
 
LMIV Contact Information: 
Patrick Duffy, MD 
Tel: (301) 761-5089 
Fax: (301) 480-1962 
Patrick.Duffy@nih.gov 
12.4.3 Unanticipated Problems 
All UPs that are also adverse events will be reported to the CSO on the NIH Problem Report 
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 not be reported to the Sponsor CSO. 
12.4.4 Pregnancy 
Pregnancy itself is not an AE. However, complications of pregnancies are AEs and may be 
SAEs. Pertinent obstetrical information for all pregnancies will be reported to the CSO and 
LMIV via fax or email within 3 business days from the site awareness of the pregnancy.  All 
pregnancies will be reported on the Pregnancy Notification/Outcome Form to the CSO. 
 
Pregnancy outcome data (e.g., delivery outcome, spontaneous, or elective termination of the 
pregnancy) will be reported to the CSO and LMIV within 3 business days of the site’s awareness 
of the outcome on a protocol-specified form.  
 
In the event of pregnancy, the following steps will be taken: 
• Discontinuation of the study agent 
• Unblind per the site unblinding procedures 
• Withdraw from the study but continue in follow-up for safety 
• Report to FMPOS Ethics Committee as an informational item 


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• Report to NIH IRB at time of Continuing Review 
• Report to DSMB, Sponsor Medical Monitor, and Site Medical Monitor 
• Advise research subject to notify the obstetrician of study vaccine exposure 
12.5 Reporting Procedures to the NIH IRB 
12.5.1 Expedited Reporting to the NIH IRB 
.  
. 
Non-compliance: Any actual or suspected non-compliance by any investigator or entity 
associated with the protocol must be reported by the NIH PI/designee within 7 calendar days of 
any investigator or individual associated with the protocol first becoming aware, unless 
otherwise indicated in this policy.  
Non-NIH IRB determinations of serious and/or continuing noncompliance about an NIH 
investigator: If the NIH is relying on a non-NIH IRB and the Reviewing IRB makes a 
determination of serious and/or continuing non-compliance regarding an NIH 
investigator, then, even if the determination has already been provided to OHSRP either 
directly or via the NIH Institutional Official (IO)/designee, the NIH PI/designee must 
report this in iRIS within 7 calendar days of any member of the research team being 
notified of the determination by the Reviewing IRB. The NIH PI must provide the 
OHSRP office of Compliance and Training with documentation from the Reviewing IRB 
unless this documentation has already been provided directly to the office by the 
Reviewing IRB or via the IO.  
Major Deviation: A deviation must be reported within 7 calendar days of an investigator 
becoming aware of an actual or suspected deviation. Although protocol deviations are also non-
compliance, these should only be reported once as deviations.  
Unanticipated Problem (UP): A UP must be reported within 7 calendar days of an investigator 
becoming aware of the actual or suspected UP.  
Death: Any death of a research subject that is possibly, probably or definitely related to the 
research must be reported within 24 hours of an investigator becoming aware of the death.  
New information that might affect the willingness of a subject to enroll or remain in the study 
should be reported within 7 calendar days of an investigator first becoming aware.  
Any suspension or termination of research activities, including holds on new enrollment, placed 
upon the research by the study sponsor, NIH or IC leadership, or any regulatory agency must be 
reported within 7 calendar days of an investigator becoming aware.  
Investigators must provide the following information to the IRB in summary format at the time 
of continuing review: Minor protocol deviations; Adverse events and Serious Adverse Events 
that do not meet the definition of an UP.  


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Investigators are required to immediately (i.e., no longer than 10 days) report SAEs or UADEs to 
the study sponsor and, if also an actual or suspected UP, to the IRB within 7 calendar days of an 
investigator becoming aware. 
 
12.5.2 Annual Reporting to the NIH IRB  
All events will be reported to the NIH IRB and FMPOS EC in summary at the time of 
Continuing Review according to NIH Policy 801. 
12.6 Investigator Reporting Responsibilities to the Local IRB 
Investigators are responsible for submitting IND Safety Reports and UP summaries that are 
received from the IND Sponsor to their local IRB/Ethics Committee (FMPOS EC). Investigators 
must also comply with all local IRB/Ethics Committee (FMPOS EC) reporting requirements, 
including expedited and annual reporting requirements. 
12.7 Follow-up of Adverse Events and Serious Adverse Events 
AEs that occur following receipt of a single vaccination are followed until the final outcome is 
known or until the end of the study follow-up period.  
 
SAEs that have not resolved by the end of the follow-up period will be followed until final 
outcome is known. If it is not possible to obtain a final outcome for an SAE (e.g., the subject is 
lost to follow-up), the reason a final outcome could not be obtained will be recorded by the 
investigator on the AE CRF (if the CRF is still open) and the SERF.  
 
SAEs that occur after the study follow-up period (6 months following the last vaccination) that 
are reported to and assessed by the Investigator to be possibly, probably, or definitely related 
must be reported to the CSO as described above. 
12.8 Sponsor’s Reporting Responsibilities 
Serious and unexpected suspected adverse reactions (SUSARs) as defined in 21 CFR 312.32 and 
determined by the IND Sponsor will be reported to FDA and all participating Investigators as 
IND Safety Reports.  
 
The IND Sponsor will also submit an IND Annual Report of the progress of the investigation to 
the FDA as defined in 21 CFR 312.33.  
 
AEs that are also UPs will be summarized by the IND Sponsor and distributed to Investigators. 


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12.9 Halting Criteria for the Study 
The PI will closely monitor study data as they become available and will make determinations 
regarding the presence and grading of AEs. The AEs will be evaluated with regard to the known 
complications associated with administration of vaccine components. If a dose of vaccine is 
considered unacceptably reactogenic (as described in the following criteria), the study will be 
halted. No new enrollments and no further vaccinations will be administered by the Investigators 
until reviewed by the DSMB and study IND Sponsor. A report of DSMB recommendations will 
be submitted to the IRBs. The following criteria will be used to define unacceptable 
reactogenicity of the malaria vaccine (AEs that are possibly, probably, or definitely related to the 
vaccine will be considered “related” and will be summarized as such): 
1. One or more subjects experience death or any life-threatening SAE, or  
2. One or more subjects experience an SAE as defined in Section 12.2 of this protocol 
that is determined to be possibly, probably, or definitely related to the vaccine, or 
3. One or more subjects are withdrawn from the study (by investigator or subject 
request) following a Grade 3 AE that is determined to be possibly, probably, or 
definitely related to the vaccine, or 
4. One or more subjects experience a hypersensitivity reaction (e.g. anaphylaxis, diffuse 
urticaria) that is determined to be possibly, probably, or definitely related to the 
vaccine, or 
5. One or more subjects experience any local or solicited AE leading to hospitalization, 
or fever >40°C (104°F) that is possibly, probably, or definitely related to the vaccine, 
or necrosiss at the injection site, within the 7 days post vaccination, or 
6. Ten percent or more of subjects in any dose Arm experience any Grade 3 solicited 
local AE (lasting 48 hours or more) that is determined to be possibly, probably, or 
definitely related to the vaccine as defined in this protocol, within the 7 days post 
vaccination, or  
7. Ten percent or more of subjects in any dose Arm experience any Grade 3 solicited 
systemic AE (lasting 48 hours or more) that is determined to be possibly, probably, or 
definitely related to the vaccine as defined in this protocol, within the 7 days post 
vaccination, or  
8. Thirty percent or more of subjects in any dose Arm experience the same Grade 2 or 
higher laboratory abnormality (see Appendix D) or ten percent or more experience 
the same Grade 3 or higher laboratory abnormality regardless of vaccine relationship, 
within the 7 days post vaccination, or  
9. Ten percent or more of subjects in any dose Arm experience the same or similar 
Grade 3 unsolicited AE that is determined to be possibly, probably, or definitely 
related to the vaccine as defined in this protocol, or 
10. Any safety issue that the study PI or IND Sponsor determines should halt the study. 
 


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If any of these halting criteria are met, request for an unblinded review by the DSMB may be 
submitted to determine if the safety issues identified were in the comparator or the test vaccines 
arm of the study. 
 
The IRBs, the NIAID, the FDA, or other government agencies may discontinue the study at any 
time. Subsequent review of serious, unexpected, and related AEs by the DSMB or IRB, the IND 
Sponsor, the FDA, and other regulatory authorities may also result in suspension of further 
administration of vaccine at the clinical site. The FDA, other regulatory authorities, and the study 
sponsor(s) retain the authority to suspend additional enrollment and administration of vaccine for 
the entire study as applicable.   
12.9.1 Reporting of Study Halting 
If a halting requirement is met, a description of the event(s) or safety issue will be reported by 
the PI or Site Investigator within 1 business day to the Sponsor CSO by fax or email and reported 
to the FDA within 3 business days. 
 
The Site Investigator will inform the PI and the local IRB that a halting rule has been met 
according to their requirements. The IND Sponsor will notify all sites that the study has been 
halted. 
12.9.2 Resumption of a Halted Study 
The IND Sponsor, in collaboration with the PI and DSMB will determine if it is safe to resume 
the study. The IND Sponsor will notify the Site Investigators of this decision. The conditions for 
resumption of the study will be defined in this notification. The Site Investigators will notify 
their local IRB(s) of the decision to resume the study. 
12.10 Halting Criteria for a Subject  
The decision to suspend administration of the study agent(s) for a single subject requires 
discontinuation of study agent administrated for the study subject(s) until a decision is made 
whether or not to continue study agent administration.   
 
The halting criteria for a single subject in this study include:  
• A subject experiences an SAE regardless of vaccine relationship, or   
• A subject experiences ≥ 2 or more Grade 3 or greater AEs (solicited local/systemic or 
unsolicited; lasting 48 hours or more) that are possibly, probably, or definitely related to 
the study agent, within the 7 days post vaccination, or 
• A subject experiences ≥ 2 or more Grade 2 or higher laboratory abnormality (see 
Appendix D) or any Grade 3 or higher laboratory abnormality that are possibly, 
probably, or definitely related to the study agent, within the 7 days post vaccination, or  


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• Any safety issue that the Site Investigator determines should pause administration of the 
study agent to a single subject or to all subjects in a specific group.   
 
The IND Sponsor, in collaboration with the PI, may also pause for an individual subject or entire 
group if a safety concern is identified during routine aggregate data analysis. 
12.10.1Reporting of Halting for a Subject  
If a pausing requirement is met, a description of the AE(s) or safety issue must be reported by the 
Site Investigator by fax or email within 2 business day to the Sponsor CSO, PI, the IRB, and 
DSMB and reported to the FDA within 3 business days 
 
The Site Investigator must inform the PI and the local IRB that a halting rule has been met 
according to their requirements. The IND Sponsor will notify all sites that the study has been 
paused. 
12.10.2 Resumption of a Halted Subject  
The IND Sponsor in collaboration with the PI and the DSMB will determine if it is safe to 
resume administration of the study agent to the subject/group. The IND Sponsor will notify the 
Site Investigators of this decision. The Site Investigators will notify their local IRB(s) of the 
decision to resume administration of the study agent prior to resumption. 
12.11 Discontinuation of Study 
The NIAID/OCRPRO as the study sponsor, the NIH IRB, and the FDA may terminate this study 
at any time.   Reasons for terminating the study may include, but are not limited to, the 
following: 
• The incidence or severity of an AE in this or other studies indicates a potential health 
hazard to subjects 
• Subject enrollment is unsatisfactory 
• Data recording is inaccurate or incomplete 
• Investigators do not adhere to the protocol or applicable regulatory guidelines in 
conducting the study 
12.12 Withdrawal of an Individual Subject 
A subject will not be considered to have completed the trial if any of the following reasons 
apply: 
1. Research terminated by Sponsor or Investigator – applies to the situation where the entire 
study is terminated by the Sponsor or Investigator, or other regulatory authority for any 
reason. 


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2. Withdrawal of consent – applies to a subject who withdraws consent to participate in the 
study for any reason. 
3. Noncompliant with protocol – applies to a subject who does not comply with protocol-
specific visits or evaluations, on a consistent basis, such that adequate follow-up is not 
possible and the subject’s safety would be compromised by continuing in the trial. This 
also applies to a subject who is lost to follow-up and is not reachable by telephone or 
other means of communication and cannot be located.   
4. Developed an AE – applies to a subject who is withdrawn from study due to an AE, 
serious or otherwise. Any grade 3 or greater AE that is assessed as possibly, probably, or 
definitely related to vaccination (other than local reactions lasting <72 hours, or systemic 
reactions lasting <24 hours) will result in withdrawal of the subject from further 
vaccinations. Subjects may also be withdrawn for any AE that would cause continued 
participation in the study to not be in the best interest of the subject, as per the 
investigator’s judgment. Any subject who is withdrawn from the study because of an AE 
related to study agent will be followed for safety until at least resolution of that AE and 
will be encouraged to remain in the safety evaluation for the duration of the study. 
5. Other – is used when previous categories do not apply and a written explanation is 
required. 
If a subject withdraws or is withdrawn prior to completion of the study, the reason for this 
decision will be recorded in the source documents and CRFs. Any subject who has received at 
least 1 dose of vaccine will be encouraged to remain in the safety evaluation for the duration of 
the study. The subject’s data will be included in the safety and immunogenicity analysis. If a 
subject fails to complete all planned vaccinations because of an AE or SAE, the subject will be 
followed until resolution or stabilization of the event. If a subject withdraws, the investigator will 
make a reasonable effort to determine the reason. 
12.12.1Replacement of Withdrawn Subjects 
Subjects who have received at least 1 vaccination and who withdraw or are terminated from the 
study prior to completion will not be replaced. Subjects withdrawn before the first vaccination 
may be replaced. 
12.13 Unblinding 
Intentional, unscheduled unblinding may occur if a subject experiences a SAE that the treating 
clinician and/or site PI believes warrants unblinding to provide appropriate clinical management 
of the subject.  The request for unblinding may be requested by the site PI or designee, Sponsor, 
Sponsor Medical Monitor, Independent Medical Monitor, and Data Safety Monitoring Board 
(DSMB).  If non-emergent, all parties should be notified to discuss prior to unblinding taking 
place.  If unblinding is requested, an “Unblinding Report” should be documented per standard 
operating procedures and submitted to the study statistician for formal unblinding.   


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If emergency unblinding is required, the PI or designee will contact the study statistician or study 
pharmacist for unblinding.  The Sponsor will be informed within 1 business day that the 
unblinding was necessary and a submitted “Unblinding Report” will be provided within 2 
business days.   
 
Subjects who are unblinded will be encouraged to remain in the study to be followed for safety. 
 
After the time of unblinding, subjects in the experimental vaccine cohort will be offered 
vaccination with ENGERIX-B, or another FDA-approved Hepatitis B containing vaccine, 
according to the usual, FDA approved schedule.   
 
Scheduled unblinding for Arms 2c, 2d, 4c will occur on Study Day 336 as already outlined in 
Appendix B and C.  Scheduled unblinding will be reported to the DSMB at the time of the next 
meeting. 
12.13.1Unblinding by Group for Interim Analysis 
If a follow-up study to explore the proof of concept of a VIMT in the field is proposed for 2018, 
group unblinding, for statistical analysis of ELISA responses, DSF, and SMFA will occur 
approximately 3 months following receipt of the final vaccine dose, as long as the individual 
subject blinding can be maintained (as determined by the unblinded statistician).  The data will 
be reviewed by study staff who have no direct contact with study participants nor conduct 
clinical assessments.  This information will only be used to determine if enough data supports 
moving forward with a PE/TBV combination study the following year.    
12.14 Safety Oversight 
12.14.1Safety Review and Communications Plan 
A Safety Review and Communication Plan (SRCP) has been developed for the protocol. The 
SRCP is an internal communications document between the Principal Investigator and the IND 
Sponsor Clinical Safety Office (CSO), which delineates the safety oversight responsibilities of 
the PI, the CSO, and other stakeholders. The SRCP also includes the overall plan for conducting 
periodic safety surveillance assessments. 
12.14.2Sponsor Medical Monitor 
A Medical Monitor, representing the IND Sponsor (OCRPRO), has been appointed for oversight 
of safety in this clinical study. The Sponsor Medical Monitor will be responsible for performing 
safety assessments as outlined in the SRCP. 


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12.14.3Independent Safety Monitor (ISM)  
An ISM in Mali will review the study prior to initiation and will be available to advise the 
Investigators on study-related medical issues and to act as a representative for the welfare of the 
subjects. The ISM will conduct independent safety monitoring and recommend appropriate 
action regarding adverse events and other safety issues.  The ISM is an expert in the field of 
oversight of clinical trials conducted in Mali and internal medicine, specifically in the population 
under study in Mali.  The ISM does not have direct involvement in the conduct of the study and 
does not have other interests with any collaborating pharmaceutical firms or their competitors.   
 
Prior to each ISM review (including DSMB meeting safety reports and at least twice yearly 
reviews), the PI will provide a safety summary report (similar to the DSMB safety reports).  
After each ISM review, a recommendation as to whether the study is to be modified, or be 
terminated will be provided in a summary report to the study PI. If the study is to continue as is, 
no report will need to be submitted by the ISM, but communication to the PI that the review has 
been completed (via in person communication, phone, or email).  All serious adverse events, all 
unanticipated problems, and all IND Safety Reports will be reported by the PI to the ISM at the 
same time they are submitted to the IRB or IND Sponsor.  The ISM will be notified immediately 
if any pausing or halting rule is met and the ISM will provide recommendation for continuation, 
modification, or termination of the study.  The PI will submit the written ISM summary report 
with the recommendations to their IRB(s) on a biannual basis or more frequently if a safety 
concern is raised. The PI will also notify the medical monitor if intentional or unintentional 
unblinding occurs. 
12.14.4Data and Safety Monitoring Board 
The NIAID Intramural DSMB will review the study prior to initiation and according to the 
schedule shown below in Table 16. The Board may convene additional reviews as necessary. 
The Board will review the study data to evaluate the safety, efficacy, study progress, and conduct 
of the study. All SAEs, all UPs, and all IND Safety Reports will be reported by the PI to the 
DSMB at the same time they are submitted to the IRB or IND Sponsor. The PI will notify the 
DSMB of any cases of intentional, unscheduled or unintentional unblinding as soon as possible. 
The PI will notify the Board at the time halting criteria are met and obtain a recommendation 
concerning continuation, modification, or termination of the study. The PI will submit the written 
DSMB summary reports with recommendations to the IRB(s).


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Table 16: Vaccination Dose Escalation Schedule and DSMB Meeting 
       Bamako/Sotuba Phase         Bancoumana/Doneguebougou Phase    
Continuous Time 
(Weeks) 
Vaccination 1  
Study Day 0 
Vaccination 2 
Study Day 28 
Vaccination 3 
Study Day 168 
Vaccination 4 
Study Day 476 
Feeding Assays 
Unblinding 
0 
Arm 1a: 16 µg Pfs25M-EPA/ASO1 (n=5) 
Arm 2a: 13 µg Pfs230D1M-EPA/ASO1 (n=5) 
 
 
 
 
 
1 
DSMB Review 
2 
Arm 1b: 47 µg Pfs25M-EPA/ASO1 (n=10) 
Arm 2b: 40 µg Pfs230D1M-EPA/ASO1 (n=10) 
Arm 3a: 16 µg Pfs25M-EPA/ASO1 + 13 µg Pfs230D1M-EPA/ASO1 (n=5) 
Arm 4a: Comparator (n=10) 
 
 
 
 
 
4 
Arm 3b: 47 µg Pfs25M-EPA/ASO1 AND 40 µg Pfs230D1M-EPA/ASO1 
(n=10) 
Arm 4b: Comparator (n=10) 
Arm 1a: (n=5) 
Arm 2a: (n=5) 
 
 
 
 
6 
 
Arm 1b: (n=10) 
Arm 2b: (n=10) 
Arm 3a: (n=5) 
Arm 4a: (n=10) 
 
 
 
 
7 
DSMB Review 
8 
 
Arm 3b: (n=10) 
Arm 4b: (n=10) 
 
 
 
 
10 
Arm 2c: 40 µg Pfs230D1M-EPA/ASO1 (n=60) 
Arm 2d: 40 µg Pfs230D1M-EPA/ASO1 fractional dosing regimen  (n=60) 
Arm 4c:Comparator (n=120) 
 
 
 
 
 
14 
 
Arm 2c: (n=60) 
Arm 2d: (n=60) 
Arm 4c: (n=120) 
 
 
 
 
24 
 
 
Arm 1a: (n=5) 
Arm 2a: (n=5) 
 
 
 
26 
 
 
Arm 1b: (n=10) 
Arm 2b: (n=10) 
Arm 3a: (n=5) 
Arm 4a: (n=10) 
 
 
 
28 
 
 
Arm 3b: (n=10) 
Arm 4b: (n=10) 
 
 
 
29 
DSMB Review 
 


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Continuous Time 
(Weeks) 
Vaccination 1  
Study Day 0 
Vaccination 2 
Study Day 28 
Vaccination 3 
Study Day 168 
Vaccination 4 
Study Day 476 
Feeding Assays 
Unblinding 
34 
 
 
Arm 2c: (n=60) 
Arm 2d: (n=60) 
Arm 4c: (n=120) 
 
 
 
35 to 47 
 
 
 
 
Arm 2c: (n=60) 
Arm 2d: (n=60) 
Arm 4c: (n=120) 
 
58 
 
 
 
 
 
Arm 2c: (n=60) 
Arm 2d: (n=60) 
Arm 4c: (n=120) 
 
                                 DSMB Review 
78 
 
 
 
Arm 2c: (n=60) 
Arm 2d: (n=60) 
Arm 4c: (n=120) 
 
 
82  to 98 
 
 
 
 
Arm 2c: (n=60) 
Arm 2d: (n=60) 
Arm 4c: (n=120) 
 
 
DSMB Review 
 


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13 Clinical Monitoring 
13.1 Site Monitoring Plan 
As per ICH-GCP 5.18 and FDA 21 CFR 312.50, clinical protocols are required to be adequately 
monitored by the study sponsor. This study monitoring will be conducted according to the 
“NIAID Intramural Clinical Monitoring Guidelines. Monitors under contract to the 
NIAID/OCRPRO will visit the clinical research site to monitor aspects of the study in 
accordance with the appropriate regulations and the approved protocol. The objectives of a 
monitoring visit will be: 1) to verify the existence of signed informed consent documents and 
documentation of the informed consent process for each monitored subject; 2) to verify the 
prompt and accurate recording of all monitored data points, and prompt reporting of all SAEs; 3) 
to compare abstracted information with individual subjects’ records and source documents 
(subjects’ charts, laboratory analyses and test results, physicians’ progress notes, nurses’ notes, 
and any other relevant original subject information); and 4) to help ensure investigators are in 
compliance with the protocol. The monitors also will inspect the clinical site regulatory files to 
ensure that regulatory requirements (Office for Human Research Protections-OHRP), FDA, and 
applicable guidelines (ICH-GCP) are being followed. During the monitoring visits, the 
investigator (and/or designee) and other study personnel will be available to discuss the study 
progress and monitoring visit. 
 
The investigator (and/or designee) will make study documents (e.g., consent forms) and pertinent 
hospital or clinical records readily available for inspection by the local IRB, the FDA, the site 
monitors, and the NIAID staff for confirmation of the study data.  
 
A specific protocol monitoring plan will be discussed with the Principal Investigator and study 
staff prior to enrollment. The plan will outline the frequency of monitoring visits based on such 
factors as study enrollment, data collection status and regulatory obligations. 
14 Statistical Considerations 
14.1 Description of Statistical Methods 
This study, like other Phase 1 studies, is exploratory rather than confirmatory; its purpose is to 
estimate AE rates and patterns of immune responses rather than to test formal statistical 
hypotheses. Estimates will be presented with their 95% confidence intervals. Descriptive 
approaches will be used to meet the protocol objectives, as well as formal statistical tests as 
outlined below. Results will be presented in tabular format, as well as graphically where 
appropriate. 


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14.2 Primary Objective 
The primary objective of this study is to assess safety and reactogenicity of Pfs25M-EPA/AS01 
and Pfs230D1M-EPA/AS01 in Malian adults. 
• 
The frequency of systemic and local AEs will be summarized. 
• 
A line listing of each clinical and laboratory AE classified as local, solicited, or other will 
be displayed in tables stratified by vaccine allocation. 
• 
AEs will be summarized by severity and relationship to vaccine 
• 
The proportion of subjects with at least 1 AE will be compared by vaccine schedule and/or 
dose group, and tests will be performed to assess whether these groups differ with respect to 
these proportions. To evaluate the difference in AEs between the initial vaccination and the 
subsequent vaccinations, a Wilcoxon signed rank test will be performed, where the response 
for each subject is the difference between the numbers of AEs in the 7 days following each 
vaccination. To compare number and severity of AEs between control and vaccine arms, 
Wilcoxon-Mann-Whitney test (also known as Wilcoxon rank sum test and the Mann-
Whitney U test) tests as well as linear regression may be used. 
• 
SAEs occurring within the study period will be listed by relationship to vaccine. 
14.3 Secondary Objectives   
The secondary objective of this study is to determine the antibody response of both vaccines and 
their combination as measured by ELISA and transmission-blocking assays as measured by 
SMFA and DSF and the effect on antibody responses of a third dose. 
 
Anti-Pfs25 antibody will be measured by ELISA on vaccination days, 2 weeks after each 
vaccination, and periodically until study completion. Antibody responses over time will be 
shown graphically with geometric mean antibodies and the associated confidence intervals based 
on the t-distribution applied to the log-transformed responses. The smaller (n=5 and n=10) 
groups are primarily included for safety, and their ELISA response will only be used in an 
exploratory nature. They will not be included in the main analysis of the secondary endpoint. We 
will compare antibody and functional assay responses within Arms over time. 
 
We will also look at differences in antibody decline over time in the vaccination groups. 
 
Graphs will display transmission-blocking assays as a function of ELISA antibody levels. 
 
Preliminary results for the SMFA and ELISA have been ascertained for Day 252 (12 weeks post-
dose 3).  They have been discussed with the scientific advisory board (SAB) and the data safety 
monitoring board (DSMB).  The conclusion to combine groups for the full booster dose was 
based on the facts that fractional (1/5 the amount of antigen and adjuvant) dosing (marginally) 
failed (p=0.0597) to be statistically significantly different than full dosing when looking at TRA 
readouts from the SMFA while being statistically significantly different in ELISA titer readouts 


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(p=0.00014).  The combined group will be given the full dose in an effort to maximize power to 
detect a signal.  The unblinding occurred as originally planned, with some of the members of the 
clinical team unblinded later than others as they completed database clean up procedures.  Some 
of the lab teams are still blinded as they are still performing assays.  They will be unblinded once 
these tasks are completed.  The DSMB has always received unblinded information throughout 
the study.  The study is being extended for one year in order to assess the kinetics of the fs230 
antibody titers with (main phase) and without (pilot phase) a booster vaccination in the second 
transmission season. The possibility for an enrollment differential between the study arms in the 
study extension does exist, but is not anticipated due to re-enrollment being very successful in a 
previous study in this area.  The loss to follow-up thus far has not appreciably been affecting one 
group more than another. 
 
14.4 Exploratory Objectives 
We will characterize and compare host immune responses, proteomic profiles, co-infection 
status, antibody repertoires, and transcriptomes to malarial antigens and to vaccine antigens in 
African adults prior and after immunization with Pfs230D1M-EPA/AS01 by mixed effects 
model or Wilcoxon signed rank test depending on the type of immune response. We will also 
make figures depicting immune response based on the study arm. 
 
We will explore the impact of Pfs230D1M-EPA/AS01 on the genotype and transcriptome profile 
of parasites isolated from study subjects via standard regression analysis. 
 
Should the study be terminated early, the investigative team will discuss with the DSMB the 
reason for termination and determine which study questions can be addressed in an unbiased 
manner with the available data. The available data will be analyzed and interpreted in light of 
early termination. 
14.5 Sample Size and Power Calculations 
SAFETY 
The arms of five subjects are sized for safety, as the higher dose is expected to be necessary for 
an adequate immune response. In these arms (Arms 1a, 2a, 3a), 5 subjects each will receive 16 
µg of Pfs25M-EPA/AS01 or 13 µg Pfs230D1M-EPA/AS01 or the combination of both. 
Vaccination arms of 5 subjects gives a probability of at least 0.80 for detecting 1 or more serious 
or severe AEs that occur with a probability of 0.275 or more per subject. 
For each dose level that has a (n=10) in Arms 1b, 2b, 3b, vaccination of 10 subjects gives a 
probability of at least 0.90 for detecting 1 or more serious or severe AEs that occur with a 
probability of 0.206 or more per subject. 


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For each dose level that has a (n=60) in Arms 2c, 2d, and 4c, vaccination of 60 subjects gives a 
probability of at least 0.90 for detecting 1 or more serious or severe AEs that occur with a 
probability of 0.038 or more per subject. 
If we combine all treated groups in Mali (Arms 1a, 1b, 2a, 2b, 3a, 3b, 2c and 2d), 165 subjects, 
we have 95% power to detect 1 or more serious or severe AEs that occur with a probability of 
0.018 or more per subject. Due to arms having varying doses, combinations and administration 
villages, this analysis would only be done to catch an AE that transcended these discrepancies. 
We will compare all AE event proportions between the control arm and treated arm by Fisher’s 
exact test. 
ELISA ANALYSIS 
There are several questions of interest based on the antibody response information after the 
2nd,3rd and for arms 2c, 2d, and 4c, a 4th (booster) doses of the respective vaccines. We are 
interested in the change in ELISA values from baseline to after a given number of doses of 
vaccine, and the change in ELISA values between doses. For this we will use Wilcoxon signed 
rank tests (WSRT) within the 60 subjects receiving a given vaccine regimen. We are also 
interested in the effect of a given number of vaccinations on the ELISA responses as compared 
to comparator. This comparison can be made by Wilcoxon Mann Whitney (WMW) test, which 
accommodates the limit of detection issues that may exist for ELISA results. 
The preliminary data from protocol #15-I-0044 on subjects who had each Pfs25 and Pfs230 
ELISA measurements after receiving two and three doses of 47µg Pfs25 + 40µg Pfs230 (using 
Alhydrogel® adjuvant, not AS01 as in this protocol) allow us to estimate the SD of the log 
transformed Pfs25 ELISA responses at baseline, post vaccination 2 and post vaccination 3. Due 
to these data still being blinded, we do not report the values for the SD and mean of the log 
transformed Pfs25 ELISA responses. 
Assuming similar values in this trial, there would be over 0.99 power to detect the difference 
between baseline and post-vaccination 2 and over 0.9 power to detect the difference between 
post vaccination 2 and post vaccination 3. 
In these same preliminary data from protocol #15-I-0044 all control subjects had undetectable 
levels of Pfs25 ELISA response post 2nd and 3rd vaccination. 
Within Arm 2c and using the background information from #15-I-0044, we have greater than 
80% power to reject a 2-sided 0.05 level WMW test if the geometric mean Pfs230 ELISA 
baseline level was 1.6-fold higher geometric mean than the level of detection in the vaccinated 
group post vaccination 2 (Note: 1.6-fold is lower than what we observed in #15-I-0044, so we 
treat these numbers as conservative). Given the similarity in SD estimate post vaccination 3, we 
should have very similar power post vaccination 3 as was calculated for post vaccination 2. 
There are three key comparisons regarding ELISA measurements and the booster dose 
(equivalently dose 4).  These include 1)  peak titers after dose 4 compared with those following 


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dose 3; 2) nadir titers  at 3 months post dose 4 compared to those post dose 3; and 3) decay rates 
after dose 4 compared to those after dose 2 and those after dose 3.   The power to detect these 
differences is modest/low due to the expected performance of the booster to perform similarly to 
the previous doses.  However, it is important to note that the primary immunogenicity endpoints 
will be comparing ELISA titers of each arm 2c and 2d versus the control arm 4c at all time 
points, which will be powered above 90%.   
 
FUNCTIONAL ASSAYS 
SMFA 
Using blinded SMFA data from #15-I-0044, a WMWT comparing 60 TRAs of the vaccine arm 
to 60 TRAs of the control, we anticipate at least 80% power to detect a difference in TRAs of 
18% (simulation-based power calculations). If TBAs are used, we will standardize the TBAs to a 
common target control mean first. The standardization will utilize the estimand that comes from 
a zero-inflated negative binomial with random effects for feed and container of mosquito. TRAs 
can be combined across feeds or calculated within feeds as they do not require the same 
standardization as that of TBAs. 
DSF 
Using the data from the primary DSF evaluation period of study #13-I-N109 and #15-I-0044 we 
were able to do power calculations for this study. The power calculations are quite sensitive to 
the infectivity rate, which varies from year to year.  Taking a weighted average of 2014 and 2015 
data we estimate infectivity was 14.8% (the percentage of subjects in the control arm that had at 
least one positive DSF).  Using this same % and comparing 108 subjects (this number assumes 
10% dropout) of the combined Arms 2c and 2d we will have ~85% power to detect 80% vaccine 
effect through the (0,1) infective/non-infective way of quantifying each DSF with 32 DSF per 
person and 30 mosquitoes per cup. The (0,1) infective/non-infective way of quantifying each 
DSF involves declaring a cup as infective if any of the 30 mosquitoes is infected and conversely 
non-infective if and only if all 30 mosquitoes are not infected. While combining arms that have 
full dosing in one and fractional dosing in the other assumes they are having similar effects, this 
assumption is deemed acceptable in order to power the analysis. If infectivity drops to 10.3%, 
then the power to detect 80% vaccine effect under same conditions is about 40%. It is important 
to note that in 2016 only 15 mosquitoes were used per cup and only 12 DSFs per person were 
conducted. Thus, we are treating the 10.3% infectivity as a “worse case scenario” for the 30 cup / 
24 DSFs set-up as proposed in this protocol.  A binomial family GEE model will be used to 
account for repeated measurements. 
On average each feed week we had about 3% positivity over all subjects. The (0,1) 
infective/non-infective way of quantifying DSF is likely an over-simplification of the true 
system. As well, this way of quantifying the DSF will have lower power when the vaccine is 
leaky rather than all or nothing. 


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For the booster (dose 4),  the combined Arms 2c and 2d will be evaluated vs. controls as the 
primary endpoint. 
 
DSF in conjunction with SMFA 
As there will be simultaneous blood draws, a unique opportunity to investigate SMFA predicting 
zero inflated DSF measurements will be pursued. Simple linear regression models will be 
explored. 
14.6 Randomization  
See Section 8.2 for more information. 
 
Enrollment into the Bamako/Sotuba, Mali portion of the study will be randomized as follows: 
• 
Arms 1a and 2a will be enrolled 1-1 with random block based on time of 
enrollment/randomization; 
• 
Arm 3a will not have a concurrent arm, so there will be no randomization for that arm; 
• 
Arms 1b, 2b and 4a will be enrolled 1-1-1 with the same style of random block based on 
time of enrollment/ randomization; 
• 
Arms 3b and 4b will be enrolled 1-1 with random block based on time of 
enrollment/randomization. 
 
Enrollment into the Bancoumana/Doneguebougou, Mali portion of the study will be randomized 
in random blocks of size no greater than 8 and stratified by village. In order to accomplish this, a 
list of all enrolled subjects will be given to the study statistician for the first randomization. As 
subjects that dropout after randomization, and are not vaccinated, cannot be replaced, 
randomization and first vaccination should occur as close in time as possible to avoid having 
subjects in the ITT efficacy and safety analyses that have never received vaccination or 
comparator. Randomization lists are not produced pre-enrollment to reduce the risk of on-site 
operational error in randomization assignment, which given the stratified nature of the 
randomization would require management of several lists. 
 
Randomization will occur between arms as follows in the Bancoumana/Doneguebougou study: 
• 
Arms 2c, 2d, and 4c will be enrolled 1-1-2 with random block sizes no greater than 8. 
 
Randomization between the arms allows for causal comparisons of ELISA and functional assay 
responses. 
15 Human Subject Protections and Ethical Obligations 
This research will be conducted in compliance with the protocol, Good Clinical Practices (GCP), 
and all applicable regulatory requirements. 


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15.1 Institutional Review Board 
A copy of the protocol, informed consent forms, and other study related information to be 
completed by subjects, such as questionnaires, diary cards, medical history forms, and any 
proposed advertising/recruitment materials or letters to the subjects will be submitted to the 
reviewing IRBs for written approval. The investigator must submit and obtain approval from the 
IRBs for all subsequent amendments to the protocol, informed consent documents, and other 
study documentation referenced above. The investigator will be responsible for obtaining IRB 
approval of the annual Continuing Review throughout the duration of the study. The 
investigators will notify the reviewing IRBs of protocol violations and SAEs as specified in the 
relevant sections of the protocol. 
15.2 Informed Consent Process 
Informed consent is a process where information is presented to enable persons to voluntarily 
decide whether or not to participate as a research subject. It is an 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 will be given the opportunity 
to ask questions and have them answered.  
 
Consent forms will be approved by all participating IRBs. The subjects will sign the informed 
consent document prior to undergoing any research procedures. The subjects may withdraw 
consent at any time throughout the course of the trial. The informed consent process will be 
documented in the subject’s research chart, as required by 21 CFR 312.62. The informed consent 
form will be signed (or fingerprinted) and personally dated by the subject and the person who 
conducted the informed consent discussion. The original signed informed consent form will be 
retained in the subject’s chart and a signed and dated copy will be provided to the subject. The 
rights and welfare of the subjects will be protected by emphasizing to them that the quality of 
their medical care will not be adversely affected if they decline to participate in this study.   
15.2.1 Mali Site Community Permission and Individual Informed Consent Process 
15.2.1.1 Community Permission 
Community permission will be obtained from village elders, family heads, and other community 
members after explanation and discussion of the study.41 The community permission process 
goes through the following steps: 
 Study investigators/personnel explain the study to village leaders, including the village 
chief, family heads, women association, and elders. 
 The village leaders then discuss the study with family heads and community members 
and relay any additional questions or concerns they may have to study personnel. 


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 The study and the informed consent process are explained in detail to heads of families 
by study investigators/personnel.    
 
At the time of community permission, the need for both husband and wife to agree to avoid 
pregnancy for the specified period if a wife chooses to volunteer will also be addressed. 
 
The individual informed consent process and form will be translated into French. The study team 
conducts careful word-for-word review of the study consent form, and will translate the consent 
orally into local languages, as the majority of potential study subjects do not read or speak 
French. Verification that the oral translations are accurate and that the potential subjects 
understand the contents of the informed consent form will be done by an independent witness 
who is not a member of the study team. An evaluation checklist is performed to make sure that 
the study is understood by the subjects before the enrolment. 
15.2.1.2 Individual Informed Consent 
Local households and families will be invited to come to the study clinic for review of the 
informed consent, and if the subject agrees to participate, the subject will sign or fingerprint (if 
illiterate) the consent form.   
 
At the consenting visit, the subject will read the consent form or have it explained in cases of 
illiteracy. Individuals in each family will be separately consented and not all individuals from a 
household need to participate.   
 
Subjects will be encouraged to ask questions, and then take a multiple-choice questionnaire 
(true/false) to evaluate consent comprehension. All incorrect responses will be reviewed with the 
subject, and he or she must verbalize understanding of all incorrect responses. A score of ≥80% 
correct is required for enrollment. For subjects scoring less than 80%, study staff may choose to 
review study details again with subject and reassess comprehension with a repeat Malaria 
Comprehension Exam. At the discretion of the Investigator, any subject whose comprehension is 
questionable, regardless of score, may be excluded from enrollment.  
 
The Malaria Comprehension Exam will be translated into French and administered orally in the 
native dialect in the case of potential subjects who cannot read. Study staff will use incorrect 
answers from the questionnaire to identify those areas of the informed consent that need further 
review with subject. This will help ensure that the subject has sufficient understanding before the 
consent form is signed. The subject may either sign the consent form immediately or later after 
further consideration. Subjects unable to read will place a fingerprint in the place of a signature. 
In addition, an independent witness will sign the consent form to attest that the consent was fully 
explained and all questions were answered.  


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15.3 Subject Confidentiality 
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, the FDA, the NIAID, the 
OHRP, or the sponsor’s designee. 
15.4 Risks 
Risks to the subjects are associated with venipuncture, immunization, drug treatment, and DSFs.  
These risks are outlined below: 
15.4.1 Venipuncture 
Risks occasionally associated with venipuncture include pain, bruising, bleeding, and infection at 
the site of venipuncture, lightheadedness, and rarely, syncope. 
15.4.2 Intramuscular Immunizations 
Possible local vaccine reactions resulting from intramuscular injection include pain, swelling, 
erythema, induration, limitation of limb movement for several days, lymphadenopathy, or 
pruritus at the injection site. Systemic reactions such as fever, chills, headache, fatigue, malaise, 
myalgia, and joint pain may also occur, and may range from mild to severe.   These side effect 
will be monitored, but are generally mild and self limiting. 
 
 
EPA has been studied in both malaria transmission vaccine studies (as noted with Pfs25H, 
Pfs25M, Pfs230D1M) and other vaccination studies 12-14,42,43.  The use of EPA has identified no 
safety issues to date.   
 
As with any vaccine, immediate hypersensitivity reactions including urticaria, anaphylaxis, or 
other IgE-mediated responses are possible. There is a theoretical possibility of risks about which 
we have no present knowledge. Subjects will be informed of any such risks should further data 
become available. 


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15.4.2.1 Immunization with Pfs25M-EPA/AS01 and/or Pfs230D1M/AS01 
As noted in Section 3.1 (for recent studies for Pfs25H-EPA/Alhydrogel® Pfs25M-
EPA/Alhydrogel®) and Section 3.2 (for recent studies for Pfs230D1M-EPA/Alhydrogel®) and 
Section 3.3 (for recent studies for Pfs25M-EPA/Alhydrogel® and Pfs230D1M-
EPA/Alhydrogel®), common solicited adverse events include injection site pain, redness, 
induration, and swelling.  Other commonly reported solicited adverse events includes headache, 
malaise, diarrhea, nausea, and fever.    
 
While several subjects in the US and the Mali studies experienced changes in hemalogic 
parameters (such as anemia, leukopenia, and neutropenia), some even moderate (Grade 2) in 
severity, an examination of trends in the US study and the unblinded portion of the Mali study 
(Group 1) has not shown any pattern deemed related to vaccination.  In addition, all neutropenia 
events that had occurred were not associated with fever and/or subsequent acute infection 
attributable to the drop in neutrophil counts. 
 
A single subject in protocol #15-I-0044, a healthy 51 year old woman, presented six days 
following receipt of her fourth vaccination while on study, with an acute onset of hemiplegia. 
She was diagnosed as having a cerebrovascular accident via CT scan and neurological 
assessment at the hospital in Bamako, Mali and hospitalized. Her symptoms worsened overnight, 
and she died the following day. This SAE was reviewed at length by the study team, independent 
safety monitor, Sponsor medical monitor, NIH IRB, FMPOS EC, and the study’s DSMB and 
was determined unrelated to vaccination. In addition, the SAE was submitted to the FDA for 
review as an informational item. Which vaccine the subject received during the study 
(comparator vaccine or test vaccine(s)) remains blinded, but given the severity of the event, at 
the recommendation of our DSMB, we are informing subjects in this study of the SAE reported 
in #15-I-0044. At this time we do not believe the arterial or venous occlusion are possible risk 
factors associated with vaccination, but want to provide as much available information to future 
volunteers as possible. Which vaccine(s) the subject received during that study will be unblinded 
in March 2017.  
 
Overall, with the exception of local reactogenicity, such as pain, swelling, erythema, tenderness 
at the site of injection and systemic symptoms such as low-grade fever and short-term flu-like 
symptoms: fatigue, myalgia, headache, malaise, MPL-based adjuvants (AS01) have been safe 
and well tolerated.  
15.4.2.2 Immunization with ENGERIX-B 
In adults, the most common (≥10%) solicited adverse events include injection site pain and 
fatigue.  Other common reported solicited adverse events (1-10%) in adults includes injection 
site erythema/induration/swelling, dizziness, and headache.  Brief fainting spells and related 


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symptoms have been reported following vaccination, most often in adolescents, that may result 
in falls and/or injuries.   
 
For complete safety details, please refer to the package insert provided. 
 
As with any vaccine, hypersensitivity, including severe allergic reaction, to any component of 
the vaccine may occur. Additionally, the tip caps of the pre-filled syringes contain natural rubber 
latex, which may cause allergic reactions, thus subjects known to be latex sensitive will be 
excluded per Section 6.5; however, those with an unknown latex sensitivity may be at risk for an 
allergic reaction. 
15.4.3 Immunization with Menactra®  
For Menactra® the most common (≥10%) solicited adverse events include injection site pain, 
redness, induration, and swelling; anorexia and diarrhea.  Other common reported solicited 
adverse events in adults includes headache, fatigue, malaise, and arthralgia.   Brief fainting spells 
and related symptoms have been reported following vaccination, most often in adolescents, that 
may result in falls and/or injuries.   
 
For complete safety details, please refer to the package insert provided. 
15.4.4 Coartem® 
Subjects in Arms 2c, 2d and 4c will all be pre-emptively treated prior to Vaccination #1, 
regardless of their malaria status, with Coartem®, which is a registered, oral, proven, and highly 
efficacious treatment.  
 
Coartem® has an acceptable safety profile. Individuals who may have any contraindication for 
the use of this drug (e.g. prolonged QTc or taking other medications that can prolong QTc, 
history of myocardial infarction) will be excluded at screening. The most common side effects 
(i.e., >30%) in adults are: headache, anorexia, dizziness, asthenia, arthralgia, and myalgia.  
Discontinuation of Coartem® 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. Coartem® should be taken with milk or another fatty food. 
 
Coartem® is a Category C pregnancy drug.  Thus all female participants will undergo pregnancy 
testing prior to receipt of the investigational dose of Coartem® if enrolled in Arms 2c, 2d, or 4c. 
Also per the package insert, Coartem® may decrease the efficacy of hormonal birth control, so 
female volunteers who are on hormonal birth control will be counseled about back up pregnancy 
prevention methods.   
 
For complete safety details, please refer to the package insert provided for Coartem®. 


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15.4.5 Direct Skin Feeds 
Risks associated with mosquito bites include mild to moderate swelling and itching at the site.  
Participants will be offered symptomatic treatment (i.e., local application of antipruritic agent) if 
these occur.  While malaria and filarial parasites are transmitted through Anopheline mosquito 
bites as part of the parasite life cycle, mosquitoes raised in an insectary that are used for DSFs 
have never been exposed to human blood (breeding stocks are maintained on human blood as 
noted in Section 9.5.2) and since these parasites are not transovarially (vertically) transmitted in 
mosquitoes, there is no risk of transmission to participants.  O’nyong’nyong virus is transmitted 
to humans by anopheles mosquitoes, but is not vertically transmitted so does not pose a risk of 
infection to subjects or the mosquito colony.  Anopheline mosquitoes can rarely transmit rift 
valley fever (RVF) virus, and RVF has been reported to transovarially infect anopheles 44. RVF 
is primarily transmitted to humans by direct contact with infected animals, although transmission 
by mosquitoes can occur, with Aedes mosquitoes being the primary vector.  The incubation 
period is 2-6 days.45 No outbreaks of RVF have been reported in Mali, and transovarial infection 
is rare and extremely unlikely to persist for generations (for example, in a lab colony maintained 
at MRTC or elsewhere).  To document the lack of viral contamination, the lab strain and field 
caught mosquitoes were tested for RVFV using molecular methods.  Testing conducted over the 
last 2 years in Mali on LMIV/MRTC clinical trials have all been negative for RVFV. Because 
routine tests for this virus are not feasible, we installed a procedure to ensure that blood meals 
used to rear the mosquitoes are free of the virus (see Section 9.5.2). 
 
This study involves direct feeds with Anopheles mosquitoes only.  Aedes and Culex mosquitoes 
transmit viral diseases such as dengue, West Nile, and yellow fever, which may be vertically 
transmitted in mosquitoes; however no Aedes and Culex mosquitoes are raised in the MRTC 
insectary, thus transmission of these viral agents to subjects is not possible through feeds 
conducted as part of this study.  As previously described (see Section 9.4.4.2), hundreds of 
subjects in Bancoumana or neighboring areas have been enrolled in DSFs.  Except for 1 case of 
definitely related Grade 2 erythema that were resolved within 48 hours and  2 cases of definitely 
related local site pruritus at the DSF site (in the same individual), there have been no other 
expected or unexpected AEs recorded as related (definitely, probably, possibly) to the feeding 
procedures. 
15.4.6 Other risks 
Women of reproductive potential will be required to agree to use birth control as outlined in 
Section 6.4.  Because this is a research study, women of reproductive potential will be asked to 
notify the site immediately upon learning of pregnancy during this study and will be tested for 
pregnancy prior to administration of the vaccine(s) and periodically throughout the course of the 
study as outlined in Appendix B.   


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15.5 Benefits 
Subjects will not receive any direct benefit from participation in this study. It is hoped that 
information gained in this study will contribute to the development of a safe and effective 
malaria vaccine. 
15.6 Compensation 
Subjects will be given in kind (such as rice and/or millet) or cash equivalent, in multiple 
installments as outlined in Table 17, to compensate for the time taken to come to the study clinic 
for study-related visits.  Preferred compensation is in kind, such as rice and/or millet, rather than 
cash, which had been decided in consultation with village elders, but case-by-case exceptions to 
receive the cash equivalent have been considered acceptable.  
The Mali Ethics Committee recommends compensating the study subject for their time lost for 
study procedures. The amount equivalent to USD $6 for each scheduled visit with laboratory 
procedures and equivalent to USD $3 for each scheduled visit without laboratory procedures, and 
equivalent to USD $6 for DSF visits is therefore proposed to compensate for time lost, since 
visits with laboratory procedures require more time than visits without and frequent DSF 
assessments are a significant time commitment to the study on behalf of the subject.   The 
estimated installments will be distributed as outlined in Table 17. 
 
 


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Table 17: Estimated Compensation Schedule1 
Study Activity 
 
US Dollar Equivalent in Rice or Millet Dispensed 
(Local Currency [CFA])2 
Bamako/Sotuba, 
low dose –  
3 vaccination 
Bamako/Sotuba, 
high dose –  
3 vaccinations 
Bancoumana/Doneguebougou, high 
dose and fractional dose –  
3 vaccinations 
Screening 
$6 (= 3000 CFA) 
Completion of 
Vaccination #1 
$24 (= 12000 CFA) 
$24 (= 12000 CFA) 
$30 (= 16000 CFA) 
Completion of 
Vaccination #2 
$39 (= 19500 CFA) 
$42 (= 21000 CFA) 
$42 (= 21000 CFA) 
Completion of 
Vaccination #3 
$30 (= 15000 CFA) 
$33 (= 16500 CFA) 
$159 (= 79500 CFA) 
Safety Follow-
up 
$24 (= 12000 CFA) 
$24 (= 12000 CFA) 
$18 (= 9000 CFA) 
Duration 
Follow-up 
$12 (= 6000 CFA) 
$12 (= 6000 CFA) 
$12 (= 6000 CFA) 
Completion of 
Vaccination #4 
N/A 
N/A 
$219 (= 109500 CFA) 
Safety Follow-
up 
N/A 
N/A 
$24 (= 12000 CFA) 
Total 
$135 (= 67500 CFA) 
$141 (= 70500 CFA) 
$510 (= 255000 CFA) 
1 Compensation installments may be paid out in 3-6 installments at the clinic’s discretion to the subjects during these specified time periods. 
2 Assuming currency exchange rate of USD $1 = 500 CFA 
 
16 Data Handling and Record Keeping 
16.1 Data Capture and Management 
In Mali, study data will be collected on paper CRFs and then put into a study specific DataFax 
electronic database.  Data from CRFs will be collected directly from subjects during study visits 
and telephone calls, or will be abstracted from subjects’ medical records. CRFs will be used as 
source.  Any type of corrections to paper CRFs must be initiated and dated by the person making 
the correction.  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, 


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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. 
16.2 Types of Data 
Source documents include all recordings of observations or notations of clinical activities and all 
reports and records necessary to confirm the data abstracted for this study.  Complete source 
documentation (laboratory test reports, hospital or medical records, progress notes, observations, 
etc.) is required for every study subject for the duration of the study. Source documentation will 
be made available for review or audit by the Sponsors, or their designees and any applicable 
Federal authorities. 
16.3 Retention of Study Records 
The investigator is responsible for retaining all essential documents listed in the ICH Good 
Clinical Practice Guideline. Study records will be maintained by the PI for a minimum of 3 years 
and in compliance with institutional, IRB, state, and federal medical records retention 
requirements, whichever is longest. All stored records will be kept confidential to the extent 
required by federal, state, and local law. 
 
Should the investigator wish to assign the study records to another party and/or move them to 
another location, the investigator will provide written notification of such intent to 
OCRPRO/NIAID with the name of the person who will accept responsibility for the transferred 
records and/or their new location. Destruction or relocation of research records will not proceed 
without written permission from NIAID/OCRPRO. 
16.4 Protocol Revisions 
No revisions to this protocol will be permitted without documented approval from the IRBs that 
granted the original approval for the study. Any change to the protocol will be submitted to the 
Sponsor and to the participating IRBs as a protocol amendment; changes not affecting risk to 
subjects may request an expedited review. In the event of a medical emergency, the Investigator 
shall perform any medical procedures that are deemed medically appropriate and will notify the 
IND Sponsor of all such occurrences. 


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Appendix A: Study Schema  
 
 
 


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Appendix B:  Schedule of Procedures/Evaluations  
 
 
Note: visits #26 and 27 are the additional blood draw visits.  
 


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Appendix B:  Schedule of Procedures 
 
  
Note: visits #26 and 27 are the additional blood draw visits 


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Appendix B:  Schedule of Procedures  
 Vaccination #4 study procedures     
 
 


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Appendix C:  Day to Day Schedule by Arm 
Bamako/Sotuba Cohort – Arms 1a, 2a, 3a (3 Vaccination; safety cohort) 
 
Study Day 0 (day of Vaccination #1) 
The following evaluations/procedures will be completed before vaccination: 
1. Verify that informed consent was obtained.  
2. Ensure that all inclusion/exclusion criteria are met.  
3. Ensure that CBC, ALT, creatinine, HIV/HCV/HBV, and urine results from screening are 
within protocol-defined limits (see Exclusion Criteria above) before vaccinating.  
4. Perform interim history and physical examination, focusing on any acute complaints.  
5. Record vital signs (blood pressure, temperature, and heart rate). 
6. During the physical examination, study staff will educate the subject regarding signs and 
symptoms of potential adverse events (AEs) and indications for use of antipyretics if fever, 
headache, or malaise occurs. 
7. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before vaccinating; a positive test will exclude the subject from the trial.  
8. For females, ensure agreement and compliance with pregnancy prevention before 
vaccinating. 
9. Obtain approximately 16 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, and anti-Pfs25M and anti-Pfs230 antibody 
ELISA.  
10. Confirm that all criteria for vaccination have been met according to inclusion/exclusion 
criteria 
11. Confirm continued eligibility to receive vaccination 
12. Notify subject of vaccine assignment. 
13. Administer the vaccine.  
 
The following evaluations/procedures will be completed following vaccination: 
1. Observe the subject for at least 30 minutes after vaccination to evaluate immediate adverse 
reactions.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Perform interim history and physical examination, focusing on any acute complaints.  
4. Record AEs and concomitant medications, if applicable. 
 
Study Day 1 (±0 days; 1 day after Vaccination #1)  
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 3 (±1 day; 3 days after Vaccination #1) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 


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4. Obtain approximately 5 mL of blood for CBC with differential and platelet count, ALT, 
creatinine. 
 
Study Day 7 (±2 days; 7 days after Vaccination #1) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 14 (±3 days; 14 days after Vaccination #1) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 11 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, and anti-Pfs25M and anti-Pfs230 antibody 
ELISA. 
  
Study Day 28 (day of Vaccination #2) 
The following evaluations/procedures will be completed before vaccination: 
1. Perform interim history and physical examination, focusing on any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. During the physical examination, study staff will educate the subject regarding signs and 
symptoms of potential adverse events (AEs) and indications for use of antipyretics if fever, 
headache, or malaise occurs. 
4. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before vaccinating; a positive test will exclude the subject from the trial.  
5. For females, ensure agreement and compliance with pregnancy prevention before 
vaccinating. 
6. Obtain approximately 11 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs25M and anti-Pfs230 antibody ELISA, 
and SMFA.  
7. Confirm continued eligibility to receive vaccination  
8. Administer the vaccine.  
 
The following evaluations/procedures will be completed following vaccination: 
1. Observe the subject for at least 30 minutes after vaccination to evaluate immediate adverse 
reactions.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Perform interim history and physical examination, focusing on any acute complaints.  
4. Record AEs and concomitant medications, if applicable. 
 
Study Day 29 (±0 days; 1 day after Vaccination #2)  
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 


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Study Day 31 (±1 day; 3 days after Vaccination #2) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 5 mL of blood for CBC with differential and platelet count, ALT, 
creatinine. 
 
Study Day 35 (±2 days; 7 days after Vaccination #2) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 42 (±3 days; 14 days after Vaccination #2) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 11 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, SMFA, and anti-Pfs25M and anti-Pfs230 
antibody ELISA. 
 
Study Day 56 (±7 days; 28 days after Vaccination #2)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 84 (±14 days; 56 days after Vaccination #2)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 112 (±21 days; 84 days after Vaccination #2) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 6 mL of blood for malaria blood smear, malaria PCR, SMFA, and anti-
Pfs25M and anti-Pfs230 antibody ELISA. 
 
Study Day 140 (±21 days; 112 days after Vaccination #2) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  


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2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 168 (day of Vaccination #3) 
The following evaluations/procedures will be completed before vaccination: 
1. Perform interim history and physical examination, focusing on any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. During the physical examination, study staff will educate the subject regarding signs and 
symptoms of potential adverse events (AEs) and indications for use of antipyretics if fever, 
headache, or malaise occurs. 
4. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before vaccinating; a positive test will exclude the subject from the trial.  
5. For females, ensure agreement and compliance with pregnancy prevention before 
vaccinating. 
6. Obtain approximately 11 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs25M and anti-Pfs230 antibody ELISA, 
and SMFA.  
7. Confirm continued eligibility to receive vaccination  
8. Administer the vaccine.  
 
The following evaluations/procedures will be completed following vaccination: 
1. Observe the subject for at least 30 minutes after vaccination to evaluate immediate adverse 
reactions.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Perform interim history and physical examination, focusing on any acute complaints.  
4. Record AEs and concomitant medications, if applicable. 
 
Study Day 169 (±0 days; 1 day after Vaccination #3)  
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 171 (±1 day; 3 days after Vaccination #3) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 5 mL of blood for CBC with differential and platelet count, ALT, 
creatinine. 
 
Study Day 175 (±2 days; 7 days after Vaccination #3) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 


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Study Day 182 (±3 days; 14 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 11 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, SMFA, and anti-Pfs25M and anti-Pfs230 
antibody ELISA. 
 
Study Day 196 (±7 days; 28 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 6 mL of blood for malaria blood smear, malaria PCR, SMFA, anti-
Pfs25M and anti-Pfs230 antibody ELISA.  
 
Study Day 224 (±7 days; 56 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 6 mL of blood for malaria blood smear, malaria PCR, SMFA, and anti-
Pfs25M and anti-Pfs230 antibody ELISA. 
 
Study Day 252 (±14 days; 84 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 280 (±21 days; 112 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable.  
4. Obtain approximately 6 mL of blood for malaria blood smear, malaria PCR, SMFA, and anti-
Pfs25M and anti-Pfs230 antibody ELISA. 
 
Study Day 308 (±21 days; 140 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 336 (±21 days; 168 days after Vaccination #3) 


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1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable.  
4. Obtain approximately 6 mL of blood for malaria blood smear, malaria PCR, SMFA, and anti-
Pfs25M and anti-Pfs230 antibody ELISA. 
 
Re-enrollment visits: 
 
Study Day 420 (-14/+90 days; 252 days after Vaccination #3);Re-screening 
1. Explain the study and informed consent document to the subject. 
2. Ensure the subject has acknowledged consent by signing or fingerprinting the informed 
consent document. Ensure that the subject receives a signed copy of the informed consent. 
3. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
4. Record vital signs (blood pressure, temperature, and heart rate). 
5. Record AEs and concomitant medications, if applicable. 
6. Obtain approximately 6 mL of blood for blood for malaria blood smear, malaria PCR, 
SMFA, and anti-Pfs230 antibody ELISA, 
 
Study Day 532 (+42 days; 364 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 6 mL of blood for blood for malaria blood smear, malaria PCR, 
SMFA, and anti-Pfs230 antibody ELISA 
5. Offer ENGERIX-B (or other approved Hepatitis B vaccine) to those subjects that received 
TBV(s) during the study. 
 
 
 
 


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Bamako/Sotuba Cohort – Arms 1b, 2b, 3b, 4a, 4b (3 Vaccinations; safety 
cohort) 
 
Study Day 0 (day of Vaccination #1) 
The following evaluations/procedures will be completed before vaccination: 
1. Verify that informed consent was obtained.  
2. Ensure that all inclusion/exclusion criteria are met.  
3. Ensure that CBC, ALT, creatinine, HIV/HCV/HBV, and urine results from screening are 
within protocol-defined limits (see Exclusion Criteria above) before vaccinating.  
4. Perform interim history and physical examination, focusing on any acute complaints.  
5. Record vital signs (blood pressure, temperature, and heart rate). 
6. During the physical examination, study staff will educate the subject regarding signs and 
symptoms of potential adverse events (AEs) and indications for use of antipyretics if fever, 
headache, or malaise occurs. 
7. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before vaccinating; a positive test will exclude the subject from the trial.  
8. For females, ensure agreement and compliance with pregnancy prevention before 
vaccinating. 
9. Obtain approximately 111 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs25M and anti-Pfs230 antibody ELISA, 
standard membrane feeding assay (SMFA), cellular assays, ex vivo assays, transcriptional 
assays, and B cell studies.  
 
NOTE: This initial blood draw prior to vaccination, given requested large volume of 
PBMCs, can be broken up into two visits prior to vaccination. 
10. Confirm that all criteria for vaccination have been met according to inclusion/exclusion 
criteria 
11. Confirm continued eligibility to receive vaccination  
12. Notify subject of vaccine assignment. 
13. Administer the vaccine.  
 
The following evaluations/procedures will be completed following vaccination: 
1. Observe the subject for at least 30 minutes after vaccination to evaluate immediate adverse 
reactions.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Perform interim history and physical examination, focusing on any acute complaints.  
4. Record AEs and concomitant medications, if applicable. 
 
Study Day 1 (±0 days; 1 day after Vaccination #1)  
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 40 mL of blood for cellular assays, ex vivo assays, transcriptional 
assays, and B cell studies.  
 
Study Day 3 (±1 day; 3 days after Vaccination #1) 


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1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 5 mL of blood for CBC with differential and platelet count, ALT, 
creatinine. 
 
Study Day 7 (±2 days; 7 days after Vaccination #1) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 14 (±3 days; 14 days after Vaccination #1) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 16 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, and anti-Pfs25M and anti-Pfs230 antibody 
ELISA, and SMFA. 
 
Study Day 28 (day of Vaccination #2) 
The following evaluations/procedures will be completed before vaccination: 
1. Perform interim history and physical examination, focusing on any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. During the physical examination, study staff will educate the subject regarding signs and 
symptoms of potential adverse events (AEs) and indications for use of antipyretics if fever, 
headache, or malaise occurs. 
4. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before vaccinating; a positive test will exclude the subject from the trial.  
5. For females, ensure agreement and compliance with pregnancy prevention before 
vaccinating. 
6. Obtain approximately 16 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs25M and anti-Pfs230 antibody ELISA, 
and standard membrane feeding assay (SMFA).  
7. Confirm continued eligibility to receive vaccination  
8. Administer the vaccine.  
 
The following evaluations/procedures will be completed following vaccination: 
1. Observe the subject for at least 30 minutes after vaccination to evaluate immediate adverse 
reactions.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Perform interim history and physical examination, focusing on any acute complaints.  
4. Record AEs and concomitant medications, if applicable. 
 
Study Day 29 (±0 days; 1 day after Vaccination #2)  


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1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 31 (±1 day; 3 days after Vaccination #2) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 5 mL of blood for CBC with differential and platelet count, ALT, 
creatinine. 
 
Study Day 35 (±2 days; 7 days after Vaccination #2) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 80 mL of blood for cellular assays, ex vivo assays, transcriptional 
assays,  and B cell studies. 
 
Study Day 42 (±3 days; 14 days after Vaccination #2) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 16 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, SMFA, and anti-Pfs25M and anti-Pfs230 
antibody ELISA. 
 
Study Day 56 (±7 days; 28 days after Vaccination #2)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 84 (±14 days; 56 days after Vaccination #2)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 112 (±21 days; 84 days after Vaccination #2) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 


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3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 11 mL of blood for malaria blood smear, malaria PCR, SMFA, and 
anti-Pfs25M and anti-Pfs230 antibody ELISA. 
 
 
Study Day 140 (±21 days; 112 days after Vaccination #2) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 168 (day of Vaccination #3) 
The following evaluations/procedures will be completed before vaccination: 
1. Perform interim history and physical examination, focusing on any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. During the physical examination, study staff will educate the subject regarding signs and 
symptoms of potential adverse events (AEs) and indications for use of antipyretics if fever, 
headache, or malaise occurs. 
4. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before vaccinating; a positive test will exclude the subject from the trial.  
5. For females, ensure agreement and compliance with pregnancy prevention before 
vaccinating. 
6. Obtain approximately 16 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs25M and anti-Pfs230 antibody ELISA, 
SMFA.  
7. Confirm continued eligibility to receive vaccination  
8. Administer the vaccine.  
 
The following evaluations/procedures will be completed following vaccination: 
1. Observe the subject for at least 30 minutes after vaccination to evaluate immediate adverse 
reactions.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Perform interim history and physical examination, focusing on any acute complaints.  
4. Record AEs and concomitant medications, if applicable. 
 
Study Day 169 (±0 days; 1 day after Vaccination #3)  
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 171 (±1 day; 3 days after Vaccination #3) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 


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4. Obtain approximately 5 mL of blood for CBC with differential and platelet count, ALT, 
creatinine. 
 
Study Day 175 (±2 days; 7 days after Vaccination #3) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 80 mL of blood for cellular assays, ex vivo assays, transcriptional 
assays, and B cell studies. 
 
Study Day 182 (±3 days; 14 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 51 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs25M and anti-Pfs230 antibody ELISA, 
SMFA, and cellular assays, ex vivo assays, transcriptional assays, and B cell studies. 
 
Study Day 196 (±7 days; 28 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 41 mL of blood for malaria blood smear, malaria PCR, SMFA, anti-
Pfs25M and anti-Pfs230 antibody ELISA, and cellular assays, ex vivo assays, transcriptional 
assays, and B cell studies 
 
Study Day 224 (±7 days; 56 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 11 mL of blood for malaria blood smear, malaria PCR, SMFA, and 
anti-Pfs25M and anti-Pfs230 antibody ELISA. 
 
Study Day 252 (±14 days; 84 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 280 (±21 days; 112 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 


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3. Record AEs and concomitant medications, if applicable.  
4. Obtain approximately 11 mL of blood for malaria blood smear, malaria PCR, SMFA, and 
anti-Pfs25M and anti-Pfs230 antibody ELISA. 
 
Study Day 308 (±21 days; 140 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 336 (±21 days; 168 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable.  
4. Obtain approximately 41 mL of blood for malaria blood smear, malaria PCR, SMFA, anti-
Pfs25M and anti-Pfs230 antibody ELISA, and cellular assays and B cell studies. 
 
Re-enrollment visits: 
 
Study Day 420 (-14 /+90 days; 252 days after Vaccination #3);Re-screening 
1. Explain the study and informed consent document to the subject. 
2. Ensure the subject has acknowledged consent by signing or fingerprinting the informed 
consent document. Ensure that the subject receives a signed copy of the informed consent. 
3. Perform basic history and focused physical examination emphasizing examination of any 
acute complaints. 
4. Record vital signs (blood pressure, temperature, and heart rate). 
5. Record AEs and concomitant medications, if applicable. 
6. Obtain approximately 6 mL of blood for blood for malaria blood smear, malaria PCR, 
SMFA, and anti-Pfs230 antibody ELISA, 
 
Study Day 532 (+42 days; 364 days after Vaccination #3) 
1. Perform basic history and focused physical examination emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 6 mL of blood for blood for malaria blood smear, malaria PCR, 
SMFA, and anti-Pfs230 antibody ELISA 
5. Offer ENGERIX-B (or other approved Hepatitis B vaccine) to those subjects that received 
TBV(s) during the study. 
 


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Bancoumana and Doneguebougou Cohort (TBV at full and fractional dosing 
versus Comparator) – Arms 2c, 2d, 4c (3 Vaccinations) 
 
Study Day -7 (±7 day; Drug Treatment Visit; -7 prior to Vaccination #1) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before vaccinating; a positive test will exclude the subject from the trial.  
5. For females, ensure agreement and compliance with pregnancy prevention before 
vaccinating. 
6. Obtain approximately 27 mL of blood for  CBC with differential and platelet count, ALT, 
creatinine, hemoglobin typing, malaria blood smear, malaria PCR, cellular assays and B cell 
assays, and transcriptional analysis. 
7. Administer Coartem (6 dosing timepoints per package insert) 
 
Study Day 0 (day of Vaccination #1) 
The following evaluations/procedures will be completed before vaccination: 
1. Verify that informed consent was obtained.  
2. Ensure that all inclusion/exclusion criteria are met.  
3. Ensure that CBC, ALT, creatinine, HIV/HCV/HBV, and urine results from screening are 
within protocol-defined limits (see Exclusion Criteria above) before vaccinating.  
4. Perform interim history and physical examination, focusing on any acute complaints.  
5. Record vital signs (blood pressure, temperature, and heart rate). 
6. During the physical examination, study staff will educate the subject regarding signs and 
symptoms of potential adverse events (AEs) and indications for use of antipyretics if fever, 
headache, or malaise occurs. 
7. Obtain urine sample for schistosomiasis testing  
8. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before vaccinating; a positive test will exclude the subject from the trial.  
9. For females, ensure agreement and compliance with pregnancy prevention before 
vaccinating. 
10. Obtain stool sample for helminth testing (Note: This can be completed within +/- 7 days of 
Study Day 0 if unable to provide on Study Day 0) 
11. Obtain approximately 57 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA, cellular assays, 
ex vivo assays, B cell assays, and transcriptional analysis.  
12. Confirm that all criteria for vaccination have been met according to inclusion/exclusion 
criteria 
13. Confirm continued eligibility to receive vaccination  
14. Assign study ID number from randomization list 
15. Administer the vaccine.  
 
The following evaluations/procedures will be completed following vaccination: 


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1. Observe the subject for at least 30 minutes after vaccination to evaluate immediate adverse 
reactions.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Perform interim history and physical examination, focusing on any acute complaints.  
4. Record AEs and concomitant medications, if applicable. 
 
Study Day 1 (±0 days; 1 day after Vaccination #1)  
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 3 (±1 day; 3 days after Vaccination #1) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 16 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, ex vivo assays, cellular assays, ex vivo assays, and B cell assays, and 
transcriptional analysis. 
 
Study Day 7 (±2 days; 7 days after Vaccination #1) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
  
Study Day 14 (±3 days; 14 days after Vaccination #1) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 11 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, and anti-Pfs230 antibody ELISA. 
 
Study Day 28 (day of Vaccination #2) 
The following evaluations/procedures will be completed before vaccination: 
1. Perform interim history and physical examination, focusing on any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. During the physical examination, study staff will educate the subject regarding signs and 
symptoms of potential adverse events (AEs) and indications for use of antipyretics if fever, 
headache, or malaise occurs. 
4. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before vaccinating; a positive test will exclude the subject from the trial.  
5. For females, ensure agreement and compliance with pregnancy prevention before 
vaccinating. 


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6. Obtain approximately 12 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA, and 
transcriptional analysis.  
7. Confirm continued eligibility to receive vaccination  
8. Confirm subject identification and study ID number 
9. Administer the vaccine.  
 
The following evaluations/procedures will be completed following vaccination: 
1. Observe the subject for at least 30 minutes after vaccination to evaluate immediate adverse 
reactions.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Perform interim history and physical examination, focusing on any acute complaints.  
4. Record AEs and concomitant medications, if applicable. 
 
Study Day 29 (±0 days; 1 day after Vaccination #2)  
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 31 (±1 day; 3 days after Vaccination #2) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 16 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, cellular assays, B cell assays, and transcriptional analysis. 
 
Study Day 35 (±2 days; 7 days after Vaccination #2) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 20 mL of blood for cellular assays, ex vivo assays, B cell assays. 
 
Study Day 42 (±3 days; 14 days after Vaccination #2) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 11 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, and anti-Pfs230 antibody ELISA. 
5. If gametocytemic, subject may be offered to participate in experimental hut. 
 
Study Day 56 (±7 days; 28 days after Vaccination #2)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  


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2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
 
Study Day 84 (±7 days; 56 days after Vaccination #2)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 112 (±14 days; 84 days after Vaccination #2) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 6 mL of blood for malaria blood smear, malaria PCR, and anti-Pfs230 
antibody ELISA. 
5. If gametocytemic, subject may be offered to participate in experimental hut. 
 
Study Day 140 (±14 days; 112 days after Vaccination #2) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 168 (day of Vaccination #3) 
The following evaluations/procedures will be completed before vaccination: 
1. Perform interim history and physical examination, focusing on any acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. During the physical examination, study staff will educate the subject regarding signs and 
symptoms of potential adverse events (AEs) and indications for use of antipyretics if fever, 
headache, or malaise occurs. 
4. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before vaccinating; a positive test will exclude the subject from the trial.  
5. For females, ensure agreement and compliance with pregnancy prevention before 
vaccinating. 
6. Obtain approximately 12 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, anti-Pfs230 antibody ELISA, and 
transcriptional analysis.   
7. Confirm continued eligibility to receive vaccination  
8. Administer the vaccine.  
 
The following evaluations/procedures will be completed following vaccination: 
1. Observe the subject for at least 30 minutes after vaccination to evaluate immediate adverse 
reactions.  
2. Record vital signs (blood pressure, temperature, and heart rate). 


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3. Perform interim history and physical examination, focusing on any acute complaints.  
4. Record AEs and concomitant medications, if applicable. 
 
Study Day 169 (±0 days; 1 day after Vaccination #3)  
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 171 (±1 day; 3 days after Vaccination #3) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 16 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, cellular assays and B cell assays, and transcriptional analysis. 
 
Study Day 175 (±2 days; 7 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 48 mL of blood for hemoglobin, malaria blood smear, malaria PCR, 
and anti-Pfs230 antibody ELISA, cellular assays, ex vivo assays, B cells assays, and 
transcriptional analysis. 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before completing feed.  
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 178 (±2 days; 10 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear, and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF 
 
Study Day 182 (±2 days; 14 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 28 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, malaria blood smear, malaria PCR, SMFA, and anti-Pfs25M and anti-Pfs230 
antibody ELISA. 


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5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before completing feed.  
6. Feeding evaluations (DSF) will also be performed at this visit. 
a) Confirm eligible for DSF 
 
Study Day 185 (±2 days; 17 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear, and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 189 (±2 days; 21 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear, and malaria PCR. 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before completing feed.  
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 192 (±2 days; 24 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 196 (±2 days; 28 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 48 mL of blood for malaria blood smear, malaria PCR, SMFA, cellular 
assays, ex vivo assays, B cells assays and anti-Pfs230 antibody ELISA and transcriptional 
analysis. 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before completing feed.  
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 


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Study Day 199 (±2 days; 31 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 203 (±2 days; 35 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before completing feed.  
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 206 (±2 days; 38 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 210 (±2 days; 42 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 8 mL of blood for malaria blood smear, malaria PCR, SMFA, and anti-
Pfs230 antibody ELISA, and transcriptional analysis. 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before completing feed.  
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 213 (±2 days; 45 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 


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5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 217 (±2 days; 49 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before completing feed.  
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 220 (±2 days; 52 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 224 (±2 days; 56 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 8 mL of blood for malaria blood smear, malaria PCR, SMFA, and anti-
Pfs230 antibody ELISA, and transcriptional analysis. 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before completing feed.  
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 227 (±2 days; 59 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 231 (±2 days; 63 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  


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2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 234 (±2 days; 66 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 238 (±2 days; 70 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 8 mL of blood for malaria blood smear, malaria PCR, SMFA, and anti-
Pfs230 antibody ELISA, and transcriptional analysis.. 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before completing feed.  
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 241 (±2 days; 73 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 245 (±2 days; 77 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 248 (±2 days; 80 days after Vaccination #3)  


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1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 252 (±2 days; 84 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 8 mL of blood for malaria blood smear, malaria PCR, SMFA, and anti-
Pfs230 antibody ELISA, and transcriptional analysis. 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before completing feed.  
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 255 (±2 days; 87 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a) Confirm eligible for DSF  
 
Study Day 280 (±14 days; 112 days after Vaccination #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 7 mL of blood for malaria blood smear, malaria PCR, SMFA, and anti-
Pfs230 antibody ELISA. 
 
Study Day 308 (±21 days; 140 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 1 mL of blood for malaria blood smear and malaria PCR. 
 
Study Day 336 (±21 days; 168 days after Vaccination #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  


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2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable.  
4. Obtain approximately 47 mL of blood for malaria blood smear, malaria PCR, SMFA, and 
anti-Pfs230 antibody ELISA, and cellular assays, ex vivo assays, B cell assays and 
transcriptional analysis. 
5. Scheduled unblinding of all subjects 
a. Offer ENGERIX-B (or other approved Hepatitis B vaccine) to those subjects that 
received TBV(s) during the study. 
 
Re-enrollment visits 
 
Study Day 462 +/-70 days (294 days after Vaccination #3);Re-screening 
1. Explain the study and informed consent document to the subject. 
2. Ensure the subject has acknowledged consent by signing or fingerprinting the informed 
consent document. Ensure that the subject receives a signed copy of the informed consent. 
3. Elicit a complete medical history, including menstrual and contraceptive history and/or 
history of surgical sterility for females, and medication use. 
4. Females of childbearing potential must be willing to use reliable contraception from at least 
21 days prior to fourth vaccination through three months after the fourth vaccination. 
5. Administer a complete physical examination, including vital signs (height, weight, blood 
pressure, temperature, and heart rate). 
6. HIV pre- and post-test counseling as indicated including follow-up contact with subject to 
report the results and referral for appropriate medical care if indicated. 
7. Obtain approximately 16 mL of blood for complete blood count (CBC) with differential and 
platelet count, ALT, creatinine (Cr), hepatitis B surface antigen, hepatitis C antibody, HIV 
antibody, malaria blood smear, malaria PCR, SMFA, and anti-Pfs230 antibody ELISA. 
8. Obtain urine (or serum) for pregnancy testing (for females) and urinalysis/urine dipstick for 
protein and blood. 
 
 
Study Day 476 ±56 days (308 days after Vaccination #3; Day of Vaccination #4) 
The following evaluations/procedures will be completed before vaccination: 
1. Verify that informed consent was obtained.  
2. Ensure that all inclusion/exclusion criteria are met.  
3. Ensure that CBC, ALT, creatinine, HIV/HCV/HBV, and urine results from re-screening are 
within protocol-defined limits (see Exclusion Criteria above) before vaccinating.  
4. Perform interim history and physical examination, focusing on any acute complaints.  
5. Record vital signs (blood pressure, temperature, and heart rate). 
6. During the physical examination, study staff will educate the subject regarding signs and 
symptoms of potential adverse events (AEs) and indications for use of antipyretics if fever, 
headache, or malaise occurs. 
7. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before vaccinating; a positive test will exclude the subject from the trial.  
8. For females, ensure agreement and compliance with pregnancy prevention before 
vaccinating. 


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9. Obtain approximately 32 mL of blood for malaria blood smear, malaria PCR, SMFA, and 
anti-Pfs230 antibody ELISA, and cellular assays, ex vivo assays, B cell assays and 
transcriptional analysis. 
10. Confirm that all criteria for vaccination have been met according to inclusion/exclusion 
criteria, including reviewing safety labs prior to vaccination. 
11. Confirm continued eligibility to receive vaccination  
12. Administer the vaccine.  
 
The following evaluations/procedures will be completed following vaccination: 
1. Observe the subject for at least 30 minutes after vaccination to evaluate immediate adverse 
reactions.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Perform interim history and physical examination, focusing on any acute complaints.  
4. Record AEs and concomitant medications, if applicable. 
 
Study Day 477 ±0 days (1 day after Vaccination #4)  
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
 
Study Day 479 ±1 day (3 days after Vaccination #4) 
1. Perform basic history and focused physical examination (including injection site), 
emphasizing examination of any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 16 mL of blood for CBC with differential and platelet count, ALT, 
creatinine, cellular assays and B cell assays, and transcriptional analysis. 
 
Study Day 483 ±2 days (7 days after Vaccination #4) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 23 mL of blood for cellular assays, ex vivo assays, B cells assays, and 
transcriptional analysis. 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before completing feed.  
6. Feeding evaluations (DSF) will also be performed at this visit. 
a) Confirm eligible for DSF 
 
Study Day 486 ±2 days (10 days after Vaccination #4) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 


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5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF 
 
Study Day 490 ±3 days (14 days after Vaccination #4) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 17 mL of blood for malaria blood smear, malaria PCR, CBC with 
differential and platelet count, ALT, creatinine, SMFA, and anti-Pfs230 antibody ELISA and 
transcriptional analysis. 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before completing feed.  
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF 
 
Study Day 493 ±2 days (17 days after Vaccination #4) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF 
 
Study Day 497 ±2 days (21 days after Vaccination #4) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before completing feed.  
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF 
 
Study Day 501 ±2 days (25 days after Vaccination #4) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF 
 
 


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Study Day 504±2 days (28 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 33 mL of blood for malaria blood smear, malaria PCR, SMFA, and 
anti-Pfs230 antibody ELISA, and cellular assays, ex vivo assays, B cell assays and 
transcriptional analysis. 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before completing feed.  
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF 
 
Study Day 507 ±2 days (31 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear, and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 511 ±2 days (35 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear, and malaria PCR. 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before completing feed.  
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 514 ±2 days (38 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 518 ±2 days (42 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 


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4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before completing feed.  
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 521 ±2 days (45 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 525 ±2 days (49 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before completing feed.  
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 528 ±2 days (52 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 532±2 days (56 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 7 mL of blood for malaria blood smear, malaria PCR, SMFA, and anti-
Pfs230 antibody ELISA 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before completing feed.  
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 


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Study Day 535 ±2 days (59 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 539 ±2 days (63 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before completing feed.  
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 542 ±2 days (66 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 546 ±2 days (70 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before completing feed.  
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 549 ±2 days (73 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 


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a. Confirm eligible for DSF  
 
 
Study Day 553 ±2 days (77 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before completing feed.  
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 556 ±2 days (80 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 560 ±2 days (84 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 7 mL of blood for malaria blood smear and malaria PCR, SMFA, and 
anti-Pfs230 antibody ELISA. 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before completing feed.  
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 563 ±2 days (87 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 567 ±2 days; (91 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  


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2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before completing feed.  
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 570 ±2 days; (94 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 574 ±2 days; (98 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before completing feed.  
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 577 ±2 days; (101 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 581 ±2 days; (105 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is negative 
before completing feed.  
6. Feeding evaluations (DSF) will also be performed at this visit. 


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a. Confirm eligible for DSF  
 
Study Day 584 ±2 days; (108 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
Study Day 588 ±2 days; (112 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. For females, obtain a urine or serum sample for β-hCG testing. Ensure that the test is 
negative before completing feed.  
5. Obtain approximately 7mL of blood for malaria blood smear and malaria PCR, SMFA, and 
anti-Pfs230 antibody ELISA. 
6. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
 
Study Day 591 ±2 days; (115 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 2 mL of blood for malaria blood smear and malaria PCR. 
5. Feeding evaluations (DSF) will also be performed at this visit. 
a. Confirm eligible for DSF  
 
 
 
Study Day 616 ±2 days; (140 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 11 mL of blood for malaria blood smear and malaria PCR, SMFA, and 
anti-Pfs230 antibody ELISA. 
 
Study Day 644 ±21 days; (168 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  


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2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 52 mL of blood for malaria blood smear, malaria PCR, SMFA, and 
anti-Pfs230 antibody ELISA, cellular assays, ex vivo assays, B cells assays and transcriptional 
analysis. 
 
 
Study Day 728 ±21 days; (252 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 11 mL of blood for malaria blood smear and malaria PCR, SMFA, and 
anti-Pfs230 antibody ELISA. 
 
Study Day 812±21 days; (336 days after Vaccination #4)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Obtain approximately 52 mL of blood for malaria blood smear, malaria PCR, SMFA, and 
anti-Pfs230 antibody ELISA, cellular assays, ex vivo assays, B cells assays and 
transcriptional analysis. 
5. Offer Menactra or equivalent vaccine 
 
 


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Appendix D:  Toxicity Table 
Local Reactogenicity Grading1 
Local Reaction to 
Injectable Product 
Mild  
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially 
Life 
Threatening 
(Grade 4) 
Pain 
Does not 
interfere 
with activity 
Repeated use of 
non-narcotic 
pain reliever > 
24 hours or 
interferes with 
activity 
Any use of 
narcotic pain 
reliever or 
prevents daily 
activity 
Emergency 
room 
(ER) visit or 
hospitalization 
Tenderness 
Mild 
discomfort to 
touch 
Discomfort with 
movement 
Significant 
discomfort at 
rest 
ER visit or 
hospitalization 
Erythema/Redness2 
2.5 – 5 cm 
5.1 – 10 cm 
> 10 cm 
Necrosis or 
exfoliative 
dermatitis 
Induration/Swelling3  
2.5 – 5 cm and 
does not 
interfere 
with activity 
5.1 – 10 cm or 
interferes with 
activity 
> 10 cm or 
prevents daily 
activity 
Necrosis 
Pruritus at Site 
Does not 
interfere 
with activity 
Repeated use of 
medication > 24 
hours or 
interferes with 
activity 
Prevents daily 
activity 
ER visit or 
hospitalization 
Limitation of Arm 
Movement 
Does not 
interfere 
with activity 
Repeated use of 
medication > 24 
hours or 
interferes with 
activity 
Prevents daily 
activity 
ER visit or 
hospitalization 
1 The definitions provided in the table are taken from the FDA Guidance for Industry “Toxicity Grading Scale for 
Healthy Adults and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials” dated September 2007. 
2 In addition to grading the measured local reaction at the greatest single diameter, the measurement should be 
recorded as a continuous variable.  
2 Induration/Swelling should be evaluated and graded using the functional scale as well as the actual measurement. 
 
 


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Vital Sign AE Grading1 
Vital Signs2  
Mild  
(Grade 1) 
Moderate 
(Grade 2) 
Severe  
(Grade 3) 
Potentially Life 
Threatening 
(Grade 4) 
Fever3 (°C) 
           (°F) 
38.0 – 38.4 
100.4 – 101.1 
38.5 – 38.9 
101.2 – 102.0 
39.0 – 40 
102.1 – 104 
> 40 
> 104 
Tachycardia - 
beats per 
minute 
101 – 115 
116 – 130 
> 130 
ER visit or 
hospitalization 
for 
arrhythmia 
Bradycardia - 
beats per 
Minute4 
50 – 54 
45 – 49 
< 45 
ER visit or 
hospitalization 
for 
arrhythmia 
Hypertension 
(systolic) - 
mm Hg 
141 – 150 
151 – 155 
> 155 
ER visit or 
hospitalization 
for 
malignant 
hypertension 
Hypertension 
(diastolic) - 
mm Hg 
91 – 95 
96 – 100 
> 100 
ER visit or 
hospitalization 
for 
malignant 
hypertension 
Hypotension 
(systolic) – 
mm Hg 
85 – 89 
80 – 84 
< 80 
ER visit or 
hospitalization 
for 
hypotensive 
shock 
1 The definitions provided in the table are taken from the FDA Guidance for Industry “Toxicity Grading Scale for 
Healthy Adults and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials” dated September 2007. 
2 Subject should be at rest for all vital sign measurements.  
2 Oral temperature; no recent hot or cold beverages or smoking.  
3 When resting heart rate is between 60 – 100 beats per minute. Use clinical judgment when characterizing  
bradycardia among some healthy subject populations, for example, conditioned athletes. 
 
 


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Systemic AE Grading1 
Systemic AEs 
Mild  
(Grade 1) 
Moderate 
(Grade 2) 
Severe  
(Grade 3) 
Potentially 
Life 
Threatening 
(Grade 4) 
Diarrhea 
2 – 3 loose 
stools or 
< 400 gms/24 
hours 
4 – 5 stools or 
400 – 800 gms/24 
hours 
6 or more watery 
stools or 
> 800gms/24 hours 
or requires 
outpatient IV 
hydration 
ER visit or 
hospitalization 
Headache 
No 
interference 
with 
activity 
Repeated use of non- 
narcotic pain 
reliever > 24 hours or 
some interference 
with activity 
Significant; any use 
of narcotic 
pain reliever or 
prevents daily 
activity 
ER visit or 
hospitalization 
Malaise 
No 
interference 
with 
activity 
Some interference 
with activity 
Significant; prevents 
daily activity 
ER visit or 
hospitalization 
Myalgia 
No 
interference 
with 
activity 
Some interference 
with activity 
Significant; prevents 
daily activity 
ER visit or 
hospitalization 
Arthralgia 
No 
interference 
with 
activity 
Some interference 
with activity 
Significant; prevents 
daily activity 
ER visit or 
hospitalization 
Nausea/ 
Vomiting 
No 
interference 
with activity 
or 1 – 2 
episodes/24 
hours 
Some interference 
with activity or > 2 
episodes/24 hours 
Prevents daily 
activity, requires 
outpatient IV 
hydration 
ER visit or 
hospitalization 
for 
hypotensive 
shock 
Urticaria 
No 
interference 
with 
activity 
Requiring PO or 
topical treatment  > 24 
hours or IV 
medications or 
steroids for ≤24 hours 
Requiring IV 
medication or 
steroids for >24 hours 
ER visit or 
hospitalization 
1 The definitions provided in the table are taken from the FDA Guidance for Industry “Toxicity Grading Scale for 
Healthy Adults and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials” dated September 2007. 


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Mali Laboratory AE Grading 
Hematology/Chemistry 
 
Hematology and 
Biochemistry values1, 2 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially 
Life 
Threatening 
(Grade 4) 
Hemoglobin (Male) - 
gm/dL  
9.5 – 10.5 
8.0 – 9.4 
6.5 – 7.9 
< 6.5 and / or 
requiring 
transfusion 
Hemoglobin (Female)  
gm/dL  
8.0 – 9.0 
7.0 – 7.9 
6.0 – 6.9 
< 6 and /or 
requiring 
transfusion 
WBC Increase – 103/µL 
11.5 – 15.0 
15.1 – 20.0 
20.1 – 25.0 
> 25.0 
WBC Decrease - 103/µL 
2.5 – 3.3 
1.5 – 2.4 
1.0 – 1.4 
< 1.0  
Neutrophil/Granulocyte 
Decrease3 - 103/µL 
0.80 – 1.00 
0.50 – 0.79 
< 0.50 
< 0.50 with 
fever 
Platelets Decreased – 
103/µL 
100 – 115 
70 – 99 
25 – 69 
< 25 
Creatinine (Male) 
µmol/L 
132.60 – 
159.11 
159.12 – 185.63 
185.64 – 
221.00 
> 221.00 and 
requires 
dialysis 
Creatinine (Female) 
µmol/L 
97.42 – 
116.94 
116.95 – 136.44 
136.45 – 
162.43 
> 162.43 and 
requires 
dialysis 
Liver Function Tests –
ALT U/L 
46.0 – 106.5 
106.6 – 209.0 
209.1 – 410.0 
> 410.0 
1 The laboratory values provided in the table are taken from the FDA Guidance for Industry “Toxicity Grading Scale for 
Healthy Adults and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials” dated September 2007.The laboratory 
values provided in the referenced tables serve as guidelines and have been adapted for institutional normal parameters (see 
below). Institutional normal reference ranges should be provided to demonstrate that they are appropriate.  
2The clinical signs or symptoms associated with laboratory abnormalities might result in characterization of the laboratory 
abnormalities as Potentially Life Threatening (Grade 4). For example, a low sodium value that falls within a grade 3 parameter 
should be recorded as a grade 4 hyponatremia event if the subject had a new seizure associated with the low sodium value.  
3 Note, neutropenias are graded and followed, but based on previous experience in African populations, should be interpreted 
with caution since lower values are more frequently observed in people of African descent 46,47.  
 
 


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Mali Adult: Institutional Normal Parameters  
 Chemistry 
Serum1 
Reference Range 
Creatinine (Female) 
- µmol/L 
< 72 
 
Creatinine (Male) -
µmol/L 
48 – 98 
 
ALT – U/L 
 
< 41 
1The laboratory values provided in the table are based on Bancoumana, Malian adults (age 18-45 years old) 
 
Hematology 
Hematology1 
Reference 
Range 
Hemoglobin (Female) - 
gm/dL 
9.1 – 13.8 
Hemoglobin (Male) - 
gm/dL 
10.8 – 15.8 
WBC – 103/µL 
3.6 – 9.0 
Absolute 
Neutrophil/Granulocyte 
Count - 103/µL 
1.3 – 4.4 
Absolute Lymphocyte 
Count - 103/µL 
1.3 – 4.4 
Platelet Count 
(Female)- 103/µL 
144 – 413 
Platelet Count (Male)- 
103/µL 
114 – 335 
1The laboratory values provided in the table are based on Bancoumana, Malian adults (age 18-45 years old) 
 
Urine Dip/Urinalysis 
Urine1 
Reference Ranges 
Protein 
None or Trace 
Blood 
(microscopic) – 
red blood cells per 
high power field 
(RBC/HPF) 
None or Trace 
 
< 5 
1The laboratory values provided in the table are based on Bancoumana, Malian adults (age 18-45 years old) 


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Appendix E: References 
 
1. 
WHO. World malaria report. Geneva, Switzerland: World Health Organization;2015. 
2. 
Sachs J, Malaney P. The economic and social burden of malaria. Nature. 
2002;415(6872):680-685. 
3. 
Agnandji ST, Lell B, Soulanoudjingar SS, et al. First results of phase 3 trial of 
RTS,S/AS01 malaria vaccine in African children. The New England journal of medicine. 
2011;365(20):1863-1875. 
4. 
Rts SCTP, Agnandji ST, Lell B, et al. A phase 3 trial of RTS,S/AS01 malaria vaccine in 
African infants. The New England journal of medicine. 2012;367(24):2284-2295. 
5. 
Birkett AJ. PATH Malaria Vaccine Initiative (MVI): perspectives on the status of malaria 
vaccine development. Human vaccines. 2010;6(1):139-145. 
6. 
Greenwood B, Targett G. Do we still need a malaria vaccine? Parasite immunology. 
2009;31(9):582-586. 
7. 
Kaslow DC, Quakyi IA, Syin C, et al. A vaccine candidate from the sexual stage of 
human malaria that contains EGF-like domains. Nature. 1988;333(6168):74-76. 
8. 
Kaslow DC. Transmission-blocking vaccines. Chemical immunology. 2002;80:287-307. 
9. 
Kubler-Kielb J, Majadly F, Wu Y, et al. Long-lasting and transmission-blocking activity 
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