Conditions: Malaria
Keywords: Field Immunity Chemoprophylaxis with Sporozoites Vaccine Malaria-Experienced
Allocation: RANDOMIZED | Intervention model: SEQUENTIAL
Enrollment: 252
Healthy volunteers: yes

Brief summary:
Background:

Malaria remains a major global health problem. Malaria is spread by the bite of mosquitos. Africa is the region of the world where most people get malaria. Sanaria PfSPZ Challenge is a malaria vaccine. Researchers want to see if the vaccine combined with partner drugs can help protect against malaria.

Objective:

To test if injections with 3 monthly doses of Sanaria PfSPZ Challenge, combined with either pyrimethamine (PYR) or chloroquine as a partner drug, is safe, tolerable, and effective.

Eligibility:

Healthy people ages 18-50 years who live in Bancoumana, Mali, or nearby

Design:

Participants will be screened with the Malaria Comprehension Exam to check their understanding of the study. They will have a medical history. They will have a physical exam. They will have blood tests, urine tests, and heart tests.

Participants will join either the pilot study or the main study.

Participants will be assigned to groups. Depending on their group, they will get at least one injection of either a placebo or the vaccine. They may have up to 3 vaccines, 4 weeks apart. The injection will be into a vein with a needle.

Participants will also take pyrimethamine or chloroquine by mouth. They will also take standard doses of antimalarial drugs by mouth.

Participants will have blood tests throughout the study.

Participants may develop a rash or injection site reaction. If this happens, photos of the site may be taken.

Participants will be observed for infection for many days after the injections.

Detailed description:
Human studies have shown that immunization by the bite of Plasmodium falciparum (Pf) sporozoite (SPZ)-infected mosquitoes (chemoprophylaxis with sporozoites \[CPS\]) or by injecting aseptic, purified, cryopreserved sporozoites (SPZ) with needle and syringe (PfSPZ chemoprophylaxis vaccination, Sanaria PfSPZ-CVac) under drug coverage with chloroquine can provide \>80% protection against homologous controlled human malaria infection (CHMI) (Roestenberg, McCall et al. 2009, Roestenberg, Teirlinck et al. 2011, Mordmuller, Surat et al. 2017). The protective immunity induced by CPS/PfSPZ-CVac is thought to target sporozoite and liver stage antigens, but the transient parasitemia observed under chloroquine prophylaxis may additionally induce immune responses targeting blood stage parasites that contribute to protection. It is important to understand the contribution of each stage in this model as transient parasitemia in the immunization process adds a safety concern and may impair the development of immunity.

We have conducted two phase 1 studies to explore whether significant protective efficacy can be achieved with exclusively pre-erythrocytic (sporozoite/liver stage) exposure in this model using PYR chemoprophylaxis. This drug, unlike chloroquine, kills the parasites in the liver, so there is no period of transient parasitemia following immunization. These studies in malaria na(SqrRoot) ve adults demonstrated that PfSPZ-CVac (with PYR administered 2 \& 3 days after injection) is safe, well-tolerated, and can prevent subpatent and patent parasitemia after PfSPZ injection. The NCT02511054 study used the same dose (5.12x104 PfSPZ) that has resulted in \>80% protection in previous trials of PfSPZ-CVac (chloroquine), showed only 2/9 vaccinees were protected against a homologous (same strain of Pf as the vaccine) CHMI. In trials using irradiated SPZ, increasing the dose of PfSPZ used for immunization has resulted in higher protective efficacy. Therefore, in an ongoing trial, NCT03083847, we have increased the PfSPZ dose about 4-fold up to 2.0x105 PfSPZ, with preliminary results show markedly improved protection against both homologous and heterologous CHMI in healthy US adults (7/8 and 4/4 study subjects uninfected, respectively). This is the first demonstration that the PfSPZ-CVac approach can confer sterile protection without blood stage exposure.

Building on these promising results from PfSPZ-CVac (pyrimethamine) in the US, this proposed study in a malaria-endemic, adult population in Bancoumana, Mali will initially assess, in a pilot phase, the safety and tolerability of still higher doses of PfSPZ Challenge (4.0x105 PfSPZ) in combination with PYR (day 0;) or chloroquine (days -2, +5). The higher PfSPZ dose is hypothesized to be necessary to achieve significant efficacy against naturally transmitted malaria in malaria-experienced populations. Once this higher dose of PfSPZ is determined to be safe and tolerable and that earlier PYR dosing prevents patent parasitemia, the main phase will explore the effect of both earlier PYR dosing and a higher dosage of PfSPZ used for immunization in the development of protective immunity against natural malaria infection. The results of this study will contribute to understanding the targets and mechanisms of immunity against Pf malaria infection, and how the degree of prior exposure to the parasite (pre-erythrocytic and/or erythrocytic stages) impacts these responses and subsequent protective efficacy in field settings.

After completion of follow up in the main phase, all participants that are still enrolled in the study will be offered a booster dose of the vaccine (4th dose) with a single dose of 4x10\^5 PfSPZ Challenge or normal saline (aligned with what they received in the main phase of the study) at approximately 11 months post 3rd vaccination. The booster dose is timed to allow administration well before the beginning of the ensuing malaria transmission season. Participants will then be followed for approximately 6 months to assess safety and vaccine efficacy through this second transmission season.

Arms:
arm 1: Receiving 400,000 PfSPZ with 75 mg pyrimethamine day 0 post PfSPZ arm 2: Receiving 400,000 PfSPZ with 75 mg pyrimethamine day 0 post PfSPZ arm 3: Receiving 400,000 PfSPZ with 75 mg pyrimethamine days 2\&amp;3 post PfSPZ arm 4: Receiving 400,000 PfSPZ with 75 mg pyrimethamine days 2\&amp;3 post PfSPZ arm 5: Receiving 400,000 PfSPZ with chloroquine 2 days prior + 5 days post PfSPZibuprofen (if necessary) arm 6: Receiving 400,000 PfSPZ with weekly chloroquineibuprofen (if necessary) arm 7: Receiving normal saline with 75 mg pyrimethamine day 0 post injection arm 8: Receiving normal saline with 75 mg pyrimethamine days 2\&amp;3 post injection arm 9: Receiving normal saline with weekly chloroquineibuprofen (if necessary) arm 10: Receiving 300,000 PfSPZ with 75 mg pyrimethamine day 0 post PfSPZ (if necessary) arm 11: Receiving 300,000 PfSPZ with 75 mg pyrimethamine day 0 post PfSPZ arm 12: Receiving 300,000 PfSPZ with 75 mg pyrimethamine days 2\&amp;3 post PfSPZ (if necessary) arm 13: Receiving 300,000 PfSPZ with 75 mg pyrimethamine days 2\&amp;3 post PfSPZ (if necessary)

Interventions: intervention 1: Chloroquine Phosphate intervention 2: PfSPZ Challenge intervention 3: Pyrimethamine intervention 4: Coartem intervention 5: ibuprofen
Intervention descriptions: intervention 1: Chloroquine phosphate is approved for suppressive treatment (prophylaxis) and for acute attacks of malaria due to P. vivax, P. malariae, P. ovale, and susceptible strains of Pf. Chloroquine is a blood-stage schizonticide, highly active against replicating forms of blood-stage drug- sensitive parasites. As such, it is routinely employed as first-line prophylaxis against development of patent parasitemia and clinical malaria in non-immune travelers to areas with chloroquine sensitive Pf. In the pilot, chloroquine administration will begin with a loading dose 2 days prior to PfSPZ Challenge and finishing with a maintenance dose 5 days after PfSPZ Challenge. In the main phase, chloroquine will be first administered as a loading dose 2 days prior to PfSPZ Challenge and continuing weekly using a maintenance dose for a total of 10 doses, finishing with a last dose 5 days after the third injection with PfSPZ Challenge. intervention 2: PfSPZ Challenge (NF54) acts as the immunogen in the PfSPZ-CVac approach to vaccinating against malaria. Participants will receive either one (in the pilot phase) or 3 (in the main phase) 400,000 or 300,000 aseptic, purified, cryopreserved, fully infectious PfSPZ injections by DVI (with concurrent administration of antimalarial drugs). Following immunization, participants in the main phase will be followed through the transmission season in order to assess vaccine efficacy. intervention 3: Pyrimethamine has been FDA approved for acute treatment and chemoprophylaxis of malaria due to susceptible strains of plasmodia.

Pyrimethamine has been shown to possess both liver and blood stage activity. In this study, we plan to use a higher dose of pyrimethamine, 75 mg orally for either one or two days each month. The pilot arms will receive either a total of 1 daily dose or 2 daily doses. The main arms will receive a total of 3 daily doses or a total of 6 doses. intervention 4: Antimalarial (Coartem ) will be administered to all participants twice during the study, approximately 2 weeks prior to the first vaccination in order to clear pre-existing parasitemia that may interfere with immunity development. The second dose will be given approximately 2 weeks prior to the 3rd vaccination in order to clear any parasitemia infection that may have happened naturally during the vaccination period and prior to the follow up period. intervention 5: Ibuprofen likewise is not a part of the investigational product/regimen, but will be provided to participants in the chloroquine arm (Arm 4) if required to mitigate typical symptoms of malaria infection such as headache, myalgia, fever and chills.

Protocol document:
Prot_SAP_000.pdf: 
Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 1 of 146
Sanaria® PfSPZ Challenge with Pyrimethamine or Chloroquine Chemoprophylaxis 
Vaccination (PfSPZ-CVac Approach): A Randomized Double Blind Placebo 
Controlled Phase I/II Trial to Determine Safety and Protective Efficacy against 
Natural Plasmodium falciparum Infection in Bancoumana and Surrounding Areas, 
Mali
                                     NIH Protocol Number:  19-I-N099
Sanaria Protocol Number: MLSPZCV3
Project Assurance:  FWA #00005897
Multi-institution:  Yes
FMPOS Protocol Number: N02019/33/CE/FMPOS
Project Assurance: FWA #00001769
Principal Investigator:
Issaka Sagara 
(MRTC/DEAP/FMPOS/USTTB)
Patrick Duffy 
(LMIV/NIAID/NIH)
Sponsored by:
National Institute of Allergy and Infectious Diseases (NIAID)
Laboratory of Malaria Immunology and Vaccinology (LMIV)
National Institutes of Health (NIH)
IND Product Sponsor: 
Sanaria Inc. 
IND: 16650 
Clinical Trial Monitoring Delegated to NIAID DCR Office of Clinical Research Policy and 
Regulatory Operations
Version: 8.0
03 January 2022
CONFIDENTIAL


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 2 of 146
Team Roster
MRTC Study Staff
Principal Investigator:
Issaka Sagara MD, MSPH, PhD
MRTC/DEAP/FMPOS/USTTB
BP 1805, Point G
Bamako, Mali
Email: isagara@icermali.org
Tel: +223-2022-8109
Clinical Associate Investigators:
Mahamadou S. Sissoko, MD, MSPH, PhD 
Mahamadoun Hamady Assadou, MD, MPH 
Mamady Kone, MD
Kourane Sissoko, MD
Sibiri Sissoko, MD, MSc
Abdoulaye Katile, MD, MSPH
Bourama Kamate, MD
Amagana Dolo, PharmD, PhD
Amatigue Zeguimé, PharmD
Kadidia Baba Cisse, BSc
Kalifa Diarra, PharmD
Balla Diarra, PharmD
Karim Bengaly, PharmD
Amadou Niangaly, PharmD


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 3 of 146
NIH Study Staff
Principal Investigator:
Patrick Duffy, MD
LMIV/NIAID/NIH
29 Lincoln Drive
Building 29B, Room 4NN06
Bethesda, MD 20892
Patrick.Duffy@nih.gov
Tel: (301) 761-5089
Fax: (301) 480-1962
Clinical Associate Investigators:
Jacquelyn K. Lane PA-C
David Cook, MD
Sara Healy, MD, MPH
Aye Diallo, PA
John Woodford, MBBS
Role: The NIH study staff will work closely with the Malian investigators and share study 
responsibilities, including making decisions about eligibility. When in Mali, NIH 
investigators may see identifiable data; and as needed, may provide consultation on clinical 
diagnosis, review medical charts, research records, and informed consent documents. At 
the NIH, coded samples and data will be stored and analyzed; however, the key to the code 
will be maintained in Mali. NIH investigators will also participate in the writing and 
dissemination of reports and study findings.
Biostatistician:
Martha Nason, PhD
Biostatistics Research Branch/NIAID/NIH
5601 Fishers Lane
Room 4C21
Rockville, MD 20852
Tel: (240) 669-5251
Role: Dr. Nason will access, interpret, and analyze data sets without identifiers and will 
participate in the writing of reports and study findings.
Sponsor Collaborators:
Stephen L. Hoffman, MD (Sanaria)
Thomas L. Richie, MD PhD (Sanaria)
Sanaria Inc.
9800 Medical Center Dr. Rockville, 
MD 20850 USA
Tel: (240) 403-2701


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 4 of 146
Clinical Trial Site:
Mali, West Africa 
Malaria Vaccine Center
MRTC/DEAP/FMPOS/USTTB
Bancoumana, Mali
Institutional Review Boards:  
National Institutes of Health (NIH) IRB
Office of IRB Operations
Office of Intramural Research
National Institutes of Health
1 Cloister Court
Bldg. 60, Room 247
Bethesda, MD 20892
La Faculté de Médecine de Pharmacie et 
d'Odontostomatologie (FMPOS) Ethics Committee
P.O Box 1805
Bamako, Mali
Data and Safety Monitoring 
Board:
The DSMB will be chartered by IND Sponsor
Mali Site Medical Monitor:
Mamadou Dembele, MD
Service de Medicine Interne
Centre Hospitalo-Universitaire du Point G
Tel: + 223 2022 5003 
Mobile: +223 7604 9387
Email: hassiramadydembele@yahoo.fr
Laboratories
Immunology Laboratories:
LMIV/NIAID/NIH
29 Lincoln Drive 
Bethesda, Maryland 20892 USA
MRTC/DEAP Immunology Laboratory
FMPOS, University of Sciences, Techniques & 
Technologies, Bamako
BP 1805, Point G
Bamako, Mali


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 5 of 146
Clinical Laboratory:
MRTC/DEAP Clinical Laboratory, 
FMPOS, USTTB
BP 1805, Point G, Bamako, Mali
Rodolphe Mérieux Laboratory
                        Bamako, Mali
Collaborating Laboratories:
NIH Intramural Sequencing Center
National Human Genome Research Institute (NHGRI)
5625 Fishers Lane 
Room 5S-16C MSC9400
Rockville, MD 20852 USA
NIH Research Technologies Branch
Genomic Technologies Section
Building 50, Room 5509
50 South Drive, MSC 8005
Bethesda, MD 20892 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
Sanaria Inc.
9800 Medical Center Drive
Suite A209
Rockville, MD 20850
Molecular Epidemiology and Drug Resistance Unit 
USTTB 
BP 1805, Point G, Bamako, Mali


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 6 of 146
Table of Contents
Team Roster ..........................................................................................................................2
Table of Contents ..................................................................................................................6
List of Tables.......................................................................................................................10
List of Figures .....................................................................................................................11
List of Appendices...............................................................................................................12
List of Abbreviations...........................................................................................................13
Précis...................................................................................................................................25
1
Introduction and Rationale............................................................................................26
1.1
Malaria Epidemiology...........................................................................................26
1.2
Malaria Life Cycle and Vaccine Strategies............................................................26
1.3
Rationale for Chemoprophylaxis Vaccination (CVac) using Pyrimethamine (PYR) 
as the Partner Drug ..........................................................................................................28
1.4
Previous Human Experience..................................................................................33
1.4.1 Whole SPZ Vaccination ........................................................................................33
1.4.2 PfSPZ Challenge (NF54) for Immunization ..........................................................34
1.4.2.1
PfSPZ-CVac under Chloroquine Prophylaxis in Malaria-Naïve Populations.35
1.4.2.2
PfSPZ-CVac (chloroquine) in Malaria Experienced Populations in Mali and 
Equatorial Guinea ............................................................................................................40
1.4.2.3
PfSPZ-CVac (chloroquine) and PfSPZ-CVac (pyrimethamine) in Malaria 
Naïve Populations............................................................................................................45
1.4.2.3.1
Main Phase.....................................................................................................47
1.5
Clinical Trial Plan .................................................................................................49
1.5.1
Rationale for a higher, one dose regimen of pyrimethamine..........................50
1.5.2
Study Plan ......................................................................................................51
2
Study Objectives...........................................................................................................52
2.1
Primary Objectives ................................................................................................52
2.2
Secondary Objective .............................................................................................52
2.3
Exploratory Objectives..........................................................................................53
3
Study Design ................................................................................................................53
3.1
Overall Design ......................................................................................................53


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 7 of 146
3.2
Pilot Study.............................................................................................................54
3.3
Main Study............................................................................................................58
4
Description of Investigational Products and Plan.........................................................59
4.1
Chloroquine Phosphate (CQ) ................................................................................59
4.2
Pyrimethamine ......................................................................................................60
4.3
PfSPZ Challenge ...................................................................................................61
4.4
Description of Intervention ...................................................................................61
4.5
Presumptive Antimalarial Treatment with Artemether/Lumefantrine...................65
5
Study Endpoints............................................................................................................65
5.1
Primary Endpoints.................................................................................................65
5.2
Secondary Endpoints.............................................................................................65
5.3
Exploratory Endpoints...........................................................................................65
5.4
Sample Size and Estimated Duration of Study ......................................................66
6
Study Population...........................................................................................................66
6.1
Description of Population and Site ........................................................................66
6.2
Recruitment...........................................................................................................66
6.3
Inclusion Criteria...................................................................................................67
6.4
Exclusion Criteria..................................................................................................67
7
Study Agents ................................................................................................................69
7.1
PfSPZ Challenge (NF54).......................................................................................69
7.2
Phosphate buffered saline and human serum albumin diluent...............................69
7.3
Storage and Handling of PfSPZ Challenge............................................................70
7.4
Control Product .....................................................................................................70
7.4.1
Storage and Handling.....................................................................................70
7.4.2
Disposition and Dispensation.........................................................................70
7.4.3
Administration and Dosage............................................................................70
7.4.4
Accountability................................................................................................70
7.5
Pyrimethamine, Chloroquine, Artemether/Lumefantrine ......................................70
7.5.1 Pyrimethamine, Artemether/Lumefantrine and Chloroquine Packaging and 
Labeling
71
7.5.2 Pyrimethamine, Chloroquine, Artemether/Lumefantrine Accountability..............71


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 8 of 146
7.5.2.1
Receipt ....................................................................................................71
7.5.2.2
Preparation and Administration ..............................................................71
7.5.2.3
Storage and Handling..............................................................................71
7.5.2.4
Return of Study Product..........................................................................71
8
STUDY SCHEDULE...................................................................................................72
8.1
Screening...............................................................................................................72
8.2
Assignment of Groups...........................................................................................73
8.3
Detailed Study Procedures ....................................................................................74
8.4
Indications for Deferral of Sanaria® PfSPZ Challenge ..........................................74
9
Study Procedures..........................................................................................................74
9.1
Photographs of Rash or Injection Site Reactions ..................................................74
9.2
Clinical Laboratory Testing...................................................................................75
9.3
Electrocardiogram.................................................................................................75
9.4
Malaria Diagnostics ..............................................................................................75
9.4.1 Malaria Blood Smears...........................................................................................75
9.4.2 Malaria qPCR........................................................................................................76
9.5
Unscheduled Blood Smear Positive Visits ............................................................76
10
Immunologic Laboratories ...........................................................................................76
11
Research Use, Storage, and Tracking of Specimens and Data.......................................78
12
Retention of Specimens for Future Use ........................................................................78
13
Data Sharing Plan.........................................................................................................79
14
Assessment of Safety ...................................................................................................79
14.1
Documenting, Recording, and Reporting Adverse Events ....................................79
14.2
Definitions for the Sponsor ...................................................................................80
14.3
Investigator Assessment of Adverse Events..........................................................82
14.3.1 Adverse Event Definitions.....................................................................................83
14.3.2 Severity .................................................................................................................85
14.3.3 Causality ...............................................................................................................85
14.4
Investigator Reporting Responsibilities to the Sponsor.........................................86
14.4.1 Adverse Events......................................................................................................86
14.4.2 Serious Adverse Events.........................................................................................87


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 9 of 146
14.4.3 Unanticipated Problems ........................................................................................87
14.4.4 Pregnancy..............................................................................................................88
14.5
Reporting Procedures to the NIH IRB and FMPOS EC ........................................88
14.5.1 Reporting to the NIH IRB .....................................................................................88
14.5.2 Reporting to the FMPOS EC.................................................................................88
14.6
Follow-up of Adverse Events and Serious Adverse Events ..................................89
14.7
Sponsor’s Reporting Responsibilities....................................................................89
14.8
Pausing Criteria for Entire Study Population ........................................................89
14.8.1 Parasitemia and Malaria Symptoms ......................................................................89
14.8.2 Reactogenicity.......................................................................................................91
14.8.3 Reporting of Study Pausing...................................................................................92
14.8.4 Resumption of a Paused Study..............................................................................92
14.9
Pausing Criteria for a Subject or Group ................................................................92
14.9.1 Reporting of Pausing for a Participant or Group...................................................93
14.9.2 Resumption of a Paused Study..............................................................................93
14.10 Withdrawal Criteria for an Individual Participant.................................................93
14.11 Replacement of Withdrawn Subjects ....................................................................94
14.12 Unblinding for the Study.......................................................................................94
14.13 Safety Oversight....................................................................................................95
14.13.1
Independent Safety Monitor (ISM) in Mali....................................................95
14.13.2
Data and Safety Monitoring Board (DSMB)..................................................95
15
Clinical Monitoring ......................................................................................................95
15.1
Site Monitoring Plan..............................................................................................95
16
Statistical Considerations..............................................................................................96
16.1
General Pilot Study ...............................................................................................96
16.2
General Main Study ..............................................................................................96
16.3
Analysis.................................................................................................................98
16.4
Sample Size and Power Calculations.....................................................................98
16.4.1
Power calculations for primary objective (safety): Pilot Phase......................98
16.4.2
Power calculations for Main Phase ................................................................99
16.4.2.1
Power calculations for primary objective (safety)...................................99


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 10 of 146
16.4.2.2
Power calculations for secondary objective (efficacy)..........................100
16.5
Randomization ....................................................................................................101
17
Human Subject Protections and Ethical Obligations...................................................101
17.1
Institutional Review Board..................................................................................101
17.2
Informed Consent Process ...................................................................................101
17.2.1 Mali Site Community Permission and Individual Informed Consent Process.....102
17.2.1.1
Community Permission................................................................................102
17.2.1.2
Individual Informed Consent .......................................................................102
17.3
Justification for Exclusion of Children ................................................................103
17.4
Justification for the Exclusion of Pregnant Women ............................................103
17.5
Subject Confidentiality........................................................................................103
17.6
Risks....................................................................................................................104
17.6.1 Venipuncture .......................................................................................................104
17.6.2 PfSPZ Challenge administered by DVI...............................................................104
17.6.3 Malaria Symptoms during immunization with PfSPZ-CVac (chloroquine) ........105
17.6.4 Medications used in the study .............................................................................107
17.6.4.1
Chloroquine Phosphate (CQ)........................................................................107
17.6.4.2
Pyrimethamine..............................................................................................108
17.6.4.3
Artemether/Lumefantrine.............................................................................109
17.6.4.4
Ibuprofen......................................................................................................109
17.7
Risk to the Community........................................................................................110
17.8
Compensation......................................................................................................110
18
Data Handling and Record Keeping............................................................................111
18.1
Data Capture and Management ...........................................................................111
18.2
Types of Data ......................................................................................................111
18.3
Retention of Study Records.................................................................................111
18.4
Protocol Revisions ..............................................................................................111
References .........................................................................................................................128
List of Tables
Table 1. Protection against blood stage versus sporozoite CHMI after CPS (chloroquine) 
immunization ................................................................................................................30


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 11 of 146
Table 2. PfSPZ-CVac (pyrimethamine) may lead to protective immunity following 
homologous CHMI.......................................................................................................32
Table 3. PfSPZ-CVac (pyrimethamine) with 2x105 PfSPZ may lead to protective immunity 
following both homologous and heterologous CHMI...................................................33
Table 4. Chronological Listing of Trials of the PfSPZ-CVac Approach to Immunization..36
Table 5. Summary of PfSPZ dose sizes during PfSPZ-CVac (chloroquine) immunization 
and the number of qPCR positive subjects following each immunization..................38
Table 6. Summary of PfSPZ dose sizes and mean parasite density after each immunization
......................................................................................................................................39
Table 7. List of AEs post PfSPZ-CVac (chloroquine) immunization with weekly 5.12x104 
PfSPZ of PfSPZ Challenge administered by DVI in NCT02773979...........................41
Table 8. Number and Percentage of subjects experiencing solicited events with 95% 
confidence intervals by symptom and treatment group...............................................43
Table 9. Parasitemia Indices (Protocol EGSPZV2)...........................................................46
Table 10. Summary of AEs by severity and frequency in pyrimethamine pilot study.......48
Table 11. Summary of AEs by severity and frequency observed in chloroquine pilot study
......................................................................................................................................49
Table 12. Addition of NSAIDS improves tolerability of PfSPZ CVac (chloroquine) .......50
Table 13: Parasitemia summary for main phase Arms 1b/4a and 2b/4b by calendar 
month post third vaccination as of 27 March 2020.....................................................55
Table 14. Chemoprophylaxis regimen and assignment.....................................................72
Table 15. Solicited Adverse Events.....................................................................................92
Table 16. Definitions for Severity of AE Grading .............................................................93
Table 17. Probability of observing 0, 1 and 2 or more break through parasitemia or 
adverse event among arms of size 4, for different true event rates...........................107
Table 18. Probability of observing 0, 1 and 2 or more events, among arms of size 90 or 
60, for different true event rates ................................................................................108
Table 19. Two-sided 95% confidence intervals based on observing a particular rate of 
safety endpoints for arms of size 90 or 60 .................................................................108
Table 20. Sample size calculations for various plausible true event rates among both 
control and vaccinated subjects .................................................................................109
Table 21: Power to detect a difference in efficacy with 81/group (after 10% loss to follow 
up)...............................................................................................................................109
Table 22: Compensation during the study .......................................................................119
List of Figures
Figure 1. Frequency of adverse events in all 4 groups after PfSPZ Challenge DVI........36
Figure 2. Maximum severity of solicited systemic symptoms per subject by day post 
treatment.......................................................................................................................41


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 12 of 146
Figure 3. Solicited Systemic Adverse Events per injection during EGSPZV2 trial..........43
Figure 4. Parasitemia Curves (Protocol EGSPZV2) .........................................................44
Figure 5. Parasitemia curves in the pyrimethamine and chloroquine groups..................49
Figure 6: Serum pyrimethamine levels in PfSPZ-CVac (pyrimethamine) study 
NCT02511054 in malaria-naïve individuals ...............................................................50
Figure 7. Pyrimethamine Pilot Study flowchart Schema ..................................................56
Figure 8. Ideal Pyrimethamine and Chloroquine Pilot Study Schema.............................57
Figure 9. Main Study Schema............................................................................................59
List of Appendices
Appendix A: Clinical Evaluation and Laboratory Procedures....................................112
Appendix B: Toxicity Table............................................................................................119
Appendix C: Cardiovascular Risk Assessment .............................................................126


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 13 of 146
List of Abbreviations
AE
adverse event
AES
asexual erythrocytic stages
AGC
absolute granulocyte count
ALT
alanine transaminase
AMA-1
Apical membrane antigen -1
ANC
absolute neutrophil count
AR
adverse reaction
AST
aspartate aminotransferase
β-hCG
β human choriogonadotropin
CBC
complete blood count
CFR
Code of Federal Regulations
CHI
Center for Human Immunology, Autoimmunity and Inflammation
cGMP
current Good Manufacturing Practices
CHMI
controlled human malaria infection
CoA
Certificate of Analysis
CPS
Chemoprophylaxis with sporozoites
CQ
chloroquine phosphate
Cr
creatinine
CRF
case report form
CSP
circumsporozoite protein
CVac
chemoprophylaxis vaccination
DMID
Division of microbiology and infectious diseases
DNA
Deoxyribonucleic acid
DSMB
Data and Safety Monitoring Board
DVI
Direct Venous Inoculation
EBA-175
erythrocyte binding antigen 175
EC
ethics committee
ECG
electrocardiogram
ELISA
enzyme-linked immunosorbent assay
ELISPOT
enzyme-linked immunosorbent spot assay
EXP-1
exported protein 1
FDA
Food and Drug Administration
FMPOS
Faculty of Medicine and Odonto-Stomatology
GCP
good clinical practice
Hb
hemoglobin
HBsAg
hepatitis B surface antigen
HBV
hepatitis B virus
HCV
hepatitis C virus
HIV
human immunodeficiency virus
HSA
human serum albumin


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 14 of 146
IB
investigator’s brochure
ICH
International Council for Harmonisation of Technical 
Requirements for Pharmaceuticals for Human Use
ICS
intracellular cytokine staining
ID
intradermal
IFA
immunofluorescence assay
IM
intramuscular
IND
Investigational New Drug
IPTp
intermittent preventive treatment in pregnancy
IRB
Institutional Review Board
ISM
Independent Safety Monitor
ITV
Infection treatment vaccination
IV
intravenous
LDH 
lactate dehydrogenase
LMIV
Laboratory of Malaria Immunology and Vaccinology (of NIAID)
LNVP
liquid nitrogen vapor phase
LSA-1
liver stage antigen 1
MCB
master cell bank
MRTC
Malaria Research and Training Center
MSP-1
merozoite surface protein 1
MSP-5
merozoite surface protein 5
N
number (typically refers to subjects or participants)
NASBA
nucleic acid sequence–based amplification
NHMI
Natural Human Malaria Infection
NIAID
National Institute of Allergy and Infectious Diseases, NIH
NIH
National Institutes of Health
NOCI
new onset of chronic illness
NS
normal saline
NSAID
non-steroidal anti-inflammatory drug, e.g., ibuprofen, naproxen
OCRPRO
Office of Clinical Research Policy and Regulatory Operations 
OHRP
Office for Human Research Protections
OHSRP
Office of Human Subjects Research Protections
PBS
phosphate-buffered saline
PCR
polymerase chain reaction
PI
principal investigator
Pf
Plasmodium falciparum
Pf7G8
Plasmodium falciparum, 7G8 clone
PfNF54
Plasmodium falciparum, NF54 strain
PfAES
Plasmodium falciparum asexual erythrocytic stages
PfSPZ
Plasmodium falciparum sporozoites
PfSPZ-CVac
PfSPZ challenge chemoprophylaxis vaccination


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 15 of 146
PfSPZ Challenge 
Aseptic, purified, cryopreserved Plasmodium falciparum  
sporozoites used for immunization and for controlled human 
malaria infection (CHMI)
POI
premature ovarian insufficiency
PYR
pyrimethamine
qPCR
quantitative polymerase chain reaction
RUNMC
Radboud University Nijmegen Medical Center
SAE
serious adverse event/serious adverse experience
SAR
suspected adverse reaction
sBS
sensitive blood smears
sc
subcutaneous
SERF
Safety Expedited Report Form
SMC
safety monitoring committee
SOP
standard operating procedure
SPZ
sporozoites
SUSAR
serious and unexpected suspected adverse reaction
UCRC
University Clinical Research Center
UP
unanticipated problem
USTTB
University of Sciences, Techniques and Technologies of Bamako
VE
vaccine efficacy
WBC
white blood cell
WCB
working cell bank
WHO
World Health Organization
WSQ
Wavelet Scalar Quantization


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 16 of 146
PROTOCOL SUMMARY
Full Title:
Sanaria® PfSPZ Challenge with Pyrimethamine or 
Chloroquine Chemoprophylaxis Vaccination (PfSPZ-
CVac Approach): A Randomized Double Blind 
Placebo Controlled Phase I/II Trial to Determine 
Safety and Protective Efficacy against Natural 
Plasmodium falciparum Infection in Bancoumana and 
Surrounding Areas, Mali
Short Title:
Mali PfSPZ-CVac (pyrimethamine)
Clinical Phase:
I/II
IND Sponsor:
Clinical Sponsor:
Sanaria Inc. 
Office of Clinical Research Policy and Regulatory 
Operations (OCRPRO) 
Conducted by:
Malaria Research and Training Center (MRTC), in 
collaboration with the Laboratory of Malaria 
Immunology and Vaccinology (LMIV)
Principal Investigators:
Issaka Sagara, MD, MPH, PhD (MRTC)
Patrick Duffy, MD  (LMIV/NIAID/NIH)


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 17 of 146
Site
MRTC (Bancoumana, Mali)
Sample Size:
N=432-440
Accrual Ceiling:
800
Study Population:
Healthy Malian adults, 18-50 years of age
Accrual Period:
Approximately April 2019 to April 2022 (Main Phase)
Approximately June 2020 to April 2023 (Booster Phase) 
Study Duration:
Start Date: Approximately April 2019
End Date: Approximately April 2023
Study subjects will be enrolled (exclusive of screening) 
for a total of approximately 1 (pilot) to 22 (main + 
booster) months
 
Study Design:
This is a randomized double-blind placebo controlled 
phase I/II study to investigate the safety, tolerability, 
and immunogenicity of direct venous inoculation (DVI) 
with three monthly doses of aseptic, purified, 
cryopreserved Plasmodium falciparum (Pf) 
sporozoites(PfSPZ) (Sanaria® PfSPZ Challenge 
(NF54)1), combined with either pyrimethamine (PYR) 
or chloroquine as a partner drug, known as PfSPZ 
Challenge chemoprophylaxis vaccination (Sanaria® 
PfSPZ-CVac). 
In brief:

Pilot Arms: to assess whether administering 
PYR on the same day as PfSPZ Challenge and 
also increasing the dose of PfSPZ Challenge to 
4x105 PfSPZ under either PYR or chloroquine 
prophylaxis is safe and tolerable 
o
Pyrimethamine arms 

Arm 1a:4x105 PfSPZ with 75 mg 
PYR day 0 post PfSPZ (n=4)

Arm 2a:4x105 PfSPZ with 75 mg 
PYR days 2 & 3 post PfSPZ 
(n=4)

Arm 5a (if necessary): 3x105 
PfSPZ with 75 mg PYR day 0 
1 Hereafter PfSPZ Challenge (NF54) will be denoted PfSPZ Challenge, unless being compared 
to another strain or clone. If a PfSPZ Challenge product is composed of a strain or clone of Pf 
other than NF54 (e.g., clone 7G8), it will be so designated [e.g., PfSPZ Challenge (7G8)]. 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 18 of 146
post PfSPZ (n=4)

Arm 6a (if necessary): 3x105 
PfSPZ with 75 mg PYR day 2&3 
post PfSPZ (n=4)
o
Chloroquine arm (4x105 PfSPZ)

Arm 3a: chloroquine 2 days 
prior + 5 days post PfSPZ (n=4)

Main Arms (n=420): to assess safety and 
tolerability of 3 exposures every 28 days of 
4x105 PfSPZ while under PYR given on either 
day 0 {or (if necessary) 3x105 PfSPZ while 
under PYR given on either day 0 (Arm 5b)} OR 
4x105 PfSPZ while under PYR given on day 2 
& 3 (Arm 2b) post vaccination {or (if 
necessary) 3x105 PfSPZ while under PYR given 
day 2 & 3 post vaccination (Arm 6b)} OR 4x105 
PfSPZ while under weekly chloroquine +/- non-
steroidal anti-inflammatory drugs (NSAIDs) 
chemoprophylaxis (Arm 3b). In addition, the 
main study will have placebo control groups 
that will receive normal saline (NS) injections 
with either PYR given on either day 0 or 2 & 3 
post injection (Arm 4a and 4b respectively) or 
weekly chloroquine chemoprophylaxis (Arm 
4c). The study will also assess development of 
protective efficacy against natural P. falciparum 
infection.
The study is to occur in two parts, a pilot study and a 
main study. In the PYR pilot arms (n=8-16, n=4 in each 
pilot arm), the goal will be to assess the safety and 
tolerability of a PfSPZ dose of 4x105 PfSPZ under a 
single dose of 75 mg of PYR chemoprophylaxis given 
on day 0 (the same day as PfSPZ injection, Arm 1a) or 
on day 2&3 post PfSPZ injection (Arm 2a) . Participants 
will be randomly enrolled in either Arm 1a or Arm 2a. If 
the regimen is safe and prevents patent parasitemia, 
these regimens will move forward to the main study. If 
there is patent parasitemia in either Arm 1a or Arm 2a, 
the dose of PfSPZ will be decreased to 3x105 PfSPZ 
with the same PYR regimen in Arms 5a or 6a 
respectively. The pyrimethamine main study will be 
conducted in the first year of the study.
The chloroquine pilot study will assess safety and 
tolerability of PfSPZ-CVac (chloroquine) with 4x105 
PfSPZ under chloroquine chemoprophylaxis at a fixed 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 19 of 146
time (Arm 3a: -2 days, +5 days post PfSPZ injection). If 
the regimen is tolerable (there are no Grade 3 AE 
symptoms lasting more than 48 hours despite adequate 
management that are deemed related to resulting 
parasitemia post PfSPZ injection), this regimen will 
move forward to the main study. If the regimen is not 
tolerable, empiric NSAIDs will be administered at days 
7 & 8 post first PfSPZ injection in the main study. In 
another similar study, addition of NSAIDs has been 
shown to decrease frequency and severity of symptoms 
related to parasitemia when administered in this 
schedule. The chloroquine main study will be conducted 
in a subsequent year of the study.
The regimens selected in the pilot study will then be 
further evaluated in the main study which is designed to 
assess safety, tolerability, immunogenicity, and 
protective efficacy of PfSPZ-CVac (pyrimethamine) and 
PfSPZ-CVac (chloroquine) against natural P.falciparum 
infection.
At the completion of the follow up period in the first 
transmission season, subjects in the main study arms 
will be allowed to participate in a booster phase that will 
assess the safety, tolerability, immunogenicity and 
protective efficacy of a 4th vaccination (booster dose). 
Arms 1b, 2b, and 3b will each receive  a single dose of 
4x105 PfSPZ Challenge whereas Arms 4a, 4b, and 4c 
will receive normal saline while under their respective 
chemoprophylaxis regimens (PYR on day 0 for Arms 
1b/4a, PYR on days 2 and 3 for Arms 2b/4b, and CQ 
on days -2 and 5 for Arms 3b/4c). The booster phase 
will extend follow-up through the subsequent 
transmission season.
 
In Detail:
The pilot phase will be open label. 
Pyrimethamine Pilot (n=8-16; 4 per arm)
In the PYR pilot, subjects will undergo immunization 
with PfSPZ-CVac (pyrimethamine) with one exposure 
to PfSPZ Challenge with 4x105 PfSPZ with PYR dosing 
on day 0 only (same day, Arm 1a) or day 2 & 3 (Arm 
2a) post PfSPZ Challenge. The goal of the pilot study is 
to first determine that dosing PYR on the same day as 
PfSPZ DVI (day 0 post DVI; earlier than previously 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 20 of 146
tested) and with a higher dose of PfSPZ (4x105, not 
previously tested) is safe, and does prevent patent 
parasitemia. Enrollment in these arms will occur 
simultaneously with 4 participants in each Arm. If there 
is breakthrough parasitemia in either Arm, the dose of 
PfSPZ will be decreased to 3x105 PfSPZ in the arm 
with the same regimen of PYR. Arm 5a will receive 
3x105 PfSPZ with PYR administered on day 0 and Arm 
6a will receive 3x105 PfSPZ with PYR administered on 
days 2 &3 post PfSPZ injection. If there is breakthrough 
parasitemia in either Arm 5a or Arm 6a, the study will 
pause for reassessment. All participants will be treated 
with standard treatment doses of 
artemether/lumefantrine approximately 2 weeks prior to 
PfSPZ DVI and at the end of their enrollment. 
Participants from the pilot study will not join the main 
study.
Chloroquine Pilot (n=4)
In the chloroquine pilot study (Arm 3a), subjects will 
undergo immunization with PfSPZ-CVac (chloroquine) 
with one exposure to PfSPZ Challenge with a 
chloroquine loading dose administered 2 days before 
PfSPZ Challenge and a maintenance dose administered 
5 days post PfSPZ Challenge. The goal of the pilot 
study is to evaluate safety and tolerability of 
administering a dose of 4x105 PfSPZ of PfSPZ 
Challenge while under chloroquine chemoprophylaxis 
(this dose has not been previously tested). If this 
regimen is not tolerable (presence of Grade 3 AE 
symptoms lasting more than 48 hours despite adequate 
management that are associated with parasitemia 
starting from day 7 post DVI), then NSAIDs will be 
administered on day 7 & 8 post DVI during the first 
vaccination only in the main study). All participants will 
be treated with standard treatment doses of 
artemether/lumefantrine approximately 2 weeks prior to 
PfSPZ DVI and at the end of their enrollment. 
Participants from the pilot study will not join the main 
study.
Main Study (n=420)
The main study will be a double-blind study, both 
investigators and participants will not be aware of the 
injection assignment (PfSPZ Challenge or normal 
saline), but they will be aware of the partner drug being 
administered. 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 21 of 146
Vaccinated participants will be randomly enrolled in 
either Arm 1b (4x105 PfSPZ + 75 mg PYR on days 0 
post DVI, n=90); Arm 2b (4x105PfSPZ + 75 mg PYR 
on days 2 & 3 post DVI, n=60); or in Arm 3b (4x105 
PfSPZ + weekly chloroquine prophylaxis +/- NSAIDs, 
n=90). 
The control Arm (Arm 4, n=180) will receive NS by 
DVI with the medications regimens to match the 
vaccinated arms. Arm 4a (n=54) will receive 75 mg 
PYR on days 0 post DVI, Arm 4b (n=36) will receive 75 
mg PYR on days 2&3 post DVI and Arm 4c (n=90) will 
receive weekly chloroquine prophylaxis +/- NSAIDs. 
Participants in the main study will receive three 
exposures to PfSPZ Challenge (Arm 1b/5b, 2b/6b and 
3b) or NS (Arm 4), separated by 4 weeks, with PYR or 
chloroquine coverage (PfSPZ-CVac). The goal in the 
main study is to assess the safety and tolerability of 
PfSPZ-CVac (pyrimethamine) regimen in which PYR is 
dosed earlier with a higher dose of PfSPZ (Arm 1b/5b) 
than previously studied. In addition we will test a 
PfSPZ-CVac (pyrimethamine) standard regimen with a 
higher dose of PfSPZ (Arm 2b/6b) . In addition, PfSPZ-
CVac (chloroquine) regimen at higher dose of PfSPZ 
will be assessed for safety and tolerability (Arm 3b).
Participants will also be enrolled in Arm 4, a placebo 
control arm to receive NS injections with either PYR or 
chloroquine chemoprophylaxis. 
 
Vaccinated participants and non-immunized controls in 
the main study together will then be monitored for 
development of parasitemia through the transmission 
season. All participants will undergo parasitemia 
clearance with artemether/lumefantrine treatment twice 
during the course of the study, approximately 2 weeks 
prior to first and third vaccinations. 
In addition, the main study arms will receive a 4th 
vaccination (booster dose) of 4x105 PfSPZ Challenge or 
NS to assess safety, tolerability, immunogenicity and 
protective efficacy of PfSPZ-CVac during a subsequent 
transmission season.  Arms 1b, 2b, and 3b will each 
receive  a single dose of 4x105 PfSPZ Challenge 
whereas Arms 4a, 4b, and 4c will receive NS while 
under their respective chemoprophylaxis regimens 
(PYR on day 0 for Arms 1b/4a, PYR on days 2 and 3 for 
Arms 2b/4b, and CQ on days -2 and 5 for Arms 3b/4c). 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 22 of 146
Study Agent/
Intervention Description:
Pyrimethamine: Arms 1, 2, 4a, 4b, 5 and 6 will receive 
orally three single strength pills (for a total of 75 mg 
PYR) per day. The regimen of PYR will be determined 
in the pilot study dosed on day 0 administered with 
either 4x105 PfSPZ (Arm 1a) or 3x105 PfSPZ (Arm 5a) 
OR on days 2 & 3 with 4x105 PfSPZ (Arm 2a) or 3x105 
PfSPZ (Arm 6a). The placebo control arms will receive 
NS injections with PYR regimen to match the vaccine 
groups, 75 mg on day 0 (Arm 4a) or on days 2 & 3 (Arm 
4b). Arms 3 (chloroquine arm) and 4c (chloroquine 
controls) will not receive PYR.
Chloroquine: Arm 3 (3a, 3b) and Arm 4c will receive a 
loading dose of approximately 1000 mg chloroquine (600 
mg base) 2 days prior to PfSPZ Challenge or NS 
injection respectively. After loading dose, Arm 3a will 
receive one additional dose of 500 mg (300 mg base) 
administered 5 days post PfSPZ Challenge injection, for 
a total of 2 doses. Arm 3b and 4c will receive 500 mg 
(300 mg base) weekly continuously with the last dose 
administered 5 days post 3rd injection. A total of 10 
doses of chloroquine will be given to Arm 3b and 4c. 
Arms 1, 2, 4a, 4b, 5 and 6 will not receive chloroquine.
PfSPZ Challenge during PfSPZ-CVac immunization:
In the pilot phase: only 1 dose of 4x105 (Arms 1a and 
2a ) OR 3x105 (Arms 5a and 6a ) of aseptic, purified, 
vialed, cryopreserved, fully infectious PfSPZ of PfSPZ 
Challenge will be administered by direct venous 
inoculation (DVI). The first day of administration of 
PfSPZ Challenge is Study Day 1 for each Arm. 
In the main phase: 3 doses of 4x105 (Arms 1b, 2b and 
3b) OR of 3x105 (Arms 5b and 6b) of aseptic, purified, 
vialed, cryopreserved, fully infectious PfSPZ of PfSPZ 
Challenge will be administered by DVI four weeks apart 
in the main phase, with a fourth dose given during the 
booster phase. The first day of administration of PfSPZ 
Challenge is Study Day 1 thus PfSPZ Challenge will be 
administered on Study Days 1, 29, 57 and 336. 
Normal saline injections:
In the placebo control arms during the main phase (Arm 
4): 3 doses of NS will be administered by DVI four 
weeks apart. The first day of administration of NS is 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 23 of 146
Study Day 1 thus NS will be administered on Study 
Days 1, 29, 57 and 336.
Primary Objectives:
  Safety
Pilot Phase

To evaluate whether PYR on day 0 prevents patent 
parasitemia post DVI administration of 4x105 PfSPZ 
or 3x105 PfSPZ (if necessary) of PfSPZ Challenge 
(Arms 1a, 2a, 5a, 6a).

To monitor the safety and tolerability of PfSPZ-CVac 
(chloroquine), post 4x105 PfSPZ of PfSPZ Challenge 
administered via DVI with chloroquine as the partner 
drug (Arm 3a)
Main Phase and Booster Phase

To monitor the safety and tolerability of PfSPZ-CVac 
(PYR), PfSPZ Challenge administered via DVI with 
PYR as the partner drug; (Arms 1b/5b, 2b/6b, 4a, 4b) 

To monitor the safety and tolerability of PfSPZ-CVac 
(chloroquine), PfSPZ Challenge administered via 
DVI with chloroquine as the partner drug; (Arms 3b, 
4c)
        
Secondary 
Objectives:
Protective Efficacy

To assess the protective efficacy of PfSPZ-CVac 
(pyrimethamine) or PfSPZ-CVac (chloroquine) 
against natural P. falciparum infection (Arm 1b/5b, 
2b/6b, 3b, 4).
Exploratory 
Objectives:
Pyrimethamine Regimen Efficacy and Immunogenicity

To evaluate whether PYR on day 0 or on day 2&3 
prevents patent and subpatent parasitemia post DVI 
administration of 4x105 OR 3x105 PfSPZ of PfSPZ 
Challenge (Arms 1, 2, 5, 6).

To assess the humoral and cell mediated immune 
responses to PfSPZ and to known pre-erythrocytic 
and blood stage antigens (All Arms)

To assess the humoral and cell mediated immune 
responses to novel pre-erythrocytic antigens (All 
Arms)


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 24 of 146

To look for immune correlates of protection

To assess the kinetics of subpatent parasitemia during 
PfSPZ-CVac immunization (Arms 1, 2, 3, 5, 6)

To describe changes in γδ T cells in malaria 
experienced individuals after PfSPZ-CVac 
immunization and malaria infection during 
transmission season (Arms 1b/5b, 2b/6b, 3b)
Primary Endpoints:
Safety
Pilot Phase

Incidence of positive sensitive blood smear (sBS) 
occurring after PfSPZ-CVac immunization starting 
on day 7 post DVI (Arms 1a, 2a, 5a, 6a).

Incidence and severity of local and systemic grade 
3 signs or symptoms lasting more than 48 hours 
despite adequate management and serious adverse 
events (SAEs) occurring after PfSPZ-CVac DVI 
(Arm 3a)
Main Phase and Booster Phase

Incidence and severity of local and systemic 
adverse events (AEs) and serious adverse events 
(SAEs) occurring after PfSPZ-CVac immunization. 
(Arms 1b/5b, 2b/6b, 4a, 4b)

Incidence of clinical malaria diagnosis occurring 
after PfSPZ-CVac immunization as defined by the 
occurrence of grade 3 signs or symptoms lasting 
more than 48 hours despite adequate management. 
(Arm 3b, 4c)
Secondary 
Endpoints:
Protective Efficacy

P. falciparum blood stage infection defined as 
detection of at least 2 P. falciparum parasites by 
microscopic examination of 0.5 µL of blood starting 
2 weeks after the 3rd vaccination for approximately 6 
months (Arm 1b/5b, 2b/6b, 3b, 4) in the Main Phase 
and then 2 weeks after the 4th vaccination for 
approximately 6 months for the Booster Phase.
Exploratory 
Pyrimethamine regimen efficacy and Immunogenicity


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 25 of 146
Endpoints:

P. falciparum blood stage infection defined as 
detection of P. falciparum parasites by quantitative 
polymerase chain reaction (qPCR) following PfSPZ 
Challenge. (Arms 1, 2, 5, 6).

Humoral immune responses after PfSPZ-CVac 
regimens by assessing antibodies to PfSPZ, Pf 
asexual erythrocytic stages (AES), and specific Pf 
sporozoite, liver and blood-stage antigens such as 
CSP, merozoite surface protein 1 (MSP-1), AMA-1 in 
subjects with no patent and/or subpatent parasitemia 
and those with patent and/or subpatent parasitemia, 
among drug groups. (All Arms)

Cellular immune responses after PfSPZ-CVac 
regimens to PfSPZ, Plasmodium falciparum asexual 
erythrocytic stages (PfAES), and specific Pf 
sporozoite, liver and blood-stage antigens, such as 
CSP, MSP-1, AMA-1, in subjects with no patent 
and/or subpatent parasitemia and those with patent 
and/or subpatent parasitemia, among drug groups. 
(All Arms) 

Cellular and humoral responses that correlate with 
protective efficacy

Prolonged prepatent period and/or reduced density 
and duration of parasitemia in those developing 
parasitemia during PfSPZ-CVac immunization (Arms 
1, 2, 3, 5, 6)

Comparison of γδ T cells before and after PfSPZ-
CVac immunization and and malaria infection during 
transmission season using ex vivo whole blood or in 
vitro staining. (All Arms)


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 26 of 146
Précis
Human studies have shown that immunization by the bite of Plasmodium falciparum (Pf) 
sporozoite (SPZ)-infected mosquitoes (chemoprophylaxis with sporozoites [CPS]) or by 
injecting aseptic, purified, cryopreserved sporozoites (SPZ) with needle and syringe 
(PfSPZ chemoprophylaxis vaccination, Sanaria® PfSPZ-CVac) under drug coverage with 
chloroquine can provide >80% protection against homologous controlled human malaria 
infection (CHMI) (Roestenberg, McCall et al. 2009, Roestenberg, Teirlinck et al. 2011, 
Mordmuller, Surat et al. 2017). The protective immunity induced by CPS/PfSPZ-CVac is 
thought to target sporozoite and liver stage antigens, but the transient parasitemia 
observed under chloroquine prophylaxis may additionally induce immune responses 
targeting blood stage parasites that contribute to protection. It is important to understand 
the contribution of each stage in this model as transient parasitemia in the immunization 
process adds a safety concern and may impair the development of immunity. 
We have conducted two phase 1 studies to explore whether significant protective efficacy 
can be achieved with exclusively pre-erythrocytic (sporozoite/liver stage) exposure in this 
model using PYR chemoprophylaxis. This drug, unlike chloroquine, kills the parasites in 
the liver, so there is no period of transient parasitemia following immunization. These 
studies in malaria naïve adults demonstrated that PfSPZ-CVac (with PYR administered 2 
& 3 days after injection) is safe, well-tolerated, and can prevent subpatent and patent 
parasitemia after PfSPZ injection. The NCT02511054 study used the same dose 
(5.12x104 PfSPZ) that has resulted in >80% protection in previous trials of PfSPZ-CVac 
(chloroquine), showed only 2/9 vaccinees were protected against a homologous (same 
strain of Pf as the vaccine) CHMI. In trials using irradiated SPZ, increasing the dose of 
PfSPZ used for immunization has resulted in higher protective efficacy. Therefore, in an 
ongoing trial, NCT03083847, we have increased the PfSPZ dose 4-fold up to 2.0x105 
PfSPZ, with preliminary results show markedly improved protection against both 
homologous and heterologous CHMI in healthy US adults (7/8 and 4/4 study subjects 
uninfected, respectively). This is the first demonstration that the PfSPZ-CVac approach 
can confer sterile protection without blood stage exposure. 
Building on these promising results from PfSPZ-CVac (pyrimethamine) in the US, this 
proposed study in a malaria-endemic, adult population in Bancoumana, Mali will initially 
assess, in a pilot phase, the safety and tolerability of still higher doses of PfSPZ 
Challenge (4.0x105 PfSPZ) in combination with PYR (day 0;) or chloroquine (days -2, 
+5). The higher PfSPZ dose is hypothesized to be necessary to achieve significant 
efficacy against naturally transmitted malaria in malaria-experienced populations. Once 
this higher dose of PfSPZ is determined to be safe and tolerable and that earlier PYR 
dosing prevents patent parasitemia, the main phase will explore the effect of both earlier 
PYR dosing and a higher dosage of PfSPZ used for immunization in the development of 
protective immunity against natural malaria infection. The results of this study will 
contribute to understanding the targets and mechanisms of immunity against Pf malaria 
infection, and how the degree of prior exposure to the parasite (pre-erythrocytic and/or 
erythrocytic stages) impacts these responses and subsequent protective efficacy in field 
settings. 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 27 of 146
After completion of follow up in the main phase, all participants that are still enrolled in 
the study will be offered a booster dose of the vaccine (4th dose) with a single dose of 
4x105 PfSPZ Challenge or normal saline (aligned with what they received in the main 
phase of the study) at approximately 11 months post 3rd vaccination. The booster dose is 
timed to allow administration well before the beginning of the ensuing malaria 
transmission season. Participants will then be followed for approximately 6 months to 
assess safety and vaccine efficacy through this second transmission season.


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 28 of 146
1
Introduction and Rationale
1.1
Malaria Epidemiology
According to the World Health Organization (WHO), global control efforts had resulted in a 
reduction in the number of malaria cases and deaths; however 435,000 people are estimated 
to have died due to malaria in 2017, 92% of which occurred in the WHO African Region 
(WHO 2018), a rate similar to what was reported in 2017. This plateau continues to 
emphasize the need for additional measures to achieve malaria elimination in endemic 
countries. Malaria-related morbidity and mortality have a major economic impact in endemic 
regions and present a significant health risk to non-immune travelers to endemic regions and 
military personnel deployed overseas. To stem the worldwide impact of this devastating 
disease, a safe and broadly effective malaria vaccine and improved antimalarial therapeutics 
are urgently required. Investigations into the development of sterilizing immunity against 
malaria infection (prevention of blood-stage parasitemia and clinical illness) are of great 
importance to guide malaria vaccine development efforts and enhance the efficacy of current 
subunit malaria vaccine candidates.
1.2
Malaria Life Cycle and Vaccine Strategies
Malaria is a vector-borne disease caused by infection with the apicomplexan protozoan 
parasite, Plasmodium. Of the five major species commonly known to infect humans the 
majority of deaths are caused by P. falciparum (Pf). The complex parasite life cycle includes 
developmental stages in obligate mammalian and insect hosts. The sporozoite (SPZ) form of 
the parasite is transmitted to humans by the bite of the parasite-infected female Anopheles 
mosquito during a blood meal. The SPZ travel in the bloodstream to the liver, invade 
hepatocytes and undergo intracellular replication for five to seven days. Arrest of parasite 
development during this clinically silent pre-erythrocytic or liver-stage would prevent 
maturation and release of parasites into the bloodstream and limit clinical impact of the 
disease. Failing this, infected hepatocytes rupture, releasing tens of thousands of merozoite 
forms into the blood, initiating the pathogenic cycle of erythrocyte invasion and replication 
(Yoeli 1965). This continuous cycle during the erythrocytic (or blood) stages is responsible 
for the clinical symptoms of malaria including fever, chills, malaise, myalgia, arthralgia, 
nausea and vomiting that may progress to severe illness including cerebral malaria, 
pulmonary edema, renal failure, shock and death.
Vaccination targeted toward the clinically silent liver-stage of infection would ideally 
provide sterile protective immunity, preventing progression to blood-stage infection and 
clinical disease, and transmission of the parasites to mosquitoes. This is the target of leading 
vaccine strategies including the partially effective recombinant circumsporozoite protein 
(CSP) based RTS,S vaccine (Mosquirix®), for which phase 3 trials were completed 
throughout Africa in 2015, and phase 4 and implementation studies are scheduled to start in 
2019. Subunit vaccines of this type utilize conserved antigenic targets to elicit protection 
against SPZ migration, hepatocyte infection and intrahepatocytic parasite replication. RTS,S 
has protected malaria-naïve adults against experimental Pf controlled human malaria 
infection (CHMI) and reduced malaria-associated clinical episodes in children living in 
malaria endemic areas; however, the level and duration of immunity seen is relatively 
modest (Stoute, Kester et al. 1998, Agnandji, Lell et al. 2011, RTS 2015). In the one study 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 29 of 146
where the incidence of parasitemia (as opposed to the incidence of clinical episodes) was 
studied in adults, a study performed in Kisumu, Kenya, RTS,S/AS01b did not induce 
statistically significant protection (Polhemus, Remich et al. 2009). Although follow up studies 
of RTS,S vaccine have shown decreases in clinical malaria in vaccinated children, at 6 
months post vaccination follow up, the levels of antibodies between vaccinated and 
unvaccinated children were not significantly different and did not predict protection against 
clinical malaria in the following 12 months (Campo, Sacarlal et al. 2014). The mechanism by 
which RTS,S and other SPZ and pre-erythrocytic vaccine strategies confer protective 
immunity against clinical episodes of malaria is still under investigation, but it is thought 
that RTS,S primarily induces protective antibody responses targeting SPZ that are dependent 
on a strong CD4+ helper T cell response, while whole SPZ-based vaccines induce protective 
CD8+ T cells in the liver that can kill developing liver stage parasites. 
In the absence of more effective subunit vaccines, malaria vaccine development has focused 
on whole organism vaccination to induce sterile immunity. Studies using mosquito bite or 
needle and syringe to administer radiation-attenuated SPZ (including PfSPZ Vaccine) for 
vaccination have induced high level (>90%) sterile immunity to Pf in humans (Hoffman, 
Goh et al. 2002, Roestenberg, McCall et al. 2009, Roestenberg, Teirlinck et al. 2011, Seder, 
Chang et al. 2013). This required a relatively large number of SPZ to induce protective 
immunity, e.g. 1000 infectious mosquito bites or five injections of 1.35x105 PfSPZ. In 
2009, a human study suggested that anti-infection immunity can be achieved with a much 
smaller parasite inoculum if non-attenuated parasites are used as the immunogen. 
Roestenberg, et al. demonstrated that wild-type PfSPZ administered 3 times by 12-15 
infectious mosquito bites (a total of 36-45 infected mosquitoes) to human subjects receiving 
concurrent antimalarial prophylaxis with the blood stage drug chloroquine can induce sterile 
protection against malaria infection upon subsequent challenge with homologous parasites 
(Roestenberg, McCall et al. 2009). More recently, the same has been shown administering 
PfSPZ by needle and syringe: three doses of 5.12x104 PfSPZ of PfSPZ Challenge (non-
attenuated PfSPZ) in the presence of chloroquine induced 100% protection (9/9 volunteers 
protected) against CHMI conducted 9-10 weeks after immunization (Mordmuller, Surat et 
al. 2017), a dose that is 8-fold less than the dose of radiation-attenuated PfSPZ needed to 
induce high level protection (Epstein, Paolino et al. 2017). The greater potency presumably 
reflects the fact that non-attenuated SPZ replicate in the liver, greatly increasing the quantity 
and duration of antigen expression, and concurrently expressing new antigens (mid and late 
liver stage antigens) that are not expressed by radiation-attenuated SPZ, which halt 
development early in the liver stages.
This method of immunization by experimental Pf infection with non-attenuated PfSPZ in 
conjunction with antimalarial prophylaxis is referred to as chemoprophylaxis with SPZ 
(CPS), infection treatment vaccination (ITV), or chemoprophylaxis vaccination (PfSPZ-
CVac) by different authors. We use the term PfSPZ-CVac in this protocol to refer to this 
vaccination concept that combines the injection by direct venous inoculation (DVI) of 
aseptic, purified, cryopreserved PfSPZ (PfSPZ Challenge) with oral administration of an 
antimalarial drug (chloroquine, PYR or others). 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 30 of 146
1.3
Rationale for Chemoprophylaxis Vaccination (CVac) using Pyrimethamine 
(PYR) as the Partner Drug
Chloroquine is a selective blood-stage schizonticide and does not kill SPZ or liver stages. 
Prophylactic doses are sufficient to eliminate all new infections with sensitive malaria strains 
as they emerge into the blood (Yayon, Vande Waa et al. 1983). Thus the non-attenuated 
sporozoites (SPZ) used for the PfSPZ-CVac approach when chloroquine is the partner drug 
develop fully in the liver and are killed only after they emerge into the blood. For this reason, 
the degree to which the protective immune response induced by PfSPZ-CVac (chloroquine) 
targets sporozoite, liver, or blood-stage antigens is unclear, and raises the question whether a 
degree of blood stage immunity is required for the high level protection that is observed. Our 
hypothesis is that blood stage exposure is not required for protection. 
The concept that sterile protective immunity to malaria induced by whole PfSPZ vaccination 
does not require blood stage immunity is supported by experimental infection studies in both 
animals and humans. In fact, in rodent malaria models, blood-stage parasites may modulate 
dendritic cell responses and suppress, rather than bolster, protective CD8+ T cell responses 
that target parasites infecting hepatocytes (Ocana-Morgner, Mota et al. 2003). The best 
evidence to date of sterile immunity without blood stage involvement comes from work of 
Bijker et al at Radboud University Nijmegen Medical Center (RUNMC). Research subjects 
were immunized by mosquito bite as had been done by Roestenberg et al, and then 
underwent CHMI by SPZ or by blood stages. There was complete protection against PfSPZ 
CHMI, but there was no protection against blood stage CHMI, indicating that the immunity 
induced by CPS was limited to pre-erythrocytic stage antigens (Bijker, Bastiaens et al. 2013). 
These results are shown in Table 1.
Table 1. Protection against blood stage versus sporozoite CHMI after CPS (chloroquine) 
immunization
^Adapted from Bijker EM, et al. 2013
A second approach to elucidating the stage-specificity of the protective immunity induced by 
the PfSPZ-CVac approach is to use a partner drug other than chloroquine to kill the parasites 
in the liver, arresting development before blood stage parasitemia occurs. This should 
eliminate the possibility that asexual blood stages contribute to the induction of sterile 
immunity. Several antimalarial drugs with activity against pre-erythrocytic stage parasites 
that have been considered for PfSPZ-CVac regimens include primaquine, proguanil, and 
azithromycin. In healthy US adults, we found that a single 45 mg dose of primaquine 
combined with weekly chloroquine is insufficient to prevent blood stage parasitemia after 
immunization with PfSPZ Challenge (Healy et al. submitted). Azithromycin has also been 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 31 of 146
assessed as the PfSPZ-CVac partner drug, and it too was unable to prevent blood stage 
parasitemia and was not protective upon PfSPZ CHMI (TÜCHMI-002 trial, Mordmuller, 
Surat et al. 2017). Atovaquone/proguanil has also been assessed, and while this did 
successfully prevent blood stage parasitemia during immunization, the protective immunity 
induced was only 20-25%, possibly because the parasites were killed too early in liver stage 
development (MALACHITE trial, Mordmüller et al, unpublished); therefore the 
atovaquone/proguanil study was unable to convincingly demonstrate that exclusive exposure 
to pre-erythrocytic stages of the parasite could induce strong, durable immunity. 
Pyrimethamine is an antimalarial drug with both liver and blood stage activity. Plasmodium 
berghei SPZ-CVac (pyrimethamine) regimens in mice have been shown to induce sterile 
protective immunity (Friesen, Borrmann et al. 2011). Our group at Laboratory of Malaria 
Immunology and Vaccinology (LMIV) was interested to investigate whether sterile 
protective immunity could likewise be induced in humans by wild-type (non-attenuated) 
PfSPZ immunizations when exposure was limited to pre-erythrocytic stages of the parasite 
life cycle, and selected PYR as the PfSPZ-CVac partner drug for these investigations. We 
also hypothesized that, as is the case with PfSPZ-CVac (chloroquine), the total immunizing 
dose of PfSPZ needed with PfSPZ-CVac (pyrimethamine) would be much lower than 
required for the radiation-attenuated PfSPZ of PfSPZ Vaccine.
We performed an initial human study (#15-I-0169; NCT02511054 ) with PYR to explore this 
question of stage-specific immunity. The first finding was that a PfSPZ-CVac 
(pyrimethamine) regimen using 3 doses of 5.12x104 PfSPZ of PfSPZ Challenge with PYR 50 
mg given on days 2 and 3 after each administration of PfSPZ Challenge can prevent 
subpatent parasitemia completely, as confirmed by negative quantitative reverse transcriptase 
polymerase chain reaction for Pf parasites. Weekly chloroquine was also administered in this 
study group as a safety back-up in case parasitemia developed. However, as the results 
showed that PYR appeared to kill all parasites during liver stage development, it was decided 
that in future studies PYR could be tested alone. A second finding, from a comparator 
chloroquine-alone arm, was that participants exposed to 5.12x104 PfSPZ experienced, as 
expected, detectable subpatent parasitemia after the first PfSPZ Challenge inoculation, with 
fewer participants being parasitemic after immunizations #2 and #3 than after immunization 
#1, presumably reflecting the development of immunity. A third finding came when the 
research subjects underwent CHMI: PfSPZ-CVac (pyrimethamine) did lead to protective 
immunity, but only 2/9 subjects in the pyrimethamine + chloroquine arm remained 
uninfected while one additional participant was significantly delayed after homologous 
CHMI. These two protected volunteers, like the volunteers in the MALACHITE trial, 
supported the concept that exclusive exposure to pre-erythrocytic stage antigens was 
sufficient to induce sterile immunity, but the expected robust protection was still not 
achieved. A fourth finding was to confirm the high level efficacy demonstrated in the prior 
PfSPZ-CVac (chloroquine) study of Mordmüller et al (Mordmuller, Surat et al. 2017), as 4/5 
(80%) subjects in the chloroquine only arm remained uninfected following homologous 
CHMI. The results of this first PYR study are presented in Table 2.


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 32 of 146
Table 2. PfSPZ-CVac (pyrimethamine) may lead to protective immunity following 
homologous CHMI
Arm
Number of 
subjects 
undergoing 
CHMI
Number 
of 
subjects 
infected
Days post 
CHMI to 
diagnosis
Median 
days post 
CHMI to 
diagnosis
Positive 
Blood 
Smear
Percent 
protected
P-
value
1a Pilot 
(PYR +CQ)
2
2
12, 14
13
1
0%
--
2 (PYR +CQ)
9
7*
8, 11, 12, 
12, 12, 14,  
18
12
3
11%
>0.7
3 (CQ)
5
1
13
13
0
80%
0.048
4
5
5
8, 9, 12, 
12, 12
12
2
0%
--
*One subject was withdrawn from study at Day 16 post CHMI. Up to day 16 post CHMI, the subject remained 
negative by both blood smear and PCR, thus never documented infected, but since that subject did not complete 
study follow up per protocol, that subject was considered to have a missing outcome for the binary analysis, but 
contributed to the time-to-event analysis being censored at day 16.
To improve on these PfSPZ-CVac (pyrimethamine) results, and to build on the concept that 
larger doses of PfSPZ lead to increased protection, as seen with PfSPZ Vaccine (radiation 
attenuated PfSPZ), a follow-up study in healthy malaria-naïve US adults was initiated (and 
remains ongoing) using larger doses of PfSPZ. This ongoing study, (National Institute of 
Allergy and Infectious Diseases [NIAID] protocol #17-I-0067, NCT03083847) increased the 
dose of PfSPZ 4-fold from 5.12x104 to 2.0x105 PfSPZ while under PYR alone or chloroquine 
alone coverage. Malaria-naïve participants were divided into three arms, Arm 2a [PfSPZ-
CVac (pyrimethamine) tested by homologous NF54 CHMI n=17]; Arm 2b [PfSPZ-CVac 
(pyrimethamine) tested by heterologous 7G8 CHMI n=10] and Arm 3 [PfSPZ-CVac 
(chloroquine) tested by heterologous 7G8 CHMI n=10]. Participants received 3 monthly 
immunizations with PfSPZ-CVac (2.0x105 PfSPZ of PfSPZ Challenge) by DVI plus 50mg 
PYR on 2 and 3 days post injection (Arm 2) or weekly chloroquine until 5 days after the 3rd 
vaccination (Arm 3)). Homologous CHMI with 3,200 PfSPZ of PfSPZ Challenge (Arm 2a) 
or heterologous CHMI with 3,200 PfSPZ of PfSPZ Challenge (7G8) (Arm 2b and 3) via DVI 
was performed approximately 12 weeks post the 3rd PfSPZ-CVac immunization. The main 
group has been divided into two cohorts and only half of the projected participants have 
completed the study at this time. The study is expected to be completed in early 2019.
The first completed cohort has now shown promising preliminary results over the first 
PfSPZ-CVac (pyrimethamine) trial described above. Despite the higher dose of PfSPZ, 50 
mg of PYR given on days 2 and 3 after PfSPZ injections was safe, well tolerated, and 
completely prevented blood stage parasite exposure as evidenced by negative quantitative 
polymerase chain reaction (qPCR) assays during follow-up period. PfSPZ-CVac 
(chloroquine) was also safe and tolerable at 1.0x105 PfSPZ, with parasitemia characteristics 
as expected; however, the higher dose of 2.0x105 PfSPZ resulted in an increase in the density 
of transient blood stage parasitemia and there were associated Grade 3 adverse events (AEs) 
on days 7-8 after injection of PfSPZ Challenge. The tolerability of the PfSPZ-CVac 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 33 of 146
(chloroquine) regimen at the 2.0x105 PfSPZ dose was subsequently much improved with 
empiric use of non-steroidal anti-inflammatory drugs (NSAIDs) administered presumptively 
on days 7-8 after PfSPZ Challenge, timed to coincide with the release of asexual blood stages 
from the liver. 
In the first cohort, upon undergoing CHMI with homologous PfSPZ Challenge parasites, 7/8 
study subjects immunized with PfSPZ-CVac (pyrimethamine) remained uninfected (see 
Table 3 below), a significant improvement from the prior study where smaller immunizing 
doses of PfSPZ had been used and only 2/9 subjects were protected. In addition, high level 
heterologous immunity was seen for the first time with a PfSPZ-CVac regimen: 4/4 
participants in PfSPZ-CVac (pyrimethamine) arm and 3/3 participants in PfSPZ-CVac 
(chloroquine) arm remained uninfected after heterologous CHMI performed approximately 
12 weeks post vaccination #3. The heterologous parasite used was the Plasmodium 
falciparum, 7G8 clone (Pf7G8), a cloned line from a parasite originally isolated in Brazil 
(Burkot, Williams et al. 1984). These results, though derived from a small sample size and 
needing confirmation, demonstrate for the first time that a PfSPZ-CVac regimen involving 
only pre-erythrocytic exposure (pyrimethamine arms) can induce high grade sterile immunity 
against both homologous and heterologous parasites for at least three months after 
completion of vaccination. The finding of heterologous protection is consistent with the 
performance of PfSPZ Vaccine in the field. 
Table 3. PfSPZ-CVac (pyrimethamine) with 2x105 PfSPZ may lead to protective immunity 
following both homologous and heterologous CHMI
Arm
Subjects
undergoing
CHMI
Subjects 
Infected
Days post 
CHMI to PCR 
diagnosis
Median days 
post CHMI 
to diagnosis
Positive 
Blood 
Smear
Percent 
protected
CVac-PYR 
(NF54)
8
1
12
12
0
87.5%
CVac-PYR 
(7G8)
4
0
n/a
n/a
0
100%
CVac-CQ 
(7G8)
3
0
n/a
n/a
0
100%
NF54 
Control
4
4
9, 11, 11, 11
11
1
0%
7G8 Control
4
4
9, 9, 12, 12
10.5
0
0%
Table 3: In order to evaluate whether increasing the dose of PfSPZ in PfSPZ-CVac (pyrimethamine) 
will result in a higher rate of protective efficacy, we designed a follow-up study, NIAID protocol 17-I-
0067, in which malaria-naïve participants were vaccinated with 2x105 PfSPZ, a 4-fold increase over the 
previous study. In this ongoing study, the main group is divided into three arms, Arm 2a [PfSPZ-CVac 
(pyrimethamine) tested by homologous NF54 CHMI n=17]; Arm 2b [PfSPZ-CVac (pyrimethamine) 
tested by heterologous 7G8 CHMI n=10] and Arm 3 [PfSPZ-CVac (chloroquine) tested by heterologous 
7G8 CHMI n=10].  Participants received 3 monthly immunizations with PfSPZ-CVac (2x105 PfSPZ of 
PfSPZ Challenge) by DVI plus 50mg pyrimethamine on 2 and 3 days post injection (Arm 2) or weekly 
chloroquine until 5 days after the 3rd vaccination (Arm 3)).  Homologous CHMI with 3,200 PfSPZ of 
PfSPZ Challenge (Arm 2a) or heterologous CHMI with 3,200 PfSPZ of PfSPZ Challenge (7G8) (Arm 2b 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 34 of 146
and 3) via DVI was performed approximately 12 weeks post the 3rd PfSPZ-CVac immunization. The 
main group has been divided into two cohorts and only half of the projected participants have 
completed the study at this time. In Arm 2a, 7/8 participants remained uninfected post homologous 
CHMI. In Arms 2b and 3, 4/4 and 3/3 participants, respectively, remained uninfected post heterologous 
CHMI. These preliminary results show for the first time that PfSPZ-CVac (pyrimethamine) or PfSPZ-
CVac (chloroquine) can achieve sterile immunity to heterologous CHMI, and can achieve sterile 
immunity in the absence of blood stage parasite exposure [PfSPZ-CVac (pyrimethamine)]. The study is 
expected to be completed in early 2019.
The potency of PfSPZ-CVac in malaria-naïve adults, now demonstrated with both 
chloroquine and PYR as the partner drugs, raises the question whether this approach might 
also provide strong protective immunity in the field, when tested against naturally acquired 
malaria infection, particularly if doses were increased further to overcome the effects of 
naturally acquired immunity. An initial study of PfSPZ-CVac (chloroquine) based on this 
hypothesis has already been done in a partnership between DMID (NIAID), the University of 
Maryland, and Malaria Research and Training Center (MRTC) at the University of Bamako in 
Mali (NCT02996695). Participants received 3 monthly immunizations with PfSPZ-CVac 
(chloroquine) [2.048x105 PfSPZ of PfSPZ Challenge] or normal saline (NS) administered by 
DVI using a double-blind design. The chloroquine was administered weekly to both vaccine 
and placebo groups, with a last chloroquine dose administered 5 days after the 3rd 
immunization. Immunization was done during the dry season, and protective immunity was 
assessed against natural transmission over 24 weeks during the ensuing wet season. Although 
previous PfSPZ-CVac studies to this point had been conducted with 5.12x104 PfSPZ, it was 
thought that increasing the dose of PfSPZ to 2.048x105 was necessary in this malaria endemic 
population in order to overcome pre-existing antimalarial immunity in Malian participants, to 
convey long-term protection over 24 weeks of follow-up, and to overcome malaria parasite 
antigenic diversity seen in malaria-endemic areas. During the 6 months of follow up post 
immunization, fewer vaccinated participants (16 of 29, or 55%) were infected than controls 
(22 of 30, or 73%), but this difference was not statistically significant in this small study 
(n=31 per group). Vaccine efficacy (VE) by the proportional method was estimated as 0.248 
(95% C.I. [-0.048, 0.543]; p = 0.100), and VE by the hazard ratio method was estimated to be 
0.336 (95% C.I. [-0.279, 0.655]; p = 0.221). Although there was no statistically significant 
difference in the incidence of malaria parasitemia between the vaccinated and the control 
groups, immunization resulted in lower infection rates, indicating that further development of 
this approach is merited, especially if still larger doses of PfSPZ might improve outcome. 
This proposed study builds on this experience. Use of PYR is attractive because it potentially 
eliminates the period of parasitemia following immunization that is a safety concern 
associated with PfSPZ-CVac (chloroquine). Our approach to testing PfSPZ-CVac 
(pyrimethamine) in endemic areas is to first test a more practical regimen in which PYR is 
administered on the same day (as opposed to administration on 2 and 3 days post injection 
that has been used in the malaria naïve population to date) as the injection with 2.0 x105 
PfSPZ in a pilot study. We also suspect that a higher dose of PfSPZ Challenge will be 
needed in both PYR and chloroquine arms in this population, owing to evidence that vaccine 
responses including PfSPZ responses are often suppressed in malaria-exposed populations, 
thus we will test that the higher dose is safe and well tolerated in the pilot study before 
proceeding to the main study. The main study will assess the efficacy of these various 
regimens against naturally transmitted malaria.


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 35 of 146
1.4
Previous Human Experience
1.4.1
Whole SPZ Vaccination 
Sanaria Inc. has developed methods to produce aseptic, purified, cryopreserved PfSPZ that are 
manufactured according to current Good Manufacturing Practices (cGMP) and are compliant 
with FDA and EMEA guidances regarding investigational products for human testing. Several 
PfSPZ-based products have been made, including non-attenuated PfSPZ [PfSPZ Challenge 
(NF54) and PfSPZ Challenge (7G8), used for CHMI and for PfSPZ-CVac immunization], 
PfSPZ Vaccine (radiation-attenuated SPZ used to immunize), PfSPZ-GA1 (a genetically 
attenuated parasite used to immunize) and PfSPZ-GA2 (a second genetically attenuated 
parasite) (Richie, Billingsley et al. 2015). The manufacturing, formulation, vialing and 
cryopreservation of each product is identical, except for the irradiation step that is added for 
PfSPZ Vaccine. 
Considering all clinical experience with these products, as of June 2018, 2040 individuals 
have received 5,403 injections with PfSPZ Challenge, PfSPZ Vaccine or PfSPZ-GA1. Safety 
and tolerability data indicate that these products are safe and well tolerated. After 
disappointing results in early trials where PfSPZ Challenge and PfSPZ Vaccine were 
administered by subcutaneous (sc), intradermal (ID), intramuscular (IM), routes, all PfSPZ-
based products are now administered by DVI. The most advanced Sanaria product, PfSPZ 
Vaccine, has been studied in 18 clinical trails, including 7 double blind, placebo-controlled 
trials conducted in African populations, and there have been no significant differences 
between vaccinees and NS placebo controls by any safety measure, including solicited AEs, 
unsolicited AEs and laboratory abnormalities. The trials conducted in malaria-naïve adults in 
the USA have not included blinded control groups, but in general, findings are consistent with 
the African trials: low rates of AEs that likely reflect background rates. In summary, during 
the course of 16 studies of PfSPZ Vaccine where immunizations have been completed (not 
counting recently initiated trials in children in Gabon and adults in Mali), 1157 research 
subjects have safely received 3786 doses by sc, ID, IM, intravenous via catheter (IV) or DVI 
routes (as high as 2.2x106 PfSPZ im and 2.7x106 PfSPZ by DVI), comprising 652 adults and 
505 children. Discounting ID, IM and IV catheter routes, this includes 448 adults age 18-65 
years, 12 children age 11-17 years, 12 children age 6-12 years, 36 children age 1-5 years, and 
400 infants age 5-11 months, who have received PfSPZ Vaccine by DVI. PfSPZ Vaccine has 
proven safe and well tolerated, with no AEs or allergic reaction clearly linked to the vaccine. 
The favorable safety and tolerability profile of PfSPZ Vaccine and other PfSPZ-based 
products has helped to accelerate their development. 
The only AEs that are clearly a result of injection with PfSPZ are the malaria-related 
symptoms that occur in conjunction with the parasitemia that develops after injection of 
PfSPZ Challenge. When PfSPZ Challenge is used to immunize as part of the PfSPZ-CVac 
approach, with chloroquine as the partner drug, these adverse parasitemia-related signs and 
symptoms occur 7-9 days after injection of PfSPZ Challenge but end quickly as the parasites 
are killed by the chloroquine, and generally are markedly reduced after the second and third 
immunizations compared to after the first, because of the rapid development of immunity. 
These side effects are discussed in more detail below.
 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 36 of 146
1.4.2
PfSPZ Challenge (NF54) for Immunization
An important role of PfSPZ Challenge (NF54) is its use as the immunogen in the PfSPZ-
CVac approach to vaccinating against malaria. To date, 8 clinical trials of PfSPZ-CVac using 
PfSPZ Challenge have been performed (or are underway), varying the route of 
administration, the dose size, the interval between doses, and the partner drug. These trials are 
summarized in Table 4 below:
Table 4. Chronological Listing of Trials of the PfSPZ-CVac Approach to Immunization
Name
Site
Institution
First 
injection
N
Route
Partner drug # doses
Highest
#
PfSPZ
Interval 
betweeen 
injections
Best protection*
1. TIP-5
NCT01728701
Nijmegen
Radboud UMC
Sep 2012
20
id
CQ
3-4
7.5x104
4 weeks
0% (0/20)
2. TÜCHMI-002
NCT02115516
Tübingen
Inst for Trop Med Apr 2014
45
DVI
CQ, or CQ + 
azithromycin*
*
3
5.12x104
4 weeks,
2 weeks,
5 days
100% (9/9)
67% (6/9)
63% (5/8)
3. 15-I-0169 
NCT02511054
Bethesda
NIAID / LMIV
Nov 2015
20
DVI
CQ, or CQ + 
pyrimethamine
3
5.12x104
4 weeks
80% (4/5)
(CQ alone)
4. MALACHITE
NCT02858817
Tübingen
Inst for Trop Med Nov 2016
21
DVI
atovaquone / 
proguanil
3
1.5x105
4 weeks
27% (3/11)
5. DMID 11-
0042  
NCT02773979
Seattle
Group Health
Jan 2017
21
DVI
CQ
3
1.024x105
7 days
5 days
0% (0/8)
75% (6/8)
6. 17-I-0067
NCT03083847
Bethesda
NIAID / LMIV
Jun 2017
51
DVI
CQ, or 
pyrimethamine
3
2.0x105
4 weeks
87.5% (7/8) 
pyrimethamine 
homologous
100% (4/4) 
pyrimethamine 
heterologous
100% (3/3) CQ 
heterologous
7. EGSPZV2
NCT02859350
Equatorial 
Guinea Malaria 
Vaccine Initiative 
(EGMVI), Baney 
Clinic, EG
2017
26
DVI
CQ
3
1.0x105
4 weeks
Pending 
(homologous 
CHMI completed, 
data cleaning in 
process).
Part A
2017
36
DVI
CQ
3
2.048x105
4 weeks
Not applicable. 
Safety study
8. DMID 11-
0042
NCT02996695
Bamako Malaria 
Research 
Training Center, 
Mali / LMIV
Part B
2017
62
DVI
CQ
3
2.048x105
4 weeks
37.7% 
(proportional), 
47.1% (time-to-
event) over 24 
weeks
Total subjects:
302 (includes placebo controls)
* Percent protected (# protected/# undergoing CHMI). **Protection was in CQ alone in groups.


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 37 of 146
1.4.2.1 PfSPZ-CVac under Chloroquine Prophylaxis in Malaria-Naïve Populations
The first trial listed was performed in The Netherlands before it was known that id 
administration was suboptimal. PfSPZ-CVac (chloroquine) was tested using the id route, and 
although the PfSPZ were safe and well tolerated, there was no protection (Bastiaens, van 
Meer et al. 2016). 
Subsequently, a randomized, placebo controlled, double-blinded, dose finding trial of PfSPZ-
CVac (chloroquine) was performed in Germany using DVI administration (TÜCHMI-002 
trial, NCT02115516) to identify the dose needed to replicate the 100% protection results see 
following mosquito-bite immunization in Roestenberg et al. In stage A of the TÜCHMI-002 
trial, three groups (n=9 in each) received three PfSPZ Challenge administrations 4 weeks 
apart by DVI with 3,200, 1.28x104 or 5.12x104 PfSPZ of PfSPZ Challenge while under 
chloroquine prophylaxis. The latter was administered weekly for a total of 10 doses, starting 
with a loading dose 2 days before the first injection of PfSPZ Challenge and ending with a last 
dose given 5 days after the third injection of PfSPZ Challenge. A fourth group (n=9) received 
placebo injections in this double blind study. Nine to ten weeks after immunization, 
homologous CHMI was performed with 3,200 PfSPZ of PfSPZ Challenge administered by 
DVI, and 3/9 (3,200 PfSPZ-CVac group), 6/9 (1.28x104 PfSPZ-CVac group) and 9/9 
(5.12x104 PfSPZ-CVac group) of the subjects respectively were protected (Mordmuller, Surat 
et al. 2017). The results of the high dose group reproduced the 100% protection seen in 
Roestenberg et al and highlighted the importance of a sufficient antigen load required for VE. 
The investigation also demonstrated that a sufficient antigen load to achieve 100% protection 
is multifold lower when compared with the irradiated PfSPZ of PfSPZ Vaccine (Seder, Chang 
et al. 2013). 
Safety data from this study after unblinding showed that AE rates between the four treatment 
groups were not significantly different from each other. The events/laboratory abnormalities 
that were solicited/studied included pain, tenderness, erythema/redness, and 
induration/swelling at the injection site; fever (°C), tachycardia, bradycardia, hypertension 
(systolic, diastolic), abnormal respiratory rate; nausea/vomiting, diarrhea, headache, fatigue, 
myalgia; and abnormal levels of sodium, potassium glucose, bilirubin, creatinine (Cr), LDH, 
alanine transaminase (ALT), aspartate aminotransferase (AST), white blood count differential 
component count including neutrophils, lymphocytes, monocytes, basophils, eosinophils, 
erythrocyte count, hemoglobin, hematocrit. Rates of AEs are shown in Figure 1. There were 
no significant differences in the rates of AEs between the three injections of PfSPZ Challenge 
(data not shown) in any of the groups.


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 38 of 146
Figure 1. Frequency of adverse events in all 4 groups after PfSPZ Challenge DVI
Figure 1.  Total number of solicited and unsolicited adverse events (including abnormal signs and laboratory 
values) recorded per volunteer during administration of 3 doses of vaccine or placebo (y axis), in each PfSPZ dose 
group (x axis).  The period covered is from day -3 to day 77.
Parasitemia was seen by qPCR in all PfSPZ-immunized volunteers after the first 
immunization with PfSPZ Challenge (except one of the volunteers in the group receiving 
3,200 PfSPZ where technical issues prevented completion of the assay for this sample), and 
in a decreasing number of volunteers after the second and third immunizations, this reduction 
especially apparent in the highest dose group (Table 5). Parasitemia typically occurred during 
the 6-10 day interval after PfSPZ Challenge administration, peaking on days 7 or 8. 
Table 5. Summary of PfSPZ dose sizes during PfSPZ-CVac (chloroquine) immunization 
and the number of qPCR positive subjects following each immunization
PfSPZ Challenge dose 
per immunization (3 
PfSPZ)
Number Subjects Positive by qPCR days 6-10
Immunization 1
Immunization 2
Immunization 3
3,200
8 / 8*
9 / 9
6 / 9
1.28x104
9 / 9
8 / 9
7 / 9
5.12x104
9/ 9
8 / 9
3 / 9
* Technical difficulties precluded assessment of one volunteer’s sample
An important, even if expected, finding of the study was that the density of parasites during the 
period of transient parasitemia increased with increasing doses of PfSPZ Challenge, 
approximately doubling with each 4-fold increase in PfSPZ dose. The geometric mean parasite 
densities (ranges) after the first immunizations in the low, medium and high dose groups in 
TÜCHMI-002 were, respectively, 462 (228-989), 711 (120-1,891) and 15,652 (5,146-36,404) 
parasites/mL (Table 6).
0
1
2
3
4
5
6
7
8
0
3200
12800
51200
Grade 1
Grade 2
Grade 3


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 39 of 146
Table 6 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 40 of 146
Table 6. Summary of PfSPZ dose sizes and mean parasite density after each immunization
* parasite equivalents per mL, including only those subjects who were positive by PCR
The highest parasitemia identified in the TÜCHMI-002 trial followed the first immunization in 
a volunteer in the high dose group: 36,404 parasites/mL. Densities above 10,000 per mL may 
be positive by thick blood smear, and may lead to malaria symptoms. Nevertheless, thick blood 
smear readings were negative in this volunteer and in all volunteers at all time points. The 
reason for the negative blood smear in the volunteer with 36,404 parasites/mL (which we 
expected should have been positive) may have related to the moribund state of the parasites 
(DNA positive by PCR, but morphology distorted on thick smear).
The volunteer experiencing the density of 36,404 had an axillary temperature to 38°C and 
symptoms of fever, fatigue and tachycardia on days 8–10 in association with this parasitemia. 
While this volunteer later developed a urinary tract infection, the investigators considered that 
the fever and symptoms were nevertheless potentially related to the parasitemia. With the 
exception of this volunteer, there were no increases above background in the frequency of 
malaria-related symptoms such as fever, headache or myalgia during the periods of 
parasitemia, in any groups.
Thus, in summary, there was no clear indication that immunization with up to 5.12x104 PfSPZ 
of PfSPZ Challenge caused any excess of abnormal signs, symptoms or laboratory values 
relative to NS placebo, with the possible exception of one volunteer in the 5.12x104 PfSPZ 
group.
In stage B of the study, condensed regimens were studied: three doses of 5.12x104 PfSPZ were 
administered to 19 volunteers divided into three cohorts, and concurrently three doses of NS 
were administered to 6 volunteers in blinded fashion (two per cohort). The timelines for the 
regimens were shortened relative to Stage A: 10 volunteers received doses of PfSPZ Challenge 
on days 0, 14 and 28, and 9 volunteers received doses of PfSPZ Challenge on days 0, 5 and 10. 
Half of the 10 volunteers on the four-week schedule received 2g extended release azithromycin 
(Zithromax UNO, Pfizer) in addition to chloroquine with the first dose of PfSPZ Challenge, to 
see whether azithromycin could prevent the development of parasitemia during immunization. 
Azithromycin was not successful in preventing parasitemia, so its use was dropped for the 
second and third immunizations. AE profiles on days 7-10 after each immunization were 
similar to those seen in Stage A, and did not suggest that parasitemias were high enough to be 
symptomatic. CHMI was conducted using PfSPZ Challenge administered by DVI 10-11 weeks 
after the last immunization, and 6/9 (67%) volunteers were protected after the 0, 14 and 28 day 
regimen, and 5/8 (63%) volunteers were protected after the 0, 5 and 10 day regimen. These 
results showed that the PfSPZ-CVac approach induced sterile immunity even after highly 
condensed regimens, although there was a reduction in VE compared to the 100% protection 
seen with the 0, 28 and 56 day regimen. 
Geometric Mean Parasite Density (range)*
Dose (PfSPZ)
Immunization 1
Immunization 2
Immunization 3
3,200
462 (228-989)
78 (2-1,236)
210 (31-2,170)
1.28x104
711 (120-1,891)
195 (13-762)
57 (14-417)
5.12x104
15,652 (5,146-
36,404)
555 (63-3,089)
42 (11-126)


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 41 of 146
There was one unanticipated safety issue. One of the volunteers receiving the 0, 14 and 28 day 
regimen of PfSPZ Challenge experienced parasitemia by PCR on day 7, peaking on day 8 and 
then falling progressively on days 9 and 10, which is the typical pattern. But then atypically, 
the subject showed increasing parasitemia on day 11, and was found to be blood smear positive 
on day 13, during which time the subject also experienced mild symptoms of malaria 
(headache, sweating). The volunteer was treated with atovaquone/proguanil on days 13-15 and 
cleared the parasitemia. A retrospective review of the day 11 blood smear with more intensive 
searching revealed trace parasites. This subject’s plasma chloroquine levels on day 13 were 
less than 2 ng/mL (certified limit of assay quantification 5 ng/mL) although trace levels of 
chloroquine were present. On questioning, the volunteer stated that he did swallow the 
chloroquine tablets. The team proposed a pharmacokinetic study of chloroquine absorption and 
metabolism in this volunteer to explain the apparent poor bioavailability. The volunteer 
initially agreed to undergo this study, and to take another dose of 10 mg/kg chloroquine with 
additional blood samplings to diagnose possible causes of the low plasma levels. However, 
when the pharmacokinetic study had been set up, the volunteer refused to participate. 
Therefore, the reason for the low chloroquine levels in this volunteer may never be definitively 
determined. The suspicion is that the volunteer kept the chloroquine tablets in his cheek and 
discarded them later. The small amount of absorption that would have taken place through the 
oral mucosa would explain the trace levels found on day 13. Post-ingestion oral examinations 
for retained tablets were not performed in this study.
The parasites from this volunteer were isolated on day 13 and tested in vitro for chloroquine 
resistance, and were found to be highly sensitive to chloroquine, consistent with all testing that 
has been performed on PfSPZ Challenge in the past. As a result of this unanticipated event 
(inadequate levels of chloroquine), subsequent studies measured levels of chloroquine prior to 
anticipated peaking of parasitemia. To date, the measured levels have been adequate. 
Chloroquine levels will thus not be measured in the current study. The safety issue in this 
volunteer is deemed related to poor compliance, and since this event, oral examinations have 
been performed to confirm chloroquine ingestion. Rigorous follow-up is always done starting 
on day 7 after immunization, since any uncontrolled parasitemia should quickly manifest as 
clinical signs and symptoms, and, because of daily follow-up, does not involve any greater risk 
than the risks of CHMI.
  
Although PfSPZ-CVac (chloroquine) with 5.12x104 PfSPZ of PfSPZ Challenge in three 
successive 4-weekly vaccinations led to 80-100% protective efficacy against homologous 
CHMI (the result depending on the intervals between injections,) it is likely that a higher dose 
of NF54 PfSPZ Challenge will be required to achieve protection against heterologous CHMI. 
As mosquitoes in endemic areas harbor multiple strains of Pf, it will be important to 
demonstrate that vaccine candidates can protect against heterologous strains. 
Therefore, a phase 1, blinded, randomized, dose escalation trial to assess the safety and 
efficacy of PfSPZ-CVac (chloroquine) using higher doses of PfSPZ was initiatiated, recruiting 
healthy malaria-naïve adults in Seattle (Clinicaltrials.gov Identifier: NCT02773979). The plan 
was for 3 study groups to receive doses of 5.12x104, 1.024x105 or 2.048x105 PfSPZ 
respectively. Each group targetted 12 subjects randomized 3:1 to PfSPZ Challenge or NS, and 
was to receive 3 vaccinations at 7 day intervals (days 0, 7, 14). This condensed weekly 
regimen, similar to (but not identical to) the 5 day interval regimen studied in Part B of the 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 42 of 146
TÛCHMI-002 trial, was selected for the Seattle trial because of the practicality of rapid 
immunization schedules and the convenience of coming in for an immunization on the same 
weekday each week for three weeks. Enrollment of successive groups for dose escalation was 
staggered, with each higher dose group to be enrolled after the lower dose group had received 
all three vaccinations. 
The first study group raised unexpected safety concerns. Subjects in the low dose group 
(5.12x104 PfSPZ at 7 day intervals) experienced a higher frequency and severity of AEs (Table 
7) in comparison to the PfSPZ-CVac (chloroquine) studies where the same dose had been 
given at 4 week, 14 day or 5 day intervals (NIAID protocol # 15-I-0169; (Mordmuller, Surat et 
al. 2017). There were 2, 7 and 1 Grade 3 (severe) AEs after vaccinations 1, 2 and 3, 
respectively, and no Grade 3 AEs had occurred in our study at National Institutes of Health 
(NIH) or in Germany. These included high grade fever, severe malaise, severe myalgia, severe 
arthralgia and severe chills, all occurring approximately 8 days after receiving the first, second 
or third injection with PfSPZ Challenge, and coinciding with periods of transient parasitemia. 
Due to the AEs, one volunteer was dropped out prior to the third immunization, reducing the 
sample size from 9 to 8. One potential explanation for the unexpected frequency of Grade 3 
AEs was that there was exaggerated cytokine release due to the administration of a large 
number of PfSPZ in the presence of blood stage parasites, which peaked in density on roughly 
the seventh and eighth days after each injection of PfSPZ Challenge. 
Table 7. List of AEs post PfSPZ-CVac (chloroquine) immunization with weekly 5.12x104 
PfSPZ of PfSPZ Challenge administered by DVI in NCT02773979
Note: X (X) X% refers to Absolute number of AEs (Number of subjects with AEs) Percent of subjects with 
AEs
* vaccinations were performed on days 3, 10 and 17, so periods of parasitemia initiated 7 days later, i.e., days 10, 
17 and 24.
** 1 subject did not receive Vax #3 but was followed for safety
*** Data only available for 6 subjects followed up to study day 25, 22 days post PfSPZ-CVac immunization #1
**** Malaria symptoms are expected around 7-8 days post injection, corresponding to the period of transient 
parasitemia
Due to these adverse findings, the design of the study was modified and the highest dose 
administered for vaccination was limited to 1.024x105 PfSPZ. The third group reverted to 5 
day intervals between injections, a scenario where PfSPZ Challenge injections #2 and #3 are 
done prior to, not concurrently with, the period of transient parasitemia resulting from the prior 
injection, which does not start until the seventh day. Consistent with the parasitemia 
hypothesis, the AE profile of this group greatly improved, with no Grade 3 AEs recorded. 
Efficacy results further supported the hypothesis that PfSPZ Challenge should not be injected 
during parasitemia: the protective efficacy of Group 1 against CHMI at week 10 was 0% (0/7 
volunteers protected), whereas for Group 3 (1.024x105 PfSPZ at 5 day intervals), protective 
efficacy was 75% (6/8 volunteers protected), similar to the results of the 5-day interval 
assessed in the TÜCHMI-002 trial (63%, see above). Group 2 received 1.024x105 PfSPZ at 7-
day intervals with better tolerability than Group 1, but did not undergo CHMI due to the 
absence of protection seen in Group 1. 
Study day*
Post CVac #1
Post CVac #2
Post CVac #3
3 - 9 (CVac #1, n=9)
0 to 6
N/A
N/A
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
10 - 16 (CVac #2, n=8)
7 to 13
0 to 6
N/A
29 (6) 75%
4 (2) 25%
2 (1) 13%
35 (7) 88%
17 - 23 (CVac #3, n=8**)
14 to 20
7 to 13
0 to 6
36 (7) 88%
20 (6) 75%
7 (2) 25%
63 (7) 88 %
24 - 25 (post CVac #3, n=6***)
21 to 22
14 to 15
7 to 8
16 (4) 67%
4(2) 33%
1 (1) 17%
21 (4) 67 %
Grade 3
Total
Days post PfSPZ Challenge Injection****
Grade 1
Grade 2


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 43 of 146
1.4.2.2 PfSPZ-CVac (chloroquine) in Malaria Experienced Populations in Mali and 
Equatorial Guinea
The good tolerability and excellent protective efficacy of PfSPZ-CVac (chloroquine) in malaria 
naïve populations from the MAVACHE trial in Germany and in Group 3 in Seattle led to a 
study to assess the development of protective efficacy against natural transmission. This was a 
randomized, double-blind placebo controlled single center study conducted by MRTC in Mali 
(Clinicaltrials.gov: NCT02996695) to evaluate the safety, tolerability, immunogenicity and 
efficacy against naturally occurring malaria in malaria-exposed Malian adults. Participants 
were randomized into two groups of 31 research subjects to receive 3 monthly injections of 
either 2.048x105 PfSPZ of PfSPZ Challenge or NS by DVI under weekly chloroquine 
prophylaxis. The last dose of chloroquine was given at 5 days post vaccination #3 followed by 
clearance with artesunate monotherapy for 7 days. Follow up to record incident natural malaria 
infection was started two weeks later. As briefly outlined in Section 1.3, during the 6 months 
of follow up post immunization, fewer vaccinated participants (16 of 29, or 55%) were infected 
than controls (22 of 30, or 73%), but this difference was not statistically significant. 
The regimen was safe and well tolerated. Reviewing the blinded data (31 vaccinees and 31 
placebo recipients combined), a total of 32 subjects experienced at least one solicited AE 
within 12 days following any dose. Of the 32 subjects, 13 experienced at least one solicited 
systemic AE, and 24 subjects experienced at least one solicited local AE, reported on the day 
of vaccination. All the local solicited AEs were mild (Table 8). All systemic AEs were mild, 
except in the case of one subject who experienced a Grade 3 (severe) fever on Day 7 after the 
first immunization. This AE, along with a Grade 2 (moderate) headache occurring in the same 
individual, were assessed by the investigator as unrelated to immunization and related to an 
intercurrent upper respiratory infection (Figure 2). There was 1 serious adverse event (SAE) 
that was not related to the study that occurred 157 days after immunization; the participant had 
a motorcycle accident that resulted in death due to cranial trauma.  


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 44 of 146
Table 8. Number and Percentage of subjects experiencing solicited events with 95% 
confidence intervals by symptom and treatment group
Figure 2. Maximum severity of solicited systemic symptoms per subject by day post 
treatment


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 45 of 146
Unblinded safety data from this Mali trial are not available at the current time. However, the 
frequency of moderate or severe AEs was low. Depending on the treatment assignment of the 
volunteer experiencing the one Grade 2 and the one Grade 3 systemic AE, the frequency was 
either 1/31 or 0/31 for Grade 2 or for Grade 3 AEs over the course of three immunizations, 
with a dose of PfSPZ (2.048x105) that was twice as high as the highest dose studied in Seattle 
(1.024x105 PfSPZ), and four times as high as the highest dose studied in the TÜCHMI-002 
trial in Germany and the chloroquine alone group in the first NIH PYR trial (5.12x104 PfSPZ). 
Regarding Grade 1 AEs, a worst case scenario (all systemic AEs occurring in the vaccine 
group and none in the placebo group), the frequency of Grade 1 AEs occurring on days 7-9 
after immunization was 6/31 (19.4%). 
These safety data indicate that the semi-immune status of the volunteers in the Mali trial 
impacted the tolerability of PfSPZ-CVac (chloroquine) immunization. Explanations could be 
(1) the effective dose of parasites was reduced due to neutralization of SPZ, elimination of 
liver stages or suppression of blood stages, leading to reduce density of parasites in the blood 
and therefore better tolerability, and/or (2) naturally acquired immunity allowed better 
tolerability of whatever density of parasitemia was present. It should be possible to better 
differentiate the contribution of each of these mechanisms once data on the density of 
parasitemia occurring after each immunization are available from the trial. In any case, the 
results of the study indicate that higher doses of PfSPZ could be safety studied in this 
population of semi-immune, malaria-exposed adults.
A second trial in Equatorial Guinea, EGSPZV2 (ClinicalTrials.gov Identifier: NCT02859350), 
has assessed the safety of PfSPZ-CVac (chloroquine) in African adults in one group of 
volunteers (out of 8 total groups in the study). The PfSPZ-CVac (chloroquine) group in this 
trial, Group 1b, started in January 2017, 3 months prior to the Mali PfSPZ-CVac (chloroquine) 
trial just discussed (protocol 15-0052). Group 1b participants, young adults age 18-35, were 
randomly assigned to received 3 injections with 1.0x105 PfSPZ of PfSPZ Challenge (NF54) 
(n=19 for the first immunization, n=18 for the second and third immunizations) or NS placebo 
(n=5 after the first two immunization, n=4 after the third immunization) at 4-week intervals, 
using a double blind design. The PfSPZ dose in vaccinees was ~2-fold higher than the highest 
dose in TÜCHMI-002 and ~half the dose of the protocol 15-0052 in Mali. All Group 1b 
volunteers were administered chloroquine using the standard regimen, a loading dose 2 days 
before the first injection of PfSPZ Challenge followed by weekly maintenance doses. Vaccine 
recipients experienced local (site of injection) AEs on six occasions after the 55 vaccinations 
(rate 0.1 AE/study subject/injection). The local AEs consisted of mild pain or mild tenderness. 
No local AEs were reported by NS recipients after their 14 vaccinations. Vaccine recipients 
experienced systemic AEs on 28 occasions (0.51 AE/study subject/injection) after the 55 
vaccinations, and placebo recipients experienced the same on 10 occasions after their 14 
vaccinations (0.71 AE/study subject/injection, a slightly higher rate than for vaccine 
recipients). The systemic adverse event rates per injection are presented in Figure 3. There was 
no statistically significant difference in rates between vaccine and placebo recipients. 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 46 of 146
Figure 3. Solicited Systemic Adverse Events per injection during EGSPZV2 trial.
The AEs included two episodes of systemic pruritus in vaccinees, both mild in severity, one 
occurring day 1 after the first immunization (day 3 after chloroquine load) and one occurring 
day 2 after the first immunization (day 4 after chloroquine load). Both resolved spontaneously 
without treatment and neither reoccurred with ongoing chloroquine administration or after the 
second or third vaccination. Both were deemed likely associated with chloroquine, which is 
reported to cause pruritus in about 50% of dark skinned persons receiving chloroquine for 
malaria treatment, noting that the prophylactic doses of chloroquine administered to volunteers 
in the EGSPZV2 trial were lower than treatment doses usually associated with pruritus. 
Vaccinees experienced 6 episodes of headache (two moderate in severity), 3 episodes of 
abdominal pain (1 moderate in severity), 2 episodes of diarrhea (1 moderate in severity), 2 
episodes of dizziness, and 1 episode of a variety of other symptoms (see Figure 3). Rates in 
placebo recipients were similar to those in vaccinees or not statistically significantly different. 
There were no concerning trends in hematological (hemoglobin, white blood cells [WBCs], 
platelets) or biochemical (Cr, ALT and AST) laboratory markers.
Parasite Densities: Twenty-two volunteers completed all three PfSPZ-CVac or placebo 
immunizations in Group 1b, 1 completed two immunizations and 1 completed only the first 
immunization. Dropping out from further immunizations was not due to AEs. PCR data (which 
remain blinded at this point) from the 22 showed that 12 were PCR positive for parasites 
during all three periods of potential parasitemia (days 6-10 after each injection of PfSPZ 
Challenge), 1 was positive during the first and third periods, 1 was positive during the first and 
second periods, and 3 were positive only during the first period. The remaining 5 out of 22 
volunteers were PCR negative during all three periods, and appear likely that 4 of these were 
the NS controls. Two additional volunteers received one or two immunizations and did not 
show parasitemia, indicating that three true-immunized volunteer did not show parasitemia 
after the first immunization. This can be deduced because the group includes 4 controls, and 7 
volunteers were PCR negative after the first dose of PfSPZ Challenge. Peak parasitemias 
occurred on days 7 or 8, after either the first or the third immunization (none peaked after the 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 47 of 146
second), with densities ranging from 0.2 to 10.3 parasites/μL (geometric mean of all peak 
parasitemias = 1.35 parasites/μL), which was lower than what was observed in the TÜCHMI-
002 trial (geomean 15.7 parasites/μL after the first immunization), noting that the dose of 
PfSPZ Challenge administered in EGSPZV2 was ~ two times higher (5.12x104 PfSPZ of 
PfSPZ Challenge in TÜCHMI-002 and 1.0x105 PfSPZ of PfSPZ Challenge in EGSPZV2). 
This reduction in the density of transient parasitemia despite a higher PfSPZ dose is likely 
related to natural acquire immunity in these volunteers, who live in a malaria-endemic area, 
and have experienced life-long exposure. Not surprisingly, given these low densities, no blood 
smears were positive. Parasitemia data are provided in Table 9 and Figure 4. It was concluded 
that lower parasite densities certainly contributed to improved tolerability, but there could 
additionally be a factor of improved tolerability at any given parasite density, relative to 
malaria-naïve individuals.
Table 9. Parasitemia Indices (Protocol EGSPZV2)
Group
Dose1
Dose 2
Dose 3
Group Name
Dose
Interval # pos / # 
imm
Median 
peak 
parasit
Range 
peak 
parasit
# pos / # 
imm
Median 
peak 
parasit
Range 
peak 
parasit
# pos / # 
imm
Median 
peak 
parasit
Range 
peak 
parasit
2b vaccinees 1.0x105 4 weeks
17/18
0.758
0-10.284
13/18
0.456
0-2.169
15/18
1.037
0-10.278
Table 9: the number of study subjects with parasitemia detected after each immunization relative to the number 
injected, the median peak parasite density (including zero values, in parasites/µL), and the range in peak density 
(including zero values, in parasites/µL).
Figure 4. Parasitemia Curves (Protocol EGSPZV2)
Figure 4: Parasite densities for all PfSPZ-CVac-immunized individuals in the EGSPZV2 trial. In each 
panel, PfSPZ Challenge was injected by DVI on day 0. Daily qPCR assays of parasite density were 
initiated on day 6. The lower limit of detection (LOD) was 0.020 parasites/µL. The volume of blood 
processed for detection of nucleic acid was 180 uL. Any undetected parasitemias are arbitrarily denoted as 
zero. No additional treatment beyond the prescribed partner drug (CQ) was administered and no 
volunteers included had recrudescent parasitemias. Note that several volunteers had a positive signal on 
day 10 or later, but these did not appear to be viable parasites as all study subjects became negative and no 
parasitemias recrudesced.
Summary of PfSPZ-CVac (chloroquine) safety concerns: In summary, PfSPZ-CVac 
(chloroquine) safety data indicate that malaria-naive adults, depending on the dose of PfSPZ 
0.00
0.01
0.10
1.00
10.00
100.00
6
7
8
9
10
Parasites per µL (log +1E-5)
Time In Days
EGSPZV2 Injection 1
341
353
379
397
401
404
407
446
458
459
508
509
510
512
515
517
518
519
525
526
527
528
529
530
0.00
0.01
0.10
1.00
10.00
100.00
6
7
8
9
10
Parasites per µL
Time In Days
EGSPZV2 Injection 2
341
353
379
397
401
404
407
446
458
459
508
509
510
512
515
517
518
519
525
526
527
528
529
530
0.00
0.01
0.10
1.00
10.00
100.00
6
7
8
9
10
Parasites per µL
Time In Days
EGSPZV2 Injection 3
341
353
379
397
401
404
407
446
458
459
508
509
510
512
515
517
518
519
525
526
527
528
529
530


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 48 of 146
Challenge administered, may experience self-limited, Grade 3 AEs during the period of 
transient parasitemia at doses on the order of 2.0x105 PfSPZ. However, initial experience from 
the ongoing PfSPZ-CVac (pyrimethamine) trial at NIH (protocol 17-I-0067) indicated that the 
Grade 3 AEs can be completely prevented by administration of an anti-pyretic/anti-
inflammatory beginning on the 7th day after administration of PfSPZ Challenge. In contrast, 
malaria-exposed Africans with naturally acquired immunity experience, at most, mild (Grade 
1) AEs, and these occur in a minority of individuals, and the rates of these AEs was similar to 
that of NS controls in the Equatorial Guinea trial, with the same result likely in Mali when the 
study is unblinded, since the rate of AEs in the combined vaccine and NS groups was very low.
The reason for the difference in tolerability profiles between malaria-naive and malaria-
exposed individuals is almost certainly naturally acquired immunity, which is known to include 
anti-toxic functionalities that reduce the symptomatology associated with a given density of 
parasitemia (Schofield and Mueller 2006, Doolan, Dobano et al. 2009), although the 
mechanisms for this clinical tolerance are not yet identified. Naturally acquired immunity also 
appears to reduce the density of parasitemia by an undetermined mechanisms, which could be 
neutralizing PfSPZ, developing liver stage parasites, and/or developing blood stages. Evidence 
for reduced parasite densities in those with naturally acquired immunity compared to malaria-
naive individuals comes from the EGSPZV2 trial, where parasite densities were ~10 times 
lower than in TÜCHMI-002 despite a 2-fold higher inoculum with PfSPZ Challenge.
The plan to increase PfSPZ Challenge doses for the PfSPZ-CVac (chloroquine) groups in the 
current study to 4.0x105, twice the dose that appeared to be excellently tolerated in Mali, 
appears reasonable at this stage and is likely to be as well tolerated as lower doses. We do 
anticipate some cases of chloroquine induced, self-limited pruritus, as has been seen in 
Equatorial Guinea, but the incidence should be low, since pruritus is more likely to occur after 
therapeutic doses of chloroquine than the lower chemoprophylactic doses used during PfSPZ-
CVac immunization (Gama, Ismael et al. 2009). Other CQ-induced side effects such as 
dizziness, blurred vision and tinnitus may also occur.
1.4.2.3 PfSPZ-CVac (chloroquine) and PfSPZ-CVac (pyrimethamine) in Malaria Naïve 
Populations
As summarized in Section 1.3, following the initial trial of PfSPZ-CVac (pyrimethamine) 
where the vaccine was safe but not sufficiently protective, a second open label, single center, 
phase 1 study has been undertaken at the NIH Clinical Center starting in 2017 (NIAID protocol 
#17-I-0067, ClinicalTrials.gov Identifier: NCT03083847). This is a phase 1 dose escalation 
study to investigate the safety and tolerability of PfSPZ-CVac (pyrimethamine) and PfSPZ-
CVac (chloroquine) with PfSPZ administered by DVI. The study was designed to explore the 
immunogenicity and protective efficacy of this regimen, given as a 3-dose series, against 
homologous Plasmodium falciparum, NF54 (PfNF54) and heterologous (Pf7G8) CHMI. As 
stated in Section 1.3, the study has generated preliminary data showing the PfSPZ-CVac 
(pyrimethamine) using doses of 2.0x105 PfSPZ of PfSPZ Challenge with each immunization 
can provide high level protection against heterologous CHMI conducted 12-13 weeks after 
immunization. This provides the core rationale for testing PfSPZ-CVac (pyrimethamine) in 
Mali under the current protocol. Safety data from the 17-I-0067 trial are presented here. 
The study is divided into a pilot phase and a main phase. The pilot phase used small cohorts 
(n=2 for each group) to study whether the sequential increase of the dose of PfSPZ Challenge 
from 5.0x104 to a target dose of 2.0x105 SPZ while on either chloroquine or PYR is safe (in 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 49 of 146
particular, no breakthrough parasitemia in the PYR group) and well tolerated (in particular, the 
transient parasitemia that occurs in the chloroquine group is well tolerated). For the PYR pilot 
(Arm 1x), participants received 2 doses of PYR (50 mg), administered on 2 and 3 days post 
PfSPZ Challenge. For the chloroquine pilot (Arm 5x), participants received 2 doses of 
chloroquine, a loading dose (1000 mg salt) 1-2 days before PfSPZ Challenge and a second 
dose (500 mg salt) 5 days post PfSPZ Challenge. The pilot phase participants received only 
one PfSPZ Challenge injection and were not enrolled in the main study and did not undergo 
CHMI. 
In the pyrimethamine pilot arms (n=8), all doses from 5.0x104 to 2.0x105 PfSPZ of 
Challenge SPZ were well tolerated. Most of the observed AEs were Grade 1 and many unlikely 
related to study participation. There was one transient Grade 3 AE of heat exhaustion in a 
participant who received the intermediate dose of 1.0x105 PfSPZ of PfSPZ Challenge that was 
not related to study participation. A second Grade 3 AE of costochondritis, unrelated to study 
participation, occurred in a subject who received 2.0x105 PfSPZ of PfSPZ Challenge. All AEs 
resolved without sequelae. Safety labs were also followed closely per protocol; there was one 
Grade 1 white count decrease and one Grade 1 ALT increase observed in one asymptomatic 
subject who received 2.0x105 PfSPZ of PfSPZ Challenge. The WBC decrease and ALT 
increase have resolved without sequelae (Table 10). 
Table 10. Summary of AEs by severity and frequency in pyrimethamine pilot study
Note: X (X) X% refers to absolute number of AEs (Number of subjects with AEs) Percent of subjects with AEs
In the chloroquine pilot arms (n=6), overall, there were more reported AEs and related AEs 
compared to the PYR pilot arms. However, the majority were Grade 1 expected AEs (Table 
11). At the dose of 1.0x105 PfSPZ of PfSPZ Challenge, neither participant had a positive blood 
smear (0/2). At the dose of 2.0x105 PfSPZ of PfSPZ Challenge, the majority of individuals 
(3/4; 75%) had a single positive blood smear with associated expected symptoms related to 
induced malaria, some of which were Grade 3 (fever and myalgia) in severity. Symptoms 
improved quickly or resolved with administration of 1 dose of NSAIDs (ibuprofen or 
acetaminophen). All subjects recovered and symptoms were at Grade 3 for less than 24 hours 
during the period of expected peak parasitemia. All chloroquine arm subjects were LMIV 
research qPCR positive for at least 2 consecutive days (day 7 and 8 post PfSPZ Challenge) as 
expected, with those experiencing the highest peak parasitemia being blood smear positive 
with associated Grade 3 symptoms. All participants were treated with Malarone® per protocol 
and completed their study participation per protocol. The summary of the AEs is outlined 
below (Table 11). 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 50 of 146
Table 11. Summary of AEs by severity and frequency observed in chloroquine pilot study
Note: X (X) X% refers to absolute number of AEs (Number of subjects with AEs) Percent of subjects with AEs.
The results of the pilot study were reviewed by the Safety Monitoring Committee (SMC) 
which recommended continuing on to the main study at the doses of 2.0x105 PfSPZ for both 
PfSPZ-CVac (pyrimethamine) and PfSPZ-CVac (chloroquine). However, the SMC 
recommended careful monitoring of Arm 3 (chloroquine arm) on the days of peak parasitemia 
and also the presumptive administration of NSAIDs on day 7-8 after each immunization, to 
help prevent Grade 2 and Grade 3 AEs. This intervention was added to the protocol as 
recommended by the SMC.
1.4.2.3.1 Main Phase
The main phase of this ongoing study is designed to assess the safety, tolerability and 
protective efficacy against CHMI of PfSPZ-CVac (pyrimethamine) and PfSPZ-CVac 
(chloroquine), using the maximum tolerated dose from the pilot trial, which for both vaccines 
was 2.0x105 PfSPZ. The main phase was divided into two cohorts, and at this time, only the 
first cohort, comprising approximately half of the planned participants, has been completed.
In the pyrimethamine arms, Arms 2a and 2b, (n=13) participants received three monthly 
injections of 2.0x105 PfSPZ of PfSPZ Challenge. There have been a total of 104 AEs; most of 
the AEs, 97/104 (93%) were Grade 1. The most commonly reported AEs were fatigue, 
palpitations, arthralgia, and myalgia. There were four Grade 2 AEs (myalgia, gastroenteritis, Cr 
increase and hemoglobin decrease). There were 2 Grade 3 AEs, a gastroenteritis that was 
deemed not related to the study and a neutrophil decrease that was considered related to the 
study. 
One participant reported three separate episodes of gastroenteritis that were all attributed to 
food that was consumed shortly before the symptoms began and not considered related to study 
products or procedures. One of the episodes was assessed as Grade 3 and one as Grade 2. 
Another participant reported a Grade 2 myalgia in her upper back after lifting a baby, six days 
after the third vaccination, not related to study products or procedures. 
Three participants reported one or more episodes of Grade 1 palpitations. Two of the 
participants experienced multiple, brief episodes of isolated palpitations that began within a 
few hours of PYR dosing. Both participants denied chest pain, shortness of breath or 
diaphoresis. These episodes were assessed by the investigator as possibly related to both PYR 
and PfSPZ Challenge due to the timing of onset. Cardiac monitoring, electrocardiogram and 
holter monitoring, did not reveal any abnormalities. A third participant experienced 
intermittent palpitations beginning 9 days after PfSPZ Challenge inoculation that persisted for 
several days associated with anxiety and evaluation by cardiology did not reveal any cardiac 
abnormalities. 
There was one reported episode of mild non-cardiac chest pain that occurred two days post 3rd 
PfSPZ Challenge injection and 1 dose of PYR, beginning approximately 12 hours after 
receiving PYR and lasting for approximately 4 hours. 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 51 of 146
Laboratory abnormalities have mostly been Grade 1. One participant had a Grade 2 decrease in 
hemoglobin deemed related to PfSPZ Challenge and/or PYR that occurred three days after 
PfSPZ Challenge injection, and a Grade 3 decrease in neutrophils that was also deemed related 
to PfSPZ Challenge and/or PYR that occurred three days after PfSPZ Challenge injection. This 
participant had pre-existing Grade 1 anemia and Grade 1-2 neutropenia. Another participant 
had a Grade 2 increase in serum Cr that occurred three days after PfSPZ Challenge injection. 
This participant had a pre-existing Grade 1 elevation in Cr. All abnormalities resolved within a 
few days without sequelae. 
The development of parasitemia was monitored by the NIH Malaria qPCR assay, which has a 
lower limit of detection of approximately 500 parasites/mL. qPCR was performed daily from 
days 6-14 post PfSPZ Challenge after first vaccination and from days 7-9 post PfSPZ 
Challenge after the 2nd and 3rd vaccinations. Participants were monitored with blood smears on 
days 6-10 after the 1st vaccination, and on days 6 and 10 after the 2nd and 3rd vaccinations. All 
NIH malaria qPCRs were performed real time for safety follow up. All results were negative 
after immunization with PfSPZ-CVac (pyrimethamine). A second, more sensitive, RNA-based 
qPCR assay, the LMIV qPCR assay, with a lower limit of detection of approximately 20 
parasites/mL, was performed retrospectively at the same time points, and confirmed that every 
timepoint was indeed negative (Figure 5 below). 
In the chloroquine arm, Arm 3 (n=5), based on the results of the Pilot Phase and the 
recommendations of the SMC, NSAIDs were administered presumptively on expected peak 
parasitemia days to improve tolerability on days 7 and 8 post-vaccination #1. Presumptive 
NSAIDs were not administered after vaccination #2 and #3. 50 AEs were reported, the 
majority (96%) were Grade 1, expected AEs. We thus observed decreased severity of AEs after 
vaccination #1 in the main phase compared to the chloroquine arms in the pilot phase of the 
study, likely due to addition of presumptive NSAID use (Table 12). 
The most commonly reported AE in the chloroquine Arm across all 3 injections was headache. 
Other commonly reported AEs include fatigue, tachycardia, arthralgia, and myalgia which are 
all expected AEs in the setting of malaria parasitemia. Most reported AEs were Grade 1. There 
were two unrelated Grade 3 SAEs described briefly below, and no Grade 2 AEs. Of note, there 
were fewer AEs reported with each subsequent injection. 
Laboratory AEs reported included one episode of decreased WBC count, three episodes of 
decreased absolute lymphocyte count (ALC) (in two different participants), and two episodes 
of decreased hemoglobin (both in the same participant), all of which were Grade 1. 
Table 12. Addition of NSAIDS improves tolerability of PfSPZ CVac (chloroquine)
PfSPZ Challenge with 
chloroquine
Grade 1
Grade 2
Grade 3
Total
Pilot Arm 5b, NO NSAIDS
19 (4) 100%
5(2) 50%
3 (2) 50%
27 (4) 100%
Main Arm 3, + NSAIDS
23 (5) 100%
0 (0) 0%
0 (0) 0%
23 (5) 100%
Note: X (X) X% refers to the absolute number of AEs (number of subjects with AEs) percent of subjects with 
AEs
Thick blood smears were completed daily from days 6-12 post PfSPZ Challenge. One blood 
smear was positive in one participant on day 8 after the first immunization with PfSPZ 
Challenge. Otherwise, all blood smears remained negative throughout. All participants had 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 52 of 146
subpatent parasitemia detectable by qPCR after the first PfSPZ Challenge administration as 
expected (Figure 5), although fewer subjects had detectable subpatent parasitemia with 
subsequent PfSPZ Challenge administrations, likely reflecting developing immunity. The 
decreasing frequency and density of parasitemia in second and third immunizations was 
expected as it has been a consistent finding in similar studies at LMIV and other centers. 
Two participants were withdrawn from the study due to SAEs. One participant experienced 
acute change in mental status that required hospitalization due to acute anticholinergic 
poisoning after ingesting the seeds of a Datura stramonium plant (also known as Jimson weed) 
for recreational use. This episode that occurred after vaccination #2 was determined to be 
possibly related to chloroquine due to the the possibility that it may have interacted with the 
toxins in the Datura plant. A second SAE occurred after vaccination #3 in a participant who 
was hospitalized after developing a spontaneous pneumothorax that required chest tube 
placement. The participant recovered completely and was discharged after 3 days. Further 
evaluation during the hospital admission showed radiographic evidence of COPD. The SAE 
was assessed as unrelated to PfSPZ Challenge or chloroquine. Both participants recovered fully 
and were withdrawn from the study prior to CHMI.
Figure 5. Parasitemia curves in the pyrimethamine and chloroquine groups
Figure 5. (1) Pyrimethamine given at 2 and 3 days post injection completely prevented blood stage 
parasitemia. (2) The expected parasitemia in the chloroquine groups diminished rapidly with 
progressive immunizations.
1.5
Clinical Trial Plan
Based on the promising preliminary results observed in the NIAID protocol 17-I-0067 described 
above, we plan to conduct a clinical trial in malaria-endemic regions of Mali to optimize and 
confirm safety, tolerability and development of protective immunity of new PfSPZ-CVac 
regimens in malaria-exposed adults against natural malaria transmission. We will investigate 
6
7
8
9
10
11
12
13
14
6
7
8
9
10
6
7
8
9
10
0
500
1000
1500
2000
2500
Days post injection
P.falciparum (Parasites/mL)
Chloroquine
Pyrimethamine
Vax #2
Vax #1
Vax #3
5/5
4/5
0/4


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 53 of 146
immunological parameters including humoral and cell-mediated responses to identify potential 
mechanisms of protection, and to explain differences in VE between PfSPZ-CVac regimens 
comparing when blood stage exposure is present (chloroquine arms) to when blood stage 
exposure may be absent (pyrimethamine arms). 
1.5.1
Rationale for a higher, one dose regimen of pyrimethamine
In the above described studies, in the PYR arms, participants have been dosed with 50 mg on 2 
and 3 days post injection with PfSPZ Challenge. This PYR regimen was sufficient in arresting 
parasite development in the liver as assessed by qPCR (Figure 5). Pharmacokinetics studies 
were conducted in the first LMIV PfSPZ-CVac (pyrimethamine) study (#15-I-0169; 
NCT02511054) described above. Samples to determine serum PYR levels were collected on 
days 6, 10 and 26 (4, 8 and 24 days post first dose of PYR) post vaccinations #1 and 2 and on 
days 6, 10 and 84 post vaccination #3. The analysis showed that PYR mean concentrations 
remained above the in vitro IC50 (2.74 ng/mL) for PfSPZ Challenge NF54 (Figure 6). This 
data suggests that if PYR is dosed earlier, 2 days earlier and on the same day as PfSPZ 
Challenge injection which is a more practical regimen, the serum levels should remain above 
therapeutic level and PYR should be effective in killing the parasites in the liver. In addition, in 
planning for a broader use of this PfSPZ-CVac (pyrimethamine) vaccine regimen such as 
incorporating with seasonal malaria chemoprophylaxis and intermittent preventive treatment in 
pregnancy (IPTp) in which adults receive an equivalent of 75mg of PYR per dose, the 
proposed study will also use one dose of 75 mg of PYR. From the available pharmacokinetic 
data, we can extrapolate that the achieved serum levels at this regimen will be sufficient to 
completely prevent the emergence of subpatent parasitemia. 
Figure 6: Serum pyrimethamine levels in PfSPZ-CVac (pyrimethamine) study 
NCT02511054 in malaria-naïve individuals
Figure 6: 50 mg of pyrimethamine was administered on days 2 and 3 post PfSPZ Challenge NF54 DVI. Blood 
samples were collected 4, 8 and 24 after the first dose of pyrimethamine at each vaccination #1 and #2. During 
vaccination #3, blood samples were collected on 4, 8 and 82 days post the first dose of pyrimethamine. Note X-
axis is time post the first dose of pyrimethamine.
1
10
100
1000
0
30
60
90
120
150
PYR Concentrations (ng/mL)
Time (Days)
PYR Mean (SD) Concentrations over Time (Days)
Male
Female
PfSPZ IC50


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 54 of 146
1.5.1
Rationale for administering a booster dose
The main phase of the study was designed to assess the safety and immunogenicity of three 
monthly doses of PfSPZ-CVac (PYR) or PfSPZ-CVac (CQ) with follow-up over an initial 
malaria transmission season.  Eligible participants on the main phase of the study will be 
offered a booster dose to extend the study through a second malaria transmission season.
A previous study of PfSPZ Vaccine (ClinicalTrials.gov Identifier NCT03510481) conducted 
by LMIV in Mali in recent years also employed a booster vaccination.  However, there have 
been no booster vaccinations given in an area of endemic malaria transmission after a primary 
series of PfSPZ CVac (PYR) or PFSPZ CVac (CQ) in the past. Administering a booster dose 
approximately 11 months after the initial course of vaccinations on this study would allow for 
an assessment of safety, immunogenicity, and efficacy of over a second malaria transmission 
season, which has never been performed before for this category of malaria vaccine.  
Understanding the role of boosting for this vaccine approach is essential to determine whether 
endemic populations who have received a primary vaccination series could benefit from 
receiving a booster dose prior to each transmission season rather than a repeated primary series 
of vaccine.  
1.5.1.1 Safety
The study began screening participants in May 2019.  The pilot phase consisted of 12 
participants who received 4.0x105 PfSPZ of PfSPZ Challenge on 23 May 2019 plus a partner 
drug of either CQ dosed on days -2 and -5 or PYR dosed on day 0 or on days +2 and +3.  All 
study medications and vaccinations were safe and very well tolerated with no evidence of 
breakthrough parasitemia by either sensitive blood smear or LMIV qPCR.  
The main phase began recruiting in May 2019 and enrolled 249 participants, of whom 240 
were randomized into either Arm 1b/4a or Arm 2b/4b and received at least one vaccination.  
All vaccinations for Arms 1b/4a and 2b/4b were completed as of 23 September 2019.  
Currently (March 2020), participants in Arms 1b/4a and 2b/4b are nearing the end of the 
malaria follow-up phase during the first transmission season.  Overall the study medications 
and vaccinations were safe and very well tolerated as summarized below.
Arm 1b/4a: PfSPZ-CVac (PYR) or normal saline with PYR administered on day 0 (n=144)
144 participants of a projected 144 received the first vaccination (or NS injection), with 140 
receiving the second and 139 receiving the third.  All participants were given 
artemether/lumefantrine approximately 7-14 days prior to first and third vaccinations per 
protocol.  All participants that remain on study are in the NHMI monitoring phase and have 
completed follow up visits through at least study day 224.  
There have been a total of 577 AEs to date. 231 (41%) were Grade 1, 339 (58%) were Grade 2, 
and 7 (1%) were Grade 3.  The most common AEs have been rhinitis (0/79 deemed related to 
study procedures, 72/79 Grade 2), elevated serum creatinine (50/65 related to study procedures, 
6/65 Grade 2 of which 5 were related to study procedures), decreased leukocyte count (38/63 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 55 of 146
related to study procedures, 5/63 Grade 2 of which 3 were related to study procedures), and 
decreased ANC count (32/52 related to study procedures, 11/52 Grade 2 of which 7 were 
related to study procedures, and 2/52 Grade 3 of which both were related to study procedures).
To date, there have been 7 Grade 3 AEs reported, of which the only two that were related to 
study procedures were the two episodes of neutropenia noted above.  In addition, there have 
been three episodes of elevated blood pressure, 1 episode of paronychia, and one episode of 
intestinal volvulus that was also an SAE (described in more detail below).  There have been no 
Grade 4 or Grade 5 AEs. 
Local site reactogenicity occurred in 3 participants (2%) including 1 episode of Grade 2 
injection site pain.  Solicited systemic AEs occurred in 13 participants (9%), 6 of which were 
Grade 2 (most commonly headaches).  
SAE: 
There was one SAE that occurred in this arm.  On 20 August 2019, participant CV-19-877-D 
presented to the study clinic with acute abdominal pain and was subsequently admitted to the 
referral hospital at Point G, Bamako and diagnosed with a sigmoid volvulus by ultrasound.  
The volvulus resolved with noninvasive medical management and the participant was 
discharged after two days.  On follow-up visits at the study clinic he continued to improve with 
resolution of all symptoms.  The volvulus was determined unlikely to be related to study 
procedures. The sponsor, DSMB and site safety monitor all agreed the participant could 
continue on study if he chose to.  He continued on the study with no further issues other than 
an episode of acute malaria contracted during the natural human malaria infection (NHMI) 
phase.
Arm 2b/4b: PfSPZ-CVac (PYR) or normal saline with PYR administered on days 2 and 3 
(n=96)
96 participants of a projected 96 received the first vaccination (or NS injection), with 95 
receiving the second and 93 receiving the third.  All participants were given 
artemether/lumefantrine approximately 7-14 days prior to first and third vaccinations per 
protocol.  All participants that remain on study are now in the NHMI monitoring phase and 
have completed follow up visits through at least study day 224.  
There have been a total of 424 AEs to date.  171 (42%) were Grade 1, 249 (57%) were Grade 
2, and 4 (1%) were Grade 3.  The most common AEs have been rhinitis (0/63 deemed related 
to study procedures, 55/60 Grade 2), elevated serum creatinine (45/56 related to study 
procedures, 9/56 Grade 2 of which 2 were related to study procedures), and decreased 
leukocyte count (36/55 related to study procedures, 2/55 Grade 2 of which 1 was related to 
study procedures), decreased ANC count (19/40 related to study procedures, 10/40 Grade 2 of 
which 5 were related to study procedures, and 1/40 Grade 3 which was related to study 
procedures).  
Local site reactogenicity occurred in 3 participants (3%), all of which were mild (Grade 1).  
Solicited systemic AEs occurred in 22 participants (22%), 13 of which were Grade 2 (most 
commonly headaches). 
To date, there have been 4 Grade 3 AEs reported, of which one episode of neutropenia as noted 
above was related to study procedures.  In addition, there was one episode of joint dislocation 
not requiring hospitalization and not related to study procedures.  There have been no Grade 4 
or Grade 5 AEs or SAEs. 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 56 of 146
1.5.1.2 Malaria infection rate 
Vaccine efficacy monitoring began after the third vaccination. Participants have been evaluated 
for malaria infection using blood smears every two weeks. The study is currently still blinded. 
Table 13: Parasitemia summary for main phase Arms 1b/4a and 2b/4b by calendar month 
post third vaccination as of 27 March 2020
BS = blood smear; Pf = Plasmodium falciparum
1.5.1.3 Futility criteria
An analysis of safety and of vaccine efficacy through the NHMI phase following the primary   
vaccination series will be conducted after all originally-planned primary and secondary 
endpoints from these objectives have been gathered.  The booster phase will proceed as 
planned unless both Arm 1b or Arm 2b fail to demonstrate statistically significant vaccine 
efficacy for the secondary endpoint, or unless the power is judged to be insufficient with the 
available numbers of participants.  
If one of the two PYR vaccination arms (Arm 1b or Arm 2b) meets all of the above criteria but 
the other does not, then a booster vaccination dose will be given in the qualifying arm (and NS 
given to its corresponding control arm) while NS will be administered to both the vaccination 
arm that failed to qualify and to its corresponding control arm.  This approach is meant to 
preserve the blinding of the study and to maximize the pooled control groups during the 
booster phase.
To assess if the power is sufficient to move forward with the booster phase, an analysis will be 
undertaken by the unblinded study statistician to perform sample size calculations for the 
booster phase.  The study will proceed unless the power to detect a vaccine efficacy of either 
50% by time-to-event analysis or 35% by proportional analysis, using the number of 
participants eligible for the booster vaccination minus 10% (to allow for loss to follow-up), is 
below 70%.  
Total + Pf BS 
post third 
vaccination
Pf + BS in unique 
individuals
% Unique individuals 
with Pf + BS
September 2019
21
15
6.4%
October 2019
79
58
25.0%
November 2019
74
29
12.5%
December 2019
26
7
3.0%
January 2020
8
1
0.4%
February 2020
9
2
0.9%
Total
217
112
48.2%


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 57 of 146
The study team will remain blinded to the analyses and the study statistician will communicate 
the results to the study team in a binary yes/no manner to either proceed or not with the booster 
dose.  If the booster phase does not proceed in either PYR arm for any reason, unblinding and 
statistical analysis may proceed.  
The same approach will be applied to the CQ group (Arms 3b/4c) at the appropriate time.
In addition, group unblinding for statistical analysis of blood smear results from the first year 
of the study (prior to the booster dose) will occur approximately 4 months following receipt of 
the booster vaccine dose, as long as 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 the 
pyrimethamine comparator arms on an accelerated vaccination schedule in a future planned 
trial.  
1.5.2
Study Plan 
The design of the proposed clinical trial entails 3 phases, a pilot phase, a main phase, and a 
booster phase: 
1) Pilot trial phase is divided in two additional parts, PYR and chloroquine phases: 
a) PYR only (Arms 1a, 2a, and if needed 5a, 6a): the pilot will simultaneously examine 
whether increasing the dose of PfSPZ Challenge to 4.0x105 PfSPZ with an increased 
dose of PYR to 75 mg given concurrently (day 0 only, Arm 1a) OR on day 2 &3 (Arm 
2a) is safe and does not result in patent parasitemia (sensitive blood smear [sBS] 
negative in 4 of 4 subjects in each Arm). If there is patent parasitemia in either Arms 
1a or 2a, the dose of PfSPZ Challenge will be decreased to 3.0x105 PfSPZ in Arms 
5a (PYR on day 0) or 6a (PYR on day 2 &3) respectively, the dose of PYR will be 
maintained. If breakthrough parasitemia occurs in Arm 5a or 6a the study will 
pause for reassessment.
b) Chloroquine only Arm (Arm 3a): to assess safety and to ensure that the increased 
dose of 4.0x105 SPZ while on weekly chloroquine regimen is tolerable (will not result 
in parasitemia related Grade 3 AEs lasting more than 48 hours despite adequate 
management). If symptoms are deemed intolerable, NSAIDs will be added to the 
regimen in the main phase of the study.(Arm 3b).
2) Main trial phase and booster phase will assess the safety, tolerability and VE of PfSPZ-CVac 
(pyrimethamine) 
a) Arm 1b/5b: 4.0x105 /3.0x105 PfSPZ with 75 mg PYR given on days 0 (concurrent 
administration) 
b) Arm 2b/6b: 4.0x105 /3.0x105 PfSPZ with 75 mg PYR given on days 2&3 post DVI 
The main trial phase and booster phase will also assess the safety, tolerability and VE of 
PfSPZ-CVac (chloroquine) 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 58 of 146
a) Arm 3b: 4.0x105 PfSPZ +weekly chloroquine prophylaxis +/- NSAIDs dose level. 
Presumptive administration of NSAIDs on days 7 and 8 after the first vaccination only 
will be added if required as determined in the pilot phase. 
The main phase and booster phase will also enroll placebo control arms that will undergo 
injections with NS and receive either PYR or chloroquine. 
a) Arm 4a: Normal saline injection with 75 mg PYR given on days 0 
b) Arm 4b: Normal saline injection with 75 mg PYR given on days 2 and 3 
c) Arm 4c: Normal saline injection while on weekly chloroquine chemoprophylaxis
VE will be measured against naturally transmitted P. falciparum infection. Participants in all 
three arms of the main study will receive three successive immunizations at 4 week intervals 
followed by assessment of parasitemia every two weeks by thick blood smears during the 
transmission season beginning 2 weeks post the 3rd immunization. Participants will also receive 
a 4th (booster) immunization at approximately 11 months after the third vaccination followed 
by assessment of parasitemia every two weeks by thick blood smears during the transmission 
season beginning 2 weeks post the 4th immunization
All participants in the main phase will undergo parasitemia clearance with 
artemether/lumefantrine treatment twice during the course of the study, approximately 1-
2 weeks prior to first and third vaccinations as outlined in Appendix A. In the 
pyrimethamine arms, the first dose of artemether/lumefantrine will be administered within 
approximately one week of the first vaccination where as in the chloroquine arms, this first 
dose will be administered within approximately two weeks of the loading dose of chloroquine. 
The first course of artemether/lumefantrine administered prior to the first vaccination aims at 
clearing naturally acquired malaria infection. In other similar studies conducted by LMIV in 
Mali, artemether/lumefantrine was administered to a total of 93 participants within 
approximately 4 days of first vaccination and there were no significant differences in safety 
measures between the vaccinated and unvaccinated participants and showed significant vaccine 
efficacy (Sissoko, Healy et al. 2017). One of the major concerns of concurrent administration 
of some antimalarials is prolongation of QTc (Traebert and Dumotier 2005).While 
pyrimethamine does not prolong QTc, chloroquine may prolong QTc (Haeusler, Chan et al. 
2018). In the chloroquine groups, when a loading dose is administered prior to the first 
vaccination, approximately two weeks interval will be maintained between the administration 
of chloroquine and artemether lumefantrine, see Appendix A.  Decreasing the interval between 
administration artemether/lumefantrine and pyrimethamine should not negatively impact the 
safety of the participants and is unlikely to affect the vaccine.
Participants in the booster phase will undergo one additional round of parasitemia clearance 
with artemether/lumefantrine approximately 1-2 weeks prior to fourth vaccinations as 
outlined in Appendix A.  
Antibody and cellular immune responses and transcriptomic analyses will be assessed 
periodically after vaccination in order to identify correlates of protection.


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 59 of 146
2
Study Objectives
2.1
Primary Objectives
Safety
Pilot Phase

To evaluate whether PYR on day 0 prevents patent parasitemia post DVI 
administration of 4x105 PfSPZ or 3x105 PfSPZ (if necessary) of PfSPZ 
Challenge (Arms 1a, 2a, 5a, 6a).

To monitor the safety and tolerability of PfSPZ-CVac (chloroquine), post 
4x105 PfSPZ Challenge administered via DVI with chloroquine as the partner 
drug (Arm 3a)
Main Phase and Booster Phase

To monitor the safety and tolerability of PfSPZ-CVac (pyrimethamine), PfSPZ 
Challenge administered via DVI with PYR as the partner drug; (Arms 1b/5b, 
2b/6b, 4a, 4b) 

To monitor the safety and tolerability of PfSPZ-CVac (chloroquine), PfSPZ 
Challenge administered via DVI with chloroquine as the partner drug; (Arms 
3b, 4c)
2.2
Secondary Objective
Protective Efficacy

To assess the protective efficacy of PfSPZ-CVac (pyrimethamine) or 
PfSPZ-CVac (chloroquine) against natural P. falciparum infection (Arm 
1b/5b, 2b/6b, 3b, 4).
2.3
Exploratory Objectives
Pyrimethamine regimen efficacy and Immunogenicity

To evaluate whether PYR on day 0 or on day 2&3 prevents patent 
parasitemia post DVI administration of 4x105 OR 3x105 PfSPZ of PfSPZ 
Challenge (Arms 1, 2, 5, 6).

To assess the humoral and cell mediated immune responses to PfSPZ and 
to known pre-erythrocytic and blood stage antigens (All Arms)

To assess the humoral and cell mediated immune responses to novel pre-
erythrocytic antigens (All Arms)

To look for immune correlates of protection

To assess the kinetics of subpatent parasitemia during PfSPZ-CVac 
immunization (Arms 1, 2, 3, 5, 6)

To describe changes in γδ T cells in malaria experienced individuals after 
PfSPZ-CVac immunization and malaria infection during transmission 
season (Arms 1b/5b, 2b/6b, 3b)


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 60 of 146
3
Study Design
3.1
Overall Design
The study proposed here by MRTC/ University of Sciences, Techniques and Technologies of 
Bamako (USTTB), Sanaria and LMIV will assess PfSPZ-CVac (pyrimethamine) and PfSPZ-
CVac (chloroquine) in a malaria experienced population in Mali. 
The design of the proposed clinical trial will entail three phases, a pilot phase and a main 
phase followed by a booster phase: 
Pilot Phase
a) Pyrimethamine Arms (1a, 2a, 5a, 6a): to assess safety and tolerability of increasing 
PfSPZ Challenge dose to 4.0x105 PfSPZ with either concurrent administration of PYR 
(day 0 post PfSPZ DVI, Arm 1a) or PYR administration on days 2 & 3 post DVI (Arm 
2a). These pilot arms will assess that these regimens will not result in patent 
parasitemia (blood smear negative). If needed, following a predefined algorithm (see 
Figure 7 and Figure 8), PYR, the dose of PfSPZ Challenge will be decreased to 
3.0x105 PfSPZin Arms 5a and 6a. 
b)  Chloroquine Arms (Arms 3a): to assess safety and tolerability of PfSPZ Challenge 
(4.0x105 PfSPZ) and chloroquine dosing on day -2 and +5 and to assure that these 
regimens will not result in Grade 3 AEs that last more than 48 hours with adequate 
management during the expected period of parasitemia or require treatment with 
another antimalarial medication. If symptoms related to patent parasitemia are deemed 
intolerable, then subjects will receive presumptive treatment with non-steroidal anti-
inflammatory drugs (NSAIDs) at the time that transient parasitemia develops 
following the first immunization and as needed thereafter in the main phase (see 
Figure 8). 
Main Phase and Booster Phase
a) Pyrimethamine Arms (Arms 1b/5b, and 2b/6b): to assess safety and tolerability of 3 
exposures every 28 days of 4x105 PfSPZ and a booster exposure while under PYR 
given on either day 0 (Arm 1b) or day 2 & 3 (Arm 2b)post DVI . Arms 5b (PYR on 
day 0) or 6b (PYR on day 2&3) that receive 3x105 PfSPZ will only be enrolled if 
there is patent parasitemia with the higher dose of PfSPZ as determined by the pilot 
phase (Figure 7). The main phase will also assess VE against natural P. falciparum 
infection (see Figure 9).
b) Chloroquine Arm (Arm 3b): to assess safety and tolerability of three exposures 
every 28 days to PfSPZ Challenge (4.0x105 PfSPZ) and chloroquine dosing on day -2, 
+5 post PfSPZ DVI and weekly thereafter (10 doses in total) and a booster exposure 
while under chloroquine dosing on day -2, +5 post PfSPZ DVI and weekly thereafter (2 
doses in total) and VE against natural P.falciparum infection (see Figure 9). NSAIDs 
may be administered on day 7 &8 post first vaccination, if required, as determined by 
the pilot phase.
c) Placebo Control Arm (Arm 4): to serve as controls enabling comparison during the 
assessment of safety, tolerability and VE of the vaccinated arms against natural 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 61 of 146
P.falciparum infection (see Figure 9). Participants will receive 3 exposures of NS 
every 28 days while under PYR given on either day 0 or day 2 & 3 post vaccination 
(Arm 4a or 4b respectively) or chloroquine dosing on day -2, +5 post injection and 
weekly thereafter for 10 doses in total (Arm 4c) plus a booster exposure of NS while 
either under PYR given on either day 0 or day 2 & 3 post vaccination (Arm 4a or 4b 
respectively) or CQ dosing on day -2, +5 post injection and weekly thereafter for 2 
doses.
3.2
Pilot Study
The overall goal of the pilot study is to assess the safety and prevention of patent parasitemia in 
concurrent administration of PYR as well as increasing dose of PfSPZ Challenge to 4.0x105 PfSPZ 
under PYR as well as the safety and tolerability of administering 4.0x105 PfSPZ under chloroquine 
prophylaxis. Participants in the pilot phase will be randomized into either Arm 1a, 2a or 3a. If other 
pilot arms are required (Arm 5a or 6a), enrollment will be sequential. Note: If enrollment into Arms 
5a and 6a occurs prior to completion of Arms 1a, 2a or 3a, the participants will be randomized into 
either Arm 5a or 6a. Appropriate regimens for further testing in the main phase will be selected from 
the pilot study results. 
In the pyrimethamine pilot, subjects will undergo immunization with PfSPZ-CVac with one 
exposure to 4.0x105 PfSPZ Challenge with 75 mg of PYR dosing on day 0 only (Arm 1a) or 
day 2&3 (Arm 2a) post PfSPZ Challenge (Figure 7). Participants will be followed with sBS 
conducted from day 6 through 12 post injection. The goal of this pilot is to determine that 
increasing the dose of PfSPZ with either standard dosing of PYR on day 2&3 (Arm 2a) and 
also earlier administration of one dose of PYR (day 0, Arm 1a) does not lead to patent 
parasitemia. Samples for assessment of subpatent parasitemia breakthrough by qPCR will also 
be collected from day 6 through day 12 post PfSPZ Challenge injection and will be run 
retrospectively. Administration of 50 mg of PYR on days 2 and 3 post 2.0x105 PfSPZ DVI in 
healthy US adults prevented subpatent and patent parasitemia as assessed by qPCR and blood 
smears respectively (Figure 5). With known pharmacokinetics of PYR, it is expected that the 
proposed dose of PYR, 75mg, will be sufficient to prevent subpatent and patent parasitemia 
even with higher dose of PfSPZ (Figure 6). Enrollment in these arms will occur 
simultaneously. If there is patent parasitemia, the dose of PfSPZ will be decreased to 3.0x105 
PfSPZ in alternate Arms. If there is patent parasitemia in Arm 1a, four participants will be 
enrolled in Arm 5a to receive 3.0x105 PfSPZ while under 75 mg of PYR on day 0. If there is 
patent parasitemia in Arm 2a, four participants will be enrolled in Arm 6a to receive 3.0x105 
PfSPZ while under 75 mg of PYR on days 2 and 3 post injection. 
Note: 

Participants to be enrolled in either Arm 5a or 6a may be enrolled and receive 
artemether/lumefantrine prior to the end of the follow up period of Arm 1a or 2a. 
However, they will not receive PfSPZ Challenge injection until the follow up period for 
Arms 1a and 2a is complete (study day 13). 

If participants in Arms 5a or 6a do not receive PfSPZ Challenge injection, they will be 
offered enrollment in the main study
If the regimen in Arm 1a and 2a are safe and there is no patent parasitemia, these regimens will 
continue to the main study. The regimens that result in patent parasitemia will not continue to 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 62 of 146
the main study. If Arm 5a and 6a result in patent parasitemia, the study will pause for 
reassessment. Samples for assessment of subpatent parasitemia breakthrough by qPCR will be 
collected from day 6 through day 12 post PfSPZ Challenge injection and will be run 
retrospectively. Subjects will be treated with standard treatment doses of 
artemether/lumefantrine at the end of their enrollment, starting 12 days post injection. Subjects 
from the pilot study will not join the main study. 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 63 of 146
Figure 7. Pyrimethamine Pilot Study flowchart Schema


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 64 of 146
Figure 8. Ideal Pyrimethamine and Chloroquine Pilot Study Schema
In the chloroquine pilot study (Arm 3a), subjects will be immunized with PfSPZ-CVac with 
one exposure to PfSPZ Challenge, with a loading dose of chloroquine administered 2 days 
before PfSPZ Challenge and maintenance dose administered 5 days post PfSPZ Challenge 
(Figure 8). The goal of the pilot study is to evaluate safety and tolerability of administering a 
dose of 4.0x105 PfSPZ of PfSPZ Challenge. If this regimen is not tolerable, that is, it results in 
grade 3 or higher signs or symptoms lasting more than 48 hours despite adequate management, 
then NSAIDs will be administered on day 7 and 8 post PfSPZ Challenge after the first 
vaccination only in the main phase. In addition, all participants will be treated with full dose of 
artemether/lumefantrine at the end of the pilot, starting at day 12 post injection or if diagnosed 
with clinical malaria per Malian guidelines prior to day 12 days post injection. Participants 
from the pilot study will not join the main study. 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 65 of 146
3.3
Main Study and Booster Phase
The main phase, which will be conducted over several years, will be a randomized double 
blind placebo controlled study. Both the participants and investigators will not be aware of the 
injection assignment (PfSPZ Challenge or normal saline) but they will be aware of partner drug 
assignments, either PYR or CQ. In the vaccinated arms, participants will be enrolled in either: 

Arm 1b/5b (4.0x105 /3.0x105 PfSPZ + 75 mg PYR on day 0 post PfSPZ Challenge, n=90) 
or

Arm 2b/6b (4.0x105 /3.0x105 PfSPZ + 75 mg PYR on days 2 & 3 post PfSPZ Challenge, 
n=60) or 

Arm 3b (4.0x105 PfSPZ + weekly chloroquine prophylaxis+/- NSAIDs, n=90) 
In the control arms, 180 participants total will be enrolled. Participants will be enrolled in 
either 

Arm 4a: Normal saline injection with 75 mg PYR given on days 0 (concurrent administration, 
n=54) or

Arm 4b: Normal saline injection with 75 mg PYR given on days 2 & 3, n=36 or

Arm 4c: Normal saline injection while on weekly chloroquine chemoprophylaxis, n=90
Note: 
1. The number of participants in each of the control arms can be redistributed if one the 
planned vaccinated arms is not enrolled. The goal would be to have the total number of 
participants in the control arm to equal the number of participants in one of the vaccinated 
arms with the highest sample size. 
2. The pyrimethamine arms will be enrolled in the first year and the chloroquine arms will be 
enrolled in a subsequent year of the study.
Participants in the vaccinated arms of the main study will receive three exposures to PfSPZ 
Challenge at 28-day intervals, with PYR or chloroquine coverage (PfSPZ-CVac, 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 66 of 146
Figure 9). The goal in the main study is to have two PYR regimens, the regimen of highest 
interest in which PYR is administered concurrently to PfSPZ Challenge (Arm 1b/5b) and a 
regimen in which a standard PYR dosing regimen is employed (Arm 2b/6b). Placebo control 
arms (Arm 4) will receive injections with NS following the same schedule as vaccinated arms 
while on PYR dosed either on day 0 or day 2 & 3 post vaccination (Arm 4a and 4b 
respectively) or chloroquine dosing on day -2, +5 post injection and weekly thereafter (Arm 4c). 
The participants in the control Arm 4, will follow the same study procedures as those in 
vaccinated Arms thus providing blinding of the of the type of injection received (PfSPZ 
Challenge or NS) for the corresponding medication dosing vaccinated arms. 
 
Participants in the main study (vaccinated and placebo controls) will receive two full doses of 
artemether/lumefantrine during the course of the study, approximately one to two weeks prior 
to the first and the 3rd injection. Administration of artemether/lumefantrine for the main phase 
can be started prior to completion of the pilot study. However, administration of chloroquine, 
pyrimethamine and PfSPZ Challenge will occur after completion of the pilot phase follow up 
(study Day 13). These participants will then be evaluated every two weeks for parasitemia 
during the malaria transmission season starting approximately 2 weeks from the third injection.


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 67 of 146
Figure 9. Main Study Schema
Booster Phase: At the completion of the follow up phase after the primary series, participants 
who received all three vaccine doses will be invited to continue participation in the study to 
receive a booster vaccine dose. This 4th dose will be administered prior to the beginning of the 
following transmission season, approximately 11 months post the 3rd vaccination. Those 
enrolled in the vaccine arms (Arms 1 and 2) will receive one injection of 9 x105 PfSPZ while 
those participants enrolled in the control arms (Arms 3a and 3b) will receive one injection of 
normal saline. Arms 1b, 2b, and 3b will each receive a single dose of 4x105 PfSPZ Challenge 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 68 of 146
whereas Arms 4a, 4b, and 4c will receive normal saline while under their respective 
chemoprophylaxis regimens (PYR on day 0 for Arms 1b/4a, PYR on days 2 and 3 for Arms 
2b/4b, and CQ on days -2 and 5 for Arms 3b/4c). Study blinding will be maintained as the 
participants transition from the main phase to the booster phase. The booster phase will extend 
malaria infection follow-up through the subsequent transmission season. If statistically 
significant protection is not obtained in both Arms 1b or 2b, the non-significant arm will be 
administered normal saline instead of PfSPZ Challenge as described in Section 1.5.1.3 (futility 
criteria). 
4
Description of Investigational Products and Plan
4.1
Chloroquine Phosphate (CQ)
Chloroquine has been widely used for treatment and prophylaxis of malaria since 1946 (Most, 
London et al. 1984). It was the treatment of choice for uncomplicated malaria for decades 
because it was safe, well tolerated, affordable and highly effective for the treatment of malaria. 
However, increasing spread of chloroquine-resistant Pf over the last two decades has severely 
limited its use (Wellems 2002). CQ is U.S Food and Drug Administration (FDA) approved for 
suppressive treatment (prophylaxis) and for acute attacks of malaria due to P. vivax, P. 
malariae, P. ovale, and susceptible strains of Pf. Chloroquine is a blood-stage schizonticide, 
highly active against replicating forms of blood-stage drug-sensitive parasites. As such, it is 
routinely employed as first-line prophylaxis against development of patent parasitemia and 
clinical malaria in non-immune travelers to areas with chloroquine sensitive Pf. Under standard 
weekly dosing, as used in this study [1000 mg (600 mg base) loading dose then 500mg (300mg 
base) weekly], routine monitoring in clinical practice for patent blood-stage infection (via 
blood smear or qPCR) is not typically conducted unless triggered by presentation for 
evaluation of malaria related symptoms. Weight based chloroquine dosing also has been 
approved for treatment and prevention of Pf malaria.
The current approved package insert for chloroquine and additional details on safety of 
chloroquine used with PfSPZ Challenge are provided in the Investigator’s Brochure (IB).
4.2
Pyrimethamine 
Pyrimethamine is a folic acid antagonist that has been commonly used as antimalarial drug for 
both treatment and prevention of malaria, usually in combination with sulfadoxine in adults, 
pregnant women, and children worldwide (Organization April 2013 (rev. January 2014)). 
Similar to chloroquine, PYR (in combination with sulfa drugs) was a drug of choice for 
malaria treatment and prophylaxis for decades because it was safe, well tolerated, affordable 
and highly effective; however, due to widespread resistance, PYR and PYR combinations have 
fallen out of favor for clinical use. Since the 1950’s, PYR has been FDA approved for acute 
treatment and chemoprophylaxis of malaria due to susceptible strains of plasmodia. Should 
circumstances arise, PYR may be used alone for acute malaria at 50 mg orally for two days. 
PYR has been shown to possess both tissue schizonticidal (i.e., liver stage specific) and blood 
schizonticidal activity against the malaria parasite in vitro and in vivo (Friesen, Borrmann et al. 
2011, Delves, Plouffe et al. 2012). 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 69 of 146
PYR, in combination with sulfadoxine, is also used for prophylaxis purposes in vulnerable 
populations in endemic countries. Per WHO recommendations, pregnant women receive IPTp, 
the recommended regimen is once a month and the adult dose contains 75 mg of PYR (WHO 
2014). In children receiving seasonal malaria chemoprophylaxis with sulfadoxine– PYR plus 
amodiaquine, the medications are dosed by age (WHO 2013). 
In addition, PYR can be used for treatment of toxoplasmosis at higher doses than utilized for 
malaria treatment or chemoprophylaxis (75-100 mg per day) and given for prolonged periods 
of time (weeks to months), usually in conjunction with sulfadoxine and folinic acid. With 
doses of PYR used for the treatment of toxoplasmosis, side effects such as anorexia and 
vomiting may occur, but can be minimized by giving the medication with meals. 
In this study, we plan to use a higher dose of PYR, 75 mg orally for either one or two days 
each month. One dose is similar to chemoprophylaxis doses used in IPTp. Administration of 75 
mg of PYR for two consecutive days only is similar to PYR dosages utilized in toxoplasmosis 
treatment, but for a significantly shorter period of time. 
The current approved package insert for PYR and additional details on safety of PYR are 
provided in the Investigator’s Brochure.
4.3
PfSPZ Challenge 
One of the strains manufactured by Sanaria Inc. is PfSPZ Challenge NF54. PfSPZ Challenge 
contains fully infectious PfSPZ purified from the salivary glands of Anopheles stephensi 
mosquitoes raised under aseptic conditions. The infectious PfSPZ are formulated in 
cryoprotectant to maintain potency for an extended period. Sanaria® PfSPZ Challenge (NF54) 
is known to be susceptible to chloroquine, PYR, atovaquone, artesunate, but not mefloquine. 
More detailed product information can be found in Sections 7.1 and the Investigator’s 
Brochure.
The clinical studies for PfSPZ Challenge are detailed in Section 1.4.2
4.4
Description of Intervention
The intervention in this study involves the injection by DVI of either 4.0x105 or 3.0x105 
aseptic, purified, cryopreserved, fully infectious PfSPZ with concurrent administration of 
antimalarial drugs to attenuate the parasites in vivo. The goal is to induce stage specific 
immunity to Pf malaria (PYR = pre-erythrocytic immunity; chloroquine = pre-erythrocytic 
immunity + possibly erythrocytic immunity). Following immunization, participants will be 
followed through the transmission season in order to assess VE. Antimalarial (Coartem®) will 
be administered to all participants twice during the study, approximately 2 weeks prior to the 
first vaccination in order to clear pre-existing parasitemia that may interfere with immunity 
development. The second dose will be given approximately 2 weeks prior to the 3rd vaccination 
(see Appendix A) in order to clear any parasitemia infection that may have happened naturally 
during the vaccination period and prior to the follow up period. 
Pilot Study

Pyrimethamine Pilot (Arms 1a, 2a, 5a and 6a): 1 dose of 4.0x105 PfSPZ Challenge 
AND either 1 dose of 75 mg PYR will be administered on day 0 (Arm 1a) or 2 doses 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 70 of 146
of 75 mg PYR administered on day 2&3 (Arm 2a) post PfSPZ Challenge. Dosing on 
day 0 will occur within +/- 1 hour of PfSPZ Challenge injection. Subjects will be 
followed daily with sensitive thick blood smears from day 6 through day 12 post 
PfSPZ Challenge. Participants will first be treated with full dose of 
artemether/lumefantrine approximately 2 weeks prior to vaccination (see Appendix 
A). In addition, all participants will be treated with full dose of 
artemether/lumefantrine at the end of the pilot, starting at day 12 post injection or if 
diagnosed with clinical malaria per Malian guidelines prior to day 12 days post 
injection. Samples for assessment of subpatent parasitemia breakthrough by qPCR 
will be collected from day 6 through day 12 post PfSPZ Challenge injection and will 
be run retrospectively. See Section 3.2 for more details. If there is patent 
parasitemia, Arm 5a or 6a will be enrolled to receive a decreased dose of PfSPZ 
Challenge, 3.0x105 PfSPZ.

Chloroquine Pilot (Arm 3a):1 dose of PfSPZ Challenge (4.0x105 PfSPZ) AND 
chloroquine beginning with a loading dose 2 days prior to PfSPZ Challenge (1000 
mg = 600 mg base) and finishing with a maintenance dose 5 days after PfSPZ 
Challenge (500 mg = 300 mg base) (Arm 3a). Subjects will be followed daily with 
blood smears from day 6 through day 12 post PfSPZ Challenge. All participants will 
first be treated with full dose of artemether/lumefantrine approximately 2 weeks 
prior to vaccination (see Appendix A). In addition, all participants will be treated 
with full dose of artemether/lumefantrine at the end of the pilot, starting at day 12 
post injection or if diagnosed with clinical malaria per Malian guidelines prior to day 
12 days post injection. See Section 3.2 for more details.
Main Study

Pyrimethamine Main (Arms 1b/5b and 2b/6b): 3 doses at 28 days intervals of 
4x105 PfSPZ AND either 1 dose of 75 mg of PYR given on day 0 (Arm 1b) or on 
day 2 & 3 (Arm 2b) post vaccination Arms 5b or 6b will be enrolled if necessary 
as determined by the pilot to receive a decreased dose of PfSPZ Challenge, 3x105 
PfSPZ. These two arms will not receive chloroquine. Dosing on each day 0 will 
occur within +/- 1 hour of PfSPZ Challenge injection. Subjects will be followed 
clinically. During the immunization phase, samples for blood smears and malaria 
qPCR will collected from 7 through 9 days post injection and assays will be 
conducted retrospectively as outlined in Appendix A. To guide clinical 
management, a blood smear will be done and read in real time if a participant 
presents with symptoms. Approximately one to two weeks prior to the first 
vaccination and the third vaccination, as outlined in Appendix A, all participants 
will be treated with a full three-day course of artemether/lumefantrine to clear any 
naturally transmitted parasitemia. All participants will again be treated with 
standard dose of artemether/lumefantrine at the time of diagnosis of clinical malaria 
per Malian ministry of health guidelines (a presence of a positive malaria test + 
clinical symptoms) (see Section 3.3 for more details). If a participant is diagnosed 
with clinical malaria and treated with artemether/lumefantrine prior to 3rd 
vaccination, scheduled dosing of artemether/lumefantrine will not be repeated if the 
treatment falls within the administration visit windows outlined in Appendix A. 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 71 of 146

Chloroquine Main (Arm 3b): 3 doses of PfSPZ Challenge (4.0x105 PfSPZ) 
separated by 28-day intervals AND chloroquine beginning with a loading dose 2 
days prior to PfSPZ Challenge (1000 mg = 600 mg base) and continuing weekly 
using a maintenance dose (500 mg = 300 mg base) for a total of 10 doses, finishing 
with a last dose 5 days after the third injection with PfSPZ Challenge. If needed as 
determined in the pilot phase, NSAIDs may be administered on days 7 & 8 post the 
first PfSPZ Challenge DVI to diminish symptoms. Subjects will be followed 
clinically. During the immunization phase, samples for blood smears and malaria 
qPCR will be collected from 7 through 9 days post injection and assays will be 
conducted retrospectively as outlined in Appendix A. To guide clinical 
management, a blood smear will be done and read in real-time if a participant 
presents with symptoms. Approximately one to two weeks prior to the first and the 
third vaccination as outlined in Appendix A, all participants will be treated with a 
full three-day course of artemether/lumefantrine to clear any naturally transmitted 
parasitemia. All participants will again be treated with standard dose of 
artemether/lumefantrine at the time of diagnosis of clinical malaria per Malian 
ministry of health guidelines (presence of a positive malaria test + clinical 
symptoms) (see Section 3.3 for more details). If a participant is diagnosed with 
clinical malaria and treated with artemether/lumefantrine prior to 3rd vaccination, 
scheduled dosing of artemether/lumefantrine will not be repeated if the treatment 
falls within the administration visit windows described in Appendix A.

Placebo Controls (Arms 4): Participants in this group will receive NS injections 
following the same schedule as vaccinated arms while on PYR dosed either on day 
0 (Arm 4a) or day 2 & 3 (Arm 4b) post vaccination or weekly chloroquine (Arm 4c). 
These participants will follow the same study procedures (Appendix A) as the 
corresponding vaccinated arm that is receiving the same drug regimen. They will 
not receive PfSPZ Challenge injections. Approximately one to two weeks prior to 
the first and the third vaccination as outlined in Appendix A, all participants will be 
treated with a full three-day course of artemether/lumefantrine to clear any naturally 
transmitted parasitemia. All participants will again be treated with standard dose of 
artemether/lumefantrine at the time of diagnosis of clinical malaria per Malian 
ministry of health guidelines (presence of a positive malaria test + clinical 
symptoms) (see Section 3.3 for more details). 
Vaccine efficacy will be evaluated against naturally transmitted Plasmodium falciparum (Pf) 
malaria infection. All participants enrolled in the main arm will be evaluated for infection by 
blood smears through the transmission season starting two weeks to approximately 6 months 
after the third vaccination. 
The chemoprophylactic agents used in the current study are PYR and chloroquine. Both PYR 
and chloroquine are commercially available FDA-approved antimalarial drugs for treatment 
and prophylaxis of Pf. Chloroquine dosing used in the study is abbreviated from the standard 
weekly suppressive prophylaxis in non-immune populations (600 mg base loading dose, then 
300 mg base weekly maintenance dose for a total of 4 weeks after Pf exposure) given that in 
this study the day of exposure to SPZ is precisely known, there is no risk of P. vivax co-
infection, and the biology of the Pf NF54 parasite is well characterized. Thus, the loading dose 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 72 of 146
will be administered 2 days prior to PfSPZ Challenge and continued weekly until 5 days after 
the third dose of PfSPZ Challenge (or the fourth dose in the booster phase as outlined below 
and in Appendix A). In prior studies, administering the last dose of chloroquine in this manner 
has been sufficient to eliminate parasitemia in all study subjects during PfSPZ-CVac 
(chloroquine) immunization (Mordmuller, Surat et al. 2017) and observed in our previous 
study, NIAID protocol #15-I-0169 and #17-I-0067 as described in Section 1.4.2. 
PYR dosing in this study will be daily dosing of 75 mg for one or two consecutive days, dosed 
on day 0 versus days 2 and 3 after either 4x105 PfSPZ or 3x105 PfSPZ of PfSPZ Challenge or 
NS (Table 14). The planned dosing of 75 mg is consistent with the doses used for IPTp or 
toxoplasmosis treatment. During the main phase, there will be a total of 3 doses (Arm 1b/ 5b) 
or 6 doses (Arm 2b/6b) of PYR received during the study. 
Booster Phase

Pyrimethamine Booster (Arms 1b and 2b): 1 additional dose (and the fourth 
cumulative dose) of 4x105 PfSPZ Challenge will be given approximately 11 months 
after third vaccinations AND 1 dose of 75 mg of PYR given on day 0 (Arm 1b) or 
on day 2 & 3 (Arm 2b) post vaccination. These two arms will not receive 
chloroquine. Dosing on each day 0 will occur within +/- 1 hour of PfSPZ Challenge 
injection. Subjects will be followed clinically. During the booster immunization 
phase, samples for blood smears and malaria qPCR will collected from 7 through 9 
days post injection and assays will be conducted retrospectively as outlined in 
Appendix A. To guide clinical management, a blood smear will be done and read 
in real time if a participant presents with symptoms. Approximately one to two 
weeks prior to the fourth vaccination, as outlined in Appendix A, all participants 
will be treated with a full three-day course of artemether/lumefantrine to clear any 
naturally transmitted parasitemia. All participants will again be treated with 
standard dose of artemether/lumefantrine at the time of diagnosis of clinical malaria 
per Malian Ministry of Health guidelines (a presence of a positive malaria test + 
clinical symptoms) (see Section 3.3 for more details). During the booster phase, 
there will be a total of 1 dose (Arm 1b) or 2 doses (Arm 2b) of PYR received.

Chloroquine Main (Arm 3b): 1 additional dose (and the fourth cumulative dose) of 
4.0x105 PfSPZ Challenge will be given approximately 11 months after third 
vaccinations AND chloroquine beginning with a loading dose 2 days prior to PfSPZ 
Challenge (1000 mg = 600 mg base) and continuing for one additional week using a 
maintenance dose (500 mg = 300 mg base) 5 days after the booster injection with 
PfSPZ Challenge for a total of 2 doses. Subjects will be followed clinically. During 
the immunization phase, samples for blood smears and malaria qPCR will be 
collected from 7 through 9 days post injection and assays will be conducted 
retrospectively as outlined in Appendix A. To guide clinical management, a blood 
smear will be done and read in real-time if a participant presents with symptoms. 
Approximately one to two weeks prior to the fourth vaccination as outlined in 
Appendix A, all participants will be treated with a full three-day course of 
artemether/lumefantrine to clear any naturally transmitted parasitemia. All 
participants will again be treated with standard dose of artemether/lumefantrine at 
the time of diagnosis of clinical malaria per Malian Ministry of Health guidelines 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 73 of 146
(presence of a positive malaria test + clinical symptoms) (see Section 3.3 for more 
details). 

Placebo Controls (Arms 4): Participants in this group will receive NS injections 
following the same schedule as vaccinated arms while on PYR dosed either on day 
0 (Arm 4a) or day 2 & 3 (Arm 4b) post vaccination or weekly chloroquine (Arm 4c). 
These participants will follow the same study procedures (Appendix A) as the 
corresponding vaccinated arm that is receiving the same drug regimen. They will 
not receive PfSPZ Challenge injections. Approximately one to two weeks prior to 
the fourth vaccination as outlined in Appendix A, all participants will be treated 
with a full three-day course of artemether/lumefantrine to clear any naturally 
transmitted parasitemia. All participants will again be treated with standard dose of 
artemether/lumefantrine at the time of diagnosis of clinical malaria per Malian 
Ministry of Health guidelines (presence of a positive malaria test + clinical 
symptoms) (see Section 3.3 for more details). 
Vaccine efficacy will be evaluated against naturally transmitted Plasmodium falciparum (Pf) 
malaria infection. All participants enrolled in the booster phase will be evaluated for infection 
by blood smears through the transmission season starting two weeks to approximately 6 
months after the fourth (booster) vaccination. 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 74 of 146
Table 14. Chemoprophylaxis regimen and assignment
Intervention
Arm Assignment
Dosing
Antimalarials
Pyrimethamine
Arm 1
Arm 2
Arm 4a &b
Arm 5
Arm 6
Three single strength pills (75 mg PYR total) given on 
day 0 (Arms 1, 4a, 5) or days 2& 3 (Arms 2, 4b, 6) 
following each administration of PfSPZ Challenge by 
DVI during PfSPZ-CVac immunization. The pilot arms 
will receive either a total of 1 daily dose (Arms 1a, 5a) or 
2 daily doses (Arms 2a, 6a). The main arms will receive a 
total of 3 daily doses (Arm 1b/5b), or 2 daily doses for a 
total of 6 daily doses (Arm 2b/6b). The booster phase will 
receive a total of 1 dose (Arm 1b), or 2 daily doses (Arm 
2b).
Chloroquine
Arm 3
Arm 4c
Loading dose of approximately 1000 mg chloroquine 
phosphate (600 mg chloroquine base) 2 days prior to first 
administration of PfSPZ Challenge by DVI during 
PfSPZ-CVac immunization. Then 500 mg (300 mg base) 
5 days after 1st injection (Arm 3a, 2 doses total) OR 
weekly until 5 days after 3rd injection (Arms 3b and 4c 
10 doses total). The booster phase will receive an 
additional loading dose 2 days prior to 4th injection of 
PfSPZ Challenge (1000 mg = 600 mg base) and one 
additional maintenance dose (500 mg = 300 mg base) 5 
days after the booster injection for a total of 2 additional 
doses.
Artemether/
lumefantrine
ALL Arms
A total of 6 doses administered twice daily of single 
tablet of artemether 80 mg/lumefantrine 480 mg (Full 
course) approximately 14 days prior to first vaccination 
and end of study participation in the pilot phase (Arms 
1a, 2a, 3a, 5a, 6a).  In the main phase, full course will be 
administered approximately 7-14 days prior to the 1st and 
3rd injection (Arms 1b/5b, 2b/6b, 3b, 4). In the booster 
phase, full course will be administered approximately 7-
14 days prior to the 4th injection (Arms 1b, 2b, 3b, 4).
A full course will also be administered at the time of 
clinical malaria diagnosis (ALL Arms)
P. falciparum sporozoites (PfSPZ), non-attenuated
PfSPZ Challenge 
(NF54) administered 
by DVI for PfSPZ-
CVac immunization
Arm 1
Arm 2
Arm 3
Arm 5
Arm 6
4.0x105 (Arms 1, 2 and 3) or 3.0x105 (Arms 5 and 6) 
aseptic, purified, cryopreserved, non-attenuated PfSPZ of 
PfSPZ Challenge (NF54) administered by DVI. 
Placebo Control


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 75 of 146
Normal saline 
administered by DVI
Arm 4
Sterile normal saline will be administered by DVI
Note: All medication administration timing will adhere to visit windows outlined in Appendix 
AAppendix A
4.5
Presumptive Antimalarial Treatment with Artemether/Lumefantrine
All participants will undergo parasitemia clearance with full course of artemether/lumefantrine 
treatment twice during the course of the study, approximately one to two weeks prior to first 
and the third vaccinations and approximately two weeks prior to fourth (booster) vaccinations.
Note: In Arms 3a, 3b and 4c, first dose of AL (Study Day -17) should be administered 14 days 
or more prior to CQ loading dose (Study Day -2)
During the Pilot Study, participants will be treated at 12 days after PfSPZ Challenge injection 
under direct observation by study staff.
In addition, during vaccination and follow up period, participants diagnosed with clinical 
malaria in accordance with the Malian Government treatment guidelines, will be treated with 
standard regimen of artemether/lumefantrine. Treated participants will continue to be 
monitored closely until any residual symptoms of malaria are mild or resolved. 
5
Study Endpoints
5.1
Primary Endpoints
Pilot Phase

Incidence of positive sBS occurring after PfSPZ-CVac immunization 
starting on day 7 post DVI (Arms 1a, 2a, 5a, 6a).

Incidence and severity of local and systemic grade 3 signs or symptoms 
lasting more than 48 hours despite adequate management and SAEs 
occurring after PfSPZ-CVac DVI (Arm 3a)
Main Phase and Booster Phase

Incidence and severity of local and systemic adverse events (AEs) and 
SAEs occurring after PfSPZ-CVac immunization. (Arms 1b/5b, 2b/6b, 4a, 
4b)

Incidence of clinical malaria diagnosis occurring after PfSPZ-CVac 
immunization as defined by the occurrence of grade 3 signs or symptoms 
lasting more than 48 hours despite adequate management. (Arm 3b, 4c)
5.2
Secondary Endpoints
Protective Efficacy


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 76 of 146
P. falciparum blood stage infection defined as detection of at least 2 P. 
falciparum parasites by microscopic examination of 0.5 µL of blood starting 2 
weeks after the 3rd vaccination in the Main Phase (Arm 1b/5b, 2b/6b, 3b, 4) and 
then 2 weeks after the 4th vaccination for approximately 6 months for the 
Booster Phase.
5.3
Exploratory Endpoints
Pyrimethamine regimen efficacy and Immunogenicity

P. falciparum blood stage infection defined as detection of P. falciparum 
parasites by qPCR following PfSPZ Challenge. (Arm 1, 2, 5, 6)

Humoral immune responses after PfSPZ-CVac regimens by assessing 
antibodies to PfSPZ, Pf asexual erythrocytic stages (AES), and specific Pf 
sporozoite, liver and blood-stage antigens such as CSP, MSP-1, AMA-1 in 
subjects with no patent and/or subpatent parasitemia and those with patent 
and/or subpatent parasitemia, among drug groups. (All Arms)

Cellular immune responses after PfSPZ-CVac regimens to PfSPZ, PfAES, 
and specific Pf sporozoite, liver and blood-stage antigens, such as CSP, 
MSP-1, AMA-1, in subjects with no patent and/or subpatent parasitemia 
and those with patent and/or subpatent parasitemia, among drug groups. 
(All Arms) 

Cellular and humoral responses that correlate with protective efficacy

Prolonged prepatent period and/or reduced density and duration of 
parasitemia in those developing parasitemia after CHMI (Arms 1, 2, 3, 5, 
6)

Comparison of γδ T cells before and after PfSPZ-CVac immunization and 
malaria infection during transmission season using ex vivo whole blood or 
in vitro staining. (All Arms)
5.4
Sample Size and Estimated Duration of Study
A total of up to 440 participants are expected to be enrolled in this trial (8, 9). Participants in 
the pilot arms will be monitored actively for about 1 month and those in the main arms will 
be monitored actively for about 10 months. Up to 800 subjects will be screened to 
accommodate possible screen failures.
6
Study Population
6.1
Description of Population and 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. Healthy adult subjects will be 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 77 of 146
recruited from the surrounding community and villages, and the demographics of the study 
population will therefore be representative of that community. 
6.2
Recruitment
Community permission will be obtained from village elders and other community members in 
Bancoumana after explanation and discussion of the study at a community meeting (see 
Section 17.2.1.1). A general announcement inviting household and family members to the 
participating clinic to learn about the study will be made at the time of community permission, 
using local radio or any traditional channel of communication. 
6.3
Inclusion Criteria
All of the following criteria must be fulfilled for a subject to participate in this trial:
1. Age ≥ 18 and ≤ 50 years (for booster phase, age ≥ 18 and ≤ 52 years)
2. Resident of Bancoumana or nearby areas 
3. In good general health and without clinically significant medical history 
4. Malaria comprehension exam completed, passed (a score of ≥80% or per 
investigator’s discretion) and reviewed prior to enrollment (not required for 
booster phase)
5. Able to provide proof of identity to the satisfaction of the study clinician completing 
the enrollment process
6. Willing to have blood samples stored for future research
7. Available for the duration of the study
8. Females of childbearing potential must be willing to use reliable contraception 
(as defined below) from 21 days prior to first PfSPZ Challenge injection to 28 
days following last PfSPZ Challenge exposure (or equivalent study day for Arm 5 
controls).  For the booster phase, this applies from 21 days prior to the booster 
vaccination to 28 days post booster vaccination.

Reliable methods of birth control include one of the following: confirmed 
pharmacologic contraceptives (parenteral) delivery; intrauterine or implantable 
device. OR

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. OR

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 urine or serum pregnancy 
test performed per protocol.
9. For booster phase only: previously or currently enrolled in protocol #19-I-N099 and 
completed all three primary vaccinations.
6.4
Exclusion Criteria


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 78 of 146
A subject will be excluded from participating in the pilot or main 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)
a. NOTE: Pregnancy is also a criterion for discontinuation of any further 
dosing or non-safety related interventions for that subject.
2. Currently breast-feeding (if female)
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)
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)
6. Infected with human immunodeficiency virus (HIV), hepatitis C virus (HCV), or 
hepatitis B (HBV) (For the booster phase: re-testing NOT required for enrollment 
unless clinically indicated)
7. Known or documented sickle cell disease by history (Note: known sickle cell trait 
is NOT exclusionary)
8. Clinically significant abnormal electrocardiogram (ECG) (For the booster phase: 
re-testing NOT required for enrollment unless clinically indicated)
9. Moderate or high risk for coronary heart disease (CHD) based on NHANES I 
cardiovascular risk assessment (Appendix C)
10. 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
11. History of receiving any other investigational product within the past 30 days
12. Participation or planned participation in a clinical trial with an investigational 
product prior to completion of the last required protocol follow-up visit
13. Medical, occupational, or family problems as a result of alcohol or illicit drug use 
during the past 12 months.
14. History of a severe allergic reaction or anaphylaxis
15. 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)
16. 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
17. Known immunodeficiency syndrome


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 79 of 146
18. Known asplenia or functional asplenia
19. Use of: 
a.
Chronic (≥14 days) oral or IV corticosteroids (excluding topical or nasal) at 
immunosuppressive doses (i.e., prednisone >10 mg/day) or 
immunosuppressive drugs within 30 days of vaccination
b. Antimalarials for example artemether, artemether-lumefantrine, artesunate, 
artesunate-amodiaquine, other than those prescribed by the investigator as 
part of the study procedures within 14 days prior to the first vaccine 
c.
Systemic antibiotics with known antimalarial activity such as 
trimethoprim-sulfamethoxazole, doxycycline, tetracycline, clindamycin, 
erythromycin, ciprofloxacin or azithromycin within 5 half-lives of the drug 
prior to the first vaccine 
20. Receipt of a live vaccine within the past 4 weeks or a killed vaccine within the 
past 2 weeks prior to Vaccination #1 and every subsequent vaccination day
21. Receipt of immunoglobulins and/or blood products within the past 6 months
22. Previous receipt of an investigational anti-infectivity malaria vaccine in the last 2 
years (this requirement is waived for the booster phase)
23. Known allergies or contraindication against: PYR, chloroquine, NSAIDs, 
artemether, lumefantrine
24. Other condition(s) that, in the opinion of the investigator, would jeopardize the 
safety or rights of a participant participating in the trial, interfere with the 
evaluation of the study objectives, or would render the subject unable to comply 
with the protocol.
7
Study Agents
7.1
PfSPZ Challenge (NF54)
Sanaria® PfSPZ Challenge consists of aseptic, purified, cryopreserved PfSPZ produced by the 
biotechnology company Sanaria Inc. (Rockville, MD, USA) (Hoffman, Billingsley et al. 2010, 
Epstein, Tewari et al. 2011, Roestenberg, Bijker et al. 2013). In brief, manufacture includes the 
production, under traditional environmental conditions, of eggs from a colony of A. stephensi 
mosquitoes housed in a controlled environmental chamber. Surface disinfection of the eggs is 
performed by exposure to chemical agents in a Class II biosafety cabinet. From this point 
forward, all materials and products are handled using aseptic methods to ensure that 
contaminating microorganisms are not introduced to and carried through the process. Surface- 
disinfected eggs are inoculated into sterile, vented flasks containing aseptic growth medium. 
The eggs hatch and develop into pupae, which are transferred to an adult mosquito container 
where the adult mosquitoes emerge. These adult mosquitoes, which have been raised under 
aseptic conditions, are fed Pf gametocyte-infected blood in an aseptic, High-Security 
Insectary. The P. falciparum gametocyte-infected blood produced from cultures of the Pf 
isolate NF54 (Pf NF54) derived from a working cell bank (WCB) of the well- characterized Pf 
NF54 isolate. Infected adult mosquitoes are maintained under aseptic conditions. Pf SPZ 
migrate to the salivary glands. The salivary glands from the PfSPZ infected mosquitoes are 
removed by hand dissection under aseptic conditions. PfSPZ are then purified from the 
salivary glands, counted, and, at a specified concentration, cryopreserved. Cryopreservation 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 80 of 146
commences with the addition of cryoprotective additives to produce the PfSPZ Challenge 
product. PfSPZ Challenge is dispensed into screw-cap vials and stored in liquid nitrogen 
vapor phase (LNVP) at 150°C to -196°C. All the procedures are described in more detail in 
the investigator’s brochure. 
7.2
Phosphate buffered saline and human serum albumin diluent
The diluent for PfSPZ Challenge is composed of phosphate-buffered saline (PBS) and human 
serum albumin (HSA). Vials of PBS and HSA will be shipped or transported to the clinical 
site, where diluent composed of PBS and HSA is prepared according to standard operating 
procedures (SOPs) provided by Sanaria. PBS that will be used will be released with a 
Certificate of Analysis (CoA) supplied by Sanaria. The PBS is stored at room temperature 
(15°C to 30°C) in a controlled room.
HSA is a licensed product, which is approved for parenteral, IV administration to humans 
and is purchased by Sanaria from CSL Behring (Kankakee, IL, USA). Every lot of HSA 
purchased is supplied with a CoA that is reviewed and approved upon receipt at Sanaria. 
The HSA lots are extensively tested to ensure that it is free of infectious agents as listed in 
the CoA and is approved for use by the FDA, US license number 1766. HSA vials are stored 
at room temperature (15°C to 30°C) in a controlled room.
7.3
Storage and Handling of PfSPZ Challenge 
Shipment of PfSPZ Challenge in LNVP is in compliance with all FDA, U.S. Department of 
Transportation, and United Nations transport guidelines for shipping bio-hazardous 
materials on dry ice and LNVP. Transfer, receipt and maintenance of PfSPZ Challenge from 
its storage site to the clinical trial site will follow SOPs provided by Sanaria. During 
transport and at the study site, the LNVP shipper will be monitored. Receipt of the PfSPZ 
Challenge will be documented on a tracking log by study staff.
7.4
Control Product
Sterile isotonic (0.9%) NS will be procured either in the US and shipped to Mali at ambient 
temperature or in Mali. Like the product, NS is a clear liquid, making it indistinguishable from 
the study product when drawn up into a syringe. NS will be used as a placebo, rather than a 
comparator vaccine being used, as currently there are no licensed vaccines available as IV 
formulations. 
7.4.1
Storage and Handling
The NS is stored at room temperature in a controlled room per product standards. 
Each NS vial can either be single use or multi-use over a period of a few hours 
(i.e. the duration of vaccine preparation on a given day).
7.4.2
Disposition and Dispensation
The clinical site must confirm and document that the vials of NS have been 
transported and stored within specified ranges. 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 81 of 146
7.4.3
Administration and Dosage
NS will be administered DVI as placebo in an equal volume to the study product.
7.4.4
Accountability
NS accountability will be maintained to document chain of custody from 
Sanaria, Inc., to study site. An inventory to account for number of vials 
dispensed for each subject injection will be recorded and kept in the study file.
7.5
Pyrimethamine, Chloroquine, Artemether/Lumefantrine 
Chloroquine, Pyrimethamine, and Artemether/Lumefantrine are commercially available 
antimalarial drugs. Package inserts for all three drugs are provided. 
CQ is supplied and manufactured in tablets of 250 mg or 500 mg (equivalent to 150 or 300 mg 
CQ base). Based on availability, CQ from the U.S, Europe or Asia may be obtained under 
approval from the Mali Ministry of Health. 
PYR is manufactured in tablets of 25 mg. Based on availability, PYR from the U.S, Europe or 
Asia may be obtained under approval from the Mali Ministry of Health.
Artemether/Lumefantrine is manufactured in tablets of 20 mg artemether and 120 mg 
lumefantrine. Artemether/Lumefantrine is readily available for standard treatment of malaria in 
Mali thus it will be obtained in Mali.
7.5.1
Pyrimethamine, Artemether/Lumefantrine and Chloroquine Packaging 
and Labeling
PYR and chloroquine tablets will be maintained in the manufacturer’s original 
packaging until prepared for dispensing in the trial.
Artemether/Lumefantrine though not drugs under study but drugs that all 
participants will receive, will be maintained in the same manner as PYR and 
chloroquine.
7.5.2
Pyrimethamine, Chloroquine, Artemether/Lumefantrine Accountability
7.5.2.1 Receipt
PYR, chloroquine, and artemether/lumefantrine, tablets will be purchased 
from reliable, commercial sources and provided to the local pharmacist and 
clinical team. Drug accountability will be managed as outlined in the 
Pharmacy Manual.
7.5.2.2 Preparation and Administration
PYR, chloroquine, and artemether/lumefantrine will be provided as tablets for 
oral administration with food. Administration is under direct observation by 
study staff according to dosing parameters. Individual doses of PYR, 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 82 of 146
chloroquine, and artemether/lumefantrine will be prepared by study staff as 
described in the Pharmacy Manual.
Note: The evening doses of artemether/lumefantrine will be given to participants 
to take at home therefore will not be under direct observation. Exceptions to 
direct observation for artemether/lumefantrine may be made at the discretion of 
the PI for extenuating circumstances, such as to accommodate religious festivals. 
7.5.2.3 Storage and Handling
PYR, chloroquine, and artemether/lumefantrine tablets will be maintained in the 
manufacturer’s original packaging and stored at the clinic under recommended 
storage conditions until prepared for dispensing.
7.5.2.4 Return of Study Product
Final accountability of drug supplies will be performed at the conclusion of 
the study. Final disposition of any remaining PYR, chloroquine, and 
artemether/lumefantrine will be determined and documented. PfSPZ 
Challenge, HSA and PBS inventory will be tracked and recorded and returned 
to Sanaria for final disposition and accountability according to Sanaria SOP.
8
STUDY SCHEDULE
8.1
Screening
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 receipt of first vaccination) and clinical 
assessments. Screening activities can occur over multiple visits if necessary.
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 actions must be completed as part of the screening process for all subjects 
within the 56 days prior to first and fourth vaccination: 

Explain the study and informed consent document to the subject. For the booster 
phase, informed consent can be signed within 90 days prior to the 4th 
vaccination.

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.


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 83 of 146

Ensure the subject has correctly answered ≥80% of the questions (see Section 
17.2.1.2) 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.

Confirm that females of childbearing potential are willing to use reliable 
contraception from 21 days prior to first PfSPZ Challenge injection through 28 
days after the third injection in the main phase. For the booster phase, 
willingness to use reliable contraception should be confirmed from at least 21 
days prior to the fourth vaccination through 28 days after the fourth vaccination. 

Administer a complete physical examination, including vital signs (height, 
weight, blood pressure, temperature, and heart rate).

Complete 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, Cr, hepatitis B testing, hepatitis C testing, 
HIV testing.  For the booster phase: HIV, hepatitis B, and hepatitis C testing 
will only be repeated if clinically indicated.

Obtain urine (or serum) for pregnancy testing (for females) and urinalysis/urine 
dipstick for protein and blood.

Obtain a 12-lead ECG.  For booster screening, an ECG will only be repeated if 
clinically indicated.
Final eligibility will be determined after all required information is obtained. The subject may 
be excluded by any of the above procedures if they meet the exclusion criteria. Acceptable 
ranges for hematological and biochemistry parameters defined for this study are given in 
Appendix B (Note: parasitemia is not an exclusion criterion.) 
If an abnormal finding is determined to be clinically significant, the subject will be informed, a 
referral letter will be issued, and the subject will be guided as to where to present for further 
investigation and medical care. Treatment for minor ailments may be provided by study 
clinicians at the study site. Decisions to exclude the subject from enrollment in the trial or to 
withdraw the subject from the trial will be at the discretion of the investigator.
If the screening visit was completed outside of the 56 days to first vaccination window, except 
for hepatitis B, hepatitis C and HIV testing, ALL remaining screening procedures listed above, 
including consent and comprehension exam, will need to be repeated and documented.
8.2
Assignment of Groups
Enrollment in the main phase occurs with receipt of the first dose of artemether/lumefantrine 
and collection of baseline study samples on study Day -17.. 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 84 of 146
In the pilot Arms, participants will be randomized into Arm 1a or 2a. Randomization will occur 
prior to enrollment, prior to or on study Day -17. If a subject was randomized into any arm but 
was not vaccinated, they may be randomized or enrolled in a subsequent Arm. If enrollment 
into Arm 5a or 6a is necessary, these participants will be enrolled sequentially. Note: If 
enrollment into Arms 5a and 6a occurs prior to completion of Arms 1a, 2a or 3a, the participants 
will be randomized into either Arm 5a or 6a. 
For the main Arms, participants in Arms 1b/5b, 2b/6b, 4a, and 4b (first year) AND 3b, and 4c 
(subsequent year) will be randomized. Randomization will occur prior to enrollment, prior to 
or on study Day -17
If a subject withdraws from the study after randomization, they can be replaced by another 
subject at random if withdrawal happens prior to receipt of first vaccination. Once a subject has 
received their first vaccination, they cannot be replaced. Note: A replacement subject may 
receive artemether/lumefantrine within 14 days of receiving first vaccination. 
On vaccination days, the vaccines associated with each randomization number will be obtained 
from the pharmacist.
For the booster phase, enrollment will occur with receipt of the first dose of 
artemether/lumefantrine prior to vaccination. Enrolled subjects cannot be replaced.
To ensure proper identification of study subjects, following subject enrollment all subjects will 
receive an identification card with their photo on it to present at the clinic with each study visit. 
Fingerprinting may also be implemented to ensure subjects are identified correctly during the 
visits. The biometric application that would be used is the Biometric Screening Log application 
developed by the data management unit of University Clinical Research Center (UCRC) of the 
University of Science, Techniques and Technology of Bamako (USTTB) in Mali.  This system 
utilizes FBI-certified fingerprint scanners to scan participant fingerprints.  In addition to 
providing identity verification during scheduled and unscheduled visits to the site at key 
stations (Identification Point, Clinic Exam Room, Lab/Phlebotomy, and Vaccination rooms), 
the software provides the following functionality:
•
ID card printing with QR code on a Zebra ID card printer
•
Participant-specific labels with 2 or 3 dimensional barcodes for samples, also printed 
from the Zebra printer
•
Dynamic visit calendar for full week
•
Daily report generated on visits that are completed, pending, missed, and scheduled
•
Weekly summary report available on study dashboard 
When the biometric system is implemented, participants’ fingerprints may be collected and 
stored.  Neurotechnology’s VeriFinger SDK fingerprint compression algorithm will be used to 
create a secure template.  This algorithm meets accuracy requirements outlined in the Wavelet 
Scalar Quantization (WSQ) Gray-Scale Fingerprint Image Compression Specification 
developed by the FBI and NIST.  PII and biometric templates are saved on a SGDBR SQL 
Server database compliant with Title 21 of the Code of Federal Regulations.  The HF SQL 
database is installed on a local server.  The server is located in a secure area with limited access 
(physical and logical access security, temperature monitoring, fire and smoke detection, 
camera surveillance).  Regular backup will be done based on data changes that will be 
encrypted and stored on local servers to prevent any leak of PII to NIH servers and or to NIH 
personnel.


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 85 of 146
8.3
Detailed Study Procedures
Detailed study procedures are outlined in Appendix A.
8.4
Indications for Deferral of Sanaria® PfSPZ Challenge 
If any one of the following AEs occurs at the time of the scheduled Sanaria® PfSPZ 
Challenge, the subject may either receive the Sanaria® PfSPZ Challenge at a later date within 
the allowable time interval specified in the protocol or withdrawn at the discretion of the 
Investigator:

Oral temperature >38.0˚C at the time of vaccination will warrant deferral of injection 
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 injection or that may complicate interpretation of the outcome variables 
following injection.
Such individual(s) will be followed in the clinic until the symptoms resolve or the window for
Sanaria® PfSPZ Challenge expires.
9
Study Procedures
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 (WBC) differential and platelet 
count

The following CBC parameters will be assessed for safety throughout the trial: 
WBC, absolute neutrophil count (ANC)/absolute granulocyte count (AGC), 
hemoglobin (Hb), and platelet count.
2. Serum creatinine (Cr)
3. Alanine aminotransferase (ALT)
4. Hepatitis B surface antigen (HBsAg) test (can include rapid diagnostics, enzyme-linked 
immunosorbent assay [ELISA], PCR if indicated) (at screening only in the main phase. 
For the booster phase, ONLY if clinically indicated)
5. HCV test (can include rapid diagnostics, ELISA, PCR if indicated) (at screening only in 
the main phase. For the booster phase, ONLY if clinically indicated)


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 86 of 146
6. HIV test (can include rapid diagnostics, ELISA, Western Blot if indicated) (at 
screening only in the main phase. For the booster phase, ONLY if clinically indicated)
7. Urine dipstick and/or Urinalysis (at screening only)
8. Urine and/or serum pregnancy testing (β-hCG)
9. Hemoglobinopathy testing (for enrolled participants only) -- testing for HbAA, HbAC, 
HbCC, HbAS, HbSS 
9.3
Electrocardiogram
Electrocardiograms (12-lead ECGs) will be performed during screening for the main phase and 
as needed throughout the study by the study site team in Mali and read by trained study 
investigators or a cardiologist. Subjects with clinically significant abnormalities will be 
excluded from the study. Subjects with QTc > 450 ms will be excluded. 
9.4
Malaria Diagnostics
9.4.1 Malaria Blood Smears
The gold standard for malaria diagnosis and evaluation of VE endpoints is the detection 
of malaria parasites on Giemsa-stained thick blood films. Blood films will be prepared 
at the specified time points or when clinically indicated and will be examined by 
technicians with a documented experience in slide reading following the SOP (ML-
005-05) established for malaria diagnosis in Sanaria PfSPZ studies slide reading within 
the MRTC (Guindo, Shott et al. 2012), and in consideration of the modifications 
suggested for CHMI trials (Laurens, Duncan et al. 2012). During the pilot study, ALL 
blood smears, regardless of the presence or absence of symptoms, will be read per 
procedures followed when evaluating symptomatic participants, depicted as sBS. Slides 
are considered positive if at least two unambiguous parasites per slide are identified and 
confirmed by a second microscopist. Positive results will be reported promptly to the 
study Principal Investigator (PI) or designee. 
Thick blood smears will be prepared from the blood remaining in the collection device, 
or (at time points when no blood collection is planned) from a finger prick sample. The 
smears will be examined microscopically.
Thick blood smears will be used for diagnosis throughout the study.
9.4.2 Malaria qPCR
While detection of parasites on thick blood smears remains the most common primary 
endpoint in human CHMI 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 (Walther, Dunachie et al. 2005, Walther, Thompson 
et al. 2006). These research molecular assays have significantly increased sensitivity for 
detection of Pf blood-stage infection approaching 20 parasites/mL, often resulting in 
diagnosis 2-4 days earlier than by paired thick blood smears (Hermsen, Telgt et al. 
2001, Schneider, Schoone et al. 2004, Andrews, Andersen et al. 2005). 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 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 87 of 146
a research qPCR that detects 18s of Pf with a detection limit of at least 20 parasites/mL 
that will be used during the study for comparison to traditional thick blood smears. 
Pf qPCR will be performed retrospectively as outlined in the study procedures (see 
Appendix A) to capture infections that remain below the detection limit for 
microscopy. For subject convenience, a finger prick sample can be used for both 
preparation of the microscopy slide and for nucleic acids preservation.
9.5
Unscheduled Blood Smear Positive Visits
Subjects with detection of Pf malaria parasites on thick blood smear after the third vaccination 
and after the fourth vaccination (booster dose) only (whether or not they were symptomatic; 
scheduled or unscheduled visits) will be asked to the clinic within 1 calendar day to provide an 
additional blood sample for the following as seen in Appendix A:

0.5 mL EDTA microtainer: for whole blood ex-vivo assays if sample not obtained 
within the last 7 days.

4 mL EDTA tube: to obtain RNA and DNA for the study of host and parasite 
transcriptome (RNA) and parasite genotype (DNA) and to obtain plasma for 
proteomics studies if sample not obtained within the last 28 days in the main study.
As indicated in Appendix A, a malaria qPCR will be collected at any unscheduled visit at 
which a blood smear sample is also collected.
10 Immunologic Laboratories
As indicated in the objectives, assays will be conducted to assess immunogenicity in addition 
to safety as described above. Laboratory assays to assess immune response to PfSPZ Challenge 
during vaccination and post CHMI will be performed at the Laboratory of Malaria 
Immunology and Vaccinology, NIAID, NIH, Sanaria Inc, and NIH Center for Human 
Immunology, Autoimmunity, and Inflammation (CHI) according to standard laboratory 
procedures.
These assays include:
1. Binding ELISA for antibodies to circumsporozoite protein (PfCSP), and 
potentially to other Pf SPZ, liver stage and blood stage antigens (which includes 
but not limited to: sporozoite surface protein 2, liver stage antigen 1, erythrocyte 
binding antigen 175 (EBA-175), merozoite surface protein 1, merozoite surface 
protein 5, and exported protein 1 [PfLSA-1, PfEBA-175 , PfMSP-1, PfMSP-5, 
EXP-1]
2. (IFN-γ) Enzyme-linked immunosorbent spot assay (ELISPOT) and multi-
parameter flow cytometry with intracellular cytokine staining (ICS) on peripheral 
blood mononuclear cells in P. falciparum liver stage antigens (CSP, LSA-1) and 
PfSPZ 
3. B and T cells studies to analyze immunologic responses 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 88 of 146
Sanaria Inc. will also assess antibodies to whole PfSPZ by immunofluorescence assay (IFA) 
and inhibition of sporozoite invasion assay and to AES parasites by IFA as described (Epstein, 
Tewari et al. 2011, Seder, Chang et al. 2013) and also by protein microarrays (Antigen 
Discovery Inc). 
Laboratory assays to assess immune responses to novel pre-erythrocytic antigens may be 
performed in the PEVA Consortium laboratories at the Laboratory of Malaria Immunology and 
Vaccinology, NIAID, NIH according to standard laboratory procedures. The target proteins are 
novel antigens that confer protection against liver stage malaria in rodent malaria models (P. 
yoelii, P. berghei) according to vaccination studies conducted by Seattle BioMed and LMIV. 
The novel antigens to be used for these laboratory assays may include PFL1995c, PFE0305w, 
LISP1 (PF14_0179), SAP1 (PF11_0480), MAL7P1.164, PF14_0113, using the identifiers in 
the PlasmoDB database (www.plasmodb.org). These antigens were initially selected on the 
basis of their gene expression during early liver stage development of P. falciparum, and 
preliminary testing shows that these antigens are immunologically recognized by individuals 
previously exposed to P. falciparum. The potential utility of these antigens as pre-erythrocytic 
vaccines has been supported by animal studies, wherein orthologues of these genes 
incorporated in DNA vaccines induce protective immunity in mice that significantly reduces 
the liver stage development of P. berghei and P. yoelii parasites. The assays included in this 
study can confirm that individuals receiving PfSPZ-CVac (pyrimethamine) develop immune 
responses to pre-erythrocytic antigens, and can provide additional data by which to assess the 
potential for these antigens to be developed as subunit vaccines to prevent infection. The long-
term objective of the PEVA consortium is to identify antigens that individually, or in 
combination with CSP or other antigens, will induce a high level of pre-erythrocytic immunity 
that is protective against P. falciparum.
The assays to be performed include:
1. Binding ELISA for antibodies to P. falciparum pre-erythrocytic antigens (PFL1995c, 
PFE0305w, LISP1 (PF14_0179), SAP1 (PF11_0480), MAL7P1.164, PF14_0113)
2.  Cellular responses, such as assays of proliferation or cytokine responses (eg, IFN-γ 
ELISPOT/ICS assay) on peripheral blood mononuclear cells stimulated with P. 
falciparum pre-erythrocytic antigens (PFL1995c, PFE0305w, LISP1 (PF14_0179), 
SAP1 (PF11_0480), MAL7P1.164, PF14_0113)
11 Research Use, Storage, and Tracking of Specimens and Data
Intended Use: Samples and data collected under this protocol will be used to study malaria 
and related diseases and possible adverse reactions (ARs) to vaccination. 
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/Bethesda, MD or at LMIV’s designated repository, 
Thermo Scientific, Rockville, MD, with the exception of retention specimens which may be 
kept at the MRTC in Mali for quality control. Samples and data will be stored using codes 
assigned by the investigators or their designees; the key to the code will remain with the 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 89 of 146
MRTC in Mali. 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 use these samples and/or data for research purposes. If the 
planned research falls within the category of “human subjects research” on the part of the NIH 
researchers, IRB review and approval will be obtained. This includes the NIH researchers 
sending out coded and linked samples or data and getting results that they can link back to their 
subjects. 
At the completion of the protocol (termination), samples and data will either be destroyed or, 
after IRB 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.
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 
PI or designee will destroy all known remaining samples and report this destruction to the 
subject and the NIH IRB and Faculty of Medicine, Pharmacy and Odonto-Stomatology Ethics 
Committee (FMPOS EC). This decision will not affect the subject’s continued participation in 
this protocol or any other protocols supported by the NIH.
12 Retention of Specimens for Future Use
Specimens collected as part of this trial will be stored for future research. These samples may 
be used to learn more about malaria infection and other diseases. These samples will not be 
sold or used to make commercial products. The subject may withdraw permission for future 
use of specimens at any time. If a subject withdraws his or her permission for future use of 
specimens, those specimens will be destroyed. All samples stored will be labeled with the 
subject’s study identification (ID) number, which cannot identify the study subject but is 
linkable to other research databases (e.g., questionnaires, clinical assessments, logbooks) 
generated by the main study. The database will contain only the study subject’s ID number. A 
master log linking the study subject ID number to the name of the subject will be maintained 
in a password protected database system with access limited to authorized research team 
members. In the event of samples being requested in the future, only the site Investigators or 
site study coordinator will have access to the log linking the study subject to the samples.
At the completion of the protocol (termination), samples and data will either be destroyed, 
stored, or transferred to another existing protocol such as “Research Use of Human 
Specimens”, NIAID Protocol #08-I-N064. In the future, other investigators may wish to study 
these samples and/or data. If so, the NIH may send samples to the other investigators. If the 
planned research falls within the category of “human subjects research” on the part of the NIH 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 90 of 146
researchers, IRB review and approval will be obtained. This includes the NIH researchers 
sending out coded and linked samples or data and getting results that they can link back to their 
subjects. 
13 Data Sharing Plan
In NIH'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 
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 will transfer materials to outside researchers in both the 
private and public sectors under a Material Transfer Agreement or Research Collaboration 
Agreement. 
14 Assessment of Safety
14.1 Documenting, Recording, and Reporting Adverse Events
At each contact with the subject, information regarding AEs will be elicited by appropriate 
questioning and examinations and will be:

immediately documented in the subject’s medical record/source document,

recorded on the electronic database, and

reported as outlined below (e.g., Investigational New Drug 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 on a study clinician to report an AE, it will be 
discussed with the PI and documented, recorded, and reported appropriately.
All events, both expected/unexpected and related/unrelated will be recorded on a source 
document. Source documents will include: progress notes, laboratory reports, consult notes, 
survey tools and data collection tools. Source documents will be reviewed in a timely manner 
by the research team. All reportable AEs that are identified will be recorded in the database. 
The start date, the stop date, the severity of each reportable event, and the PI’s or designee’s 
judgment of the AEs relationship and expectedness to the study agent/intervention will also 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 91 of 146
be recorded in database. 
14.2 Definitions for the Sponsor
Adverse Event (AE)
Any untoward medical occurrence in a human subject, including any abnormal sign, symptom, 
or disease, temporally associated with the subject’s participation in research, whether or not 
considered related to the subject’s participation in 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)
A Serious Adverse Event (SAE) is defined as any adverse event that

results in death;

is life-threatening (places the subject at immediate risk of death from the event as it 
occurred);

results in inpatient hospitalization or prolongation of existing hospitalization;

results in a persistent or significant disability/incapacity

results in a congenital anomaly/birth defect; or

based upon appropriate medical judgment, may jeopardize the subject’s health and may 
require medical or surgical intervention to prevent one of the other outcomes listed in 
this definition. (examples of such events include allergic bronchospasm requiring 
intensive treatment in the emergency room or at home, blood dyscrasias or convulsions 
that do not result in inpatient hospitalization, or the development of drug dependency 
or drug abuse)
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.
Protocol Deviation 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 92 of 146
Any change, divergence, or departure from the IRB approved study procedures in a research 
protocol. Protocol deviations are designated as major  or minor.
      Major Deviations - Deviations from the IRB approved protocol that have, or may have the 
potential to, negatively impact, the rights, welfare or safety of the subject, or to substantially 
negatively impact the scientific integrity or validity of the study. 
      Minor Deviations - Deviations that do not have the potential to negatively impact the 
rights, safety, or welfare of subjects or others, or the scientific integrity or validity of the study.
Non-compliance: Failure of investigator(s) to follow the applicable laws, regulations, or 
institutional policies governing the protection of human subjects in research or the 
requirements or determinations of the IRB, whether intentional or not 
Unanticipated Problem (UP): Any event, incident, experience, or outcome that meets all 
three of the following criteria would be considered a UP: 
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
2. related or possibly related to participation in the research; and
3. suggests that the research places subjects or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) than was previously 
known or recognized.
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. These events may involve a greater risk of social or 
economic harm to subjects or others rather than physical/psychological harm. Such events 
would be considered a non-serious UP. Examples of an UPnonAE include a breach 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.
14.3 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 (except for malaria, when in addition each 
laboratory value will be separately recorded) will be recorded as the AE. Assessment of safety 
will include clinical observation and monitoring of hematological, chemical, and immunologic 
parameters. Subjects will be closely monitored for at least 30 minutes following the first dose 
of any medication and after each administration of PfSPZ Challenge. Additionally, subjects 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 93 of 146
will return to the clinic for clinical assessments, almost daily during the peak parasitemia 
period from day 7 through 14 post vaccination in the pilot Arms. In the main arms, after first 
vaccination, participants will be followed approximately every other day on days 7 through 14. 
After subsequent vaccinations, participants will be seen on every other day on days 7 through 9 
post vaccination (Appendix A). 
 
The type of information to be collected includes solicited symptoms (direct questioning about 
known possible side effects of the product), unsolicited symptoms (open-ended questioning 
such as “do you have any other symptoms”), signs (fever, tenderness, etc.), start date, severity 
and duration of symptoms, laboratory abnormalities. There will also be an assessment of the 
degree of relatedness of AEs to investigational product or other study procedures. The 
information to be captured is summarized below:
Local reactogenicity related to PfSPZ Challenge injection – solicited questions include pain, 
tenderness, redness, swelling, induration, bruising, and pruritus at the injection site:

Collected for 7 days post administration of PfSPZ Challenge

Collected from the subject via solicited questions at each study visit 
Systemic reactogenicity related to PfSPZ Challenge injection – solicited questions include 
rash, urticaria, pruritus, edema, headache, fever, chills, malaise, myalgia, arthralgia, 
palpitations, shortness of breath, dizziness/vertigo and non-musculoskeletal chest pain:

Collected for 7 days post administration of PfSPZ Challenge

Collected from the subject via symptom solicited questions at each study visit 
Systemic symptoms related to parasitemia/malaria infection – solicited questions include 
headache, fever, chills, rigors, sweats, malaise, dizziness, palpitations, shortness of breath, 
myalgia, arthralgia, nausea, vomiting, abdominal pain, diarrhea, back pain, non-
musculoskeletal chest pain:

Collected from 7-14 days post administration of PfSPZ Challenge during the 
immunization phase via solicited questions at study visits
Systemic symptoms related to chloroquine – solicited questions include headache, nausea, 
vomiting, diarrhea, abdominal pain, anorexia, dizziness/vertigo tinnitus, change in vision, sleep 
disturbance, pruritus, rash due to photosensitivity, rash, peripheral neuropathy, anxiety, muscle 
weakness, weight loss

Collected from initial loading dose of chloroquine until 6 days post last 
chloroquine administration

Collected from the subject via symptom solicited questions at each study visit 
Systemic symptoms related to PYR – solicited questions include nausea, vomiting, diarrhea, 
abdominal pain, anorexia, rash 

Collected for 6 days post PYR administration

Collected from the subject via symptom solicited questions at each study visit 
Unsolicited AEs will be assessed throughout the study at each study visit (telephone and clinic 
follow up) until the final study visit. After that period only unsolicited AEs, SAEs, UPs, and 
NOCIs will be recorded.


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 94 of 146
All AEs will be graded for severity and assessed for relationship to the study product. 
Reactions will be graded as described in this protocol. A study clinician will be available and 
on call 24 hours a day during the study period. Subjects will be provided contact information 
and contact cards once enrolled with 24 hour access numbers to the study team. Should a 
subject call a study clinician to report an AE, it will be fully documented in the subject’s case 
report form (CRF), study chart, and discussed with the PI.
All local and systemic reactions will be captured on the appropriate source documents and 
database. Those assessed as serious will be further reported on the Sponsor’s SAE/UP report 
form. AEs judged to be possibly, probably, or definitely related to the study product will be 
followed to adequate resolution. All concomitant medications will be collected throughout the 
study.
14.3.1 Adverse Event Definitions
A solicited adverse event is defined as a predefined event that is an expected event related to 
the investigational intervention. These include reactogenicity events following administration 
of PfSPZ Challenge and events that can be reasonably expected as part of this study as 
defined in prior similar studies (Chulay, Schneider et al. 1986, Church, Le et al. 1997, 
Epstein, Rao et al. 2007, Roestenberg, McCall et al. 2009), including local and systemic signs 
and symptoms related to the PfSPZ Challenge and/or diagnosis of malaria or antimalarial 
chemoprophylaxis.
Solicited AEs will be captured by direct questioning during the study. Solicited AEs to be 
recorded as endpoints for this study are provided in Table 15. Solicited AEs that are possibly 
related to chloroquine and/or PYR will be collected for 6 days following each drug 
administration. Solicited AEs that are possibly related to PfSPZ Challenge injection will be 
captured at each visit for 7 days (local and systemic symptoms). Symptoms related to malaria 
secondary to PfSPZ Challenge will be solicited at each visit from 7 to 14 days (systemic 
symptoms) following each injection of PfSPZ Challenge during the PfSPZ-CVac 
immunization phase and booster phase.
Additionally, the following rare side effects have been reported with both chloroquine and 
PYR; these will not be solicited AEs, but relationship will be determined on a subject by 
subject basis.
Chloroquine: severe changes in mood, psychosis, electrocardiographic change, and 
hypotension.
PYR: severe hypersensitivity reactions (such as Stevens-Johnson syndrome, toxic 
epidermal necrolysis, erythema multiforme, anaphylaxis), pulmonary eosinophilia, 
megaloblastic anemia, leukopenia, thrombocytopenia, pancytopenia, atrophic glossitis, 
cardiac rhythm disorders.
Table 15. Solicited Adverse Events
Solicited Adverse Events


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 95 of 146
PfSPZ Challenge (local)
Injection pain/tenderness
Injection swelling/edema
Injection pruritus
Injection erythema/redness
Injection induration 
Bruising
PfSPZ Challenge (systemic)
Rash
Headache
Myalgia
Urticaria
Fever
Arthralgias
Generalized pruritus
Chills/Rigors/Sweats
Dizziness/ Vertigo
Generalized edema
Malaise/Fatigue
Chest Pain (non-skeletal)
Palpitations
Shortness of Breath
PfSPZ Challenge (malaria)
Headache
Malaise/ Fatigue
Myalgia
Fever
Nausea
Arthralgias
Chills/Rigors/Sweats
Vomiting
Back Pain
 Chest Pain (non-skeletal)
Abdominal Pain
Diarrhea
Shortness of Breath
Palpitations
Dizziness/Vertigo
Chloroquine phosphate
Anorexia
Diarrhea
Sleep Disturbance
Nausea
Dizziness/Vertigo
Pruritus
Vomiting
Tinnitus
Photosensitivity
Abdominal Pain
Change in Vision
Peripheral Neuropathy
Headache
Malaise/Fatigue
Muscle Ache or Weakness
Anxiety
Weight Loss
Rash
Pyrimethamine
Nausea
Diarrhea
Anorexia
Vomiting
Abdominal Pain
Rash
Laboratory Results
ALT (increased ALT)
Hgb (decreased Hgb)
WBC (increased WBC, decreased
WBC)
           Cr (increased Cr)
Platelets (decreased 
platelets, increased 
platelets)
ANC (decreased neutrophil 
count)
ALC (increased lymphocytes, 
decreased lymphocytes)
14.3.2 Severity
Severity of AEs will be assessed by the investigator in 
Appendix B. AEs not included in the 
Appendix B will be graded for severity using the followings definitions as seen in Table 16.
Table 16. Definitions for Severity of AE Grading
Severity
Definition
Grade 1 (Mild)
No interference with activity, may use 1 dose 
of an over the counter medication
Grade 2 (Moderate)
Repeated use of non-narcotic pain reliever > 
24 hours or some interference with activity


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 96 of 146
Grade 3 (Severe)
Activities of daily living limited to <50% of 
baseline, medical evaluation/therapy required
Grade 4 (Life-Threatening)
Extreme limitation in activity, significant 
assistance required; immediate medical 
intervention or therapy required to prevent 
death
Grade 5
Death
14.3.3 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. 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 97 of 146
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. 
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 as related (including possibly, probably, or definitely) or unrelated 
(including unlikely or not).
14.4 Investigator Reporting Responsibilities to the Sponsor
14.4.1
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.
14.4.2
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 Sanaria, Inc. 
Regulatory Affairs Office by fax or e-mail attachment within 3 business day after the 
clinical site becomes aware of the event. Deaths and immediately life-threatening SAEs 
will be reported within 1 business day after the clinical site becomes aware of the event. 
Sanaria Inc.
SAE Fax: 240-306-0596
Individuals:
1. Stephen L. Hoffman, M.D.
Tel: 240-403-2701 (office) Tel: 240-299-3178 (mobile)
Email:  slhoffman@sanaria.com
2. Thomas L Richie, M.D., Ph.D.
Tel: 240-403-2727 (office)


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 98 of 146
Tel: 301-466-7943 (mobile)
Email: trichie@sanaria.com
3. Tooba Murshedkar, MS
SAE Fax: 240-306-0596
Email:  tmurshedkar@sanaria.com
In Mali – the clinical site investigator will also notify the LMIV PI and the site medical 
monitor in Mali by email, fax, or telephone within 1 working day of notification of an 
SAE occurrence.
LMIV Contact Information:
Patrick Duffy, MD
Tel: (301) 761-5089
Fax: (301) 480 1962
Email: Patrick.Duffy@nih.gov
Independent Safety Monitor (ISM)
Mamadou Dembele, MD
Service Médicine Interne
Centre Hospitalo-Universitaire du Point G
+ 223 2022 5003 or mobile: +223 7604 93 87
Email: hassiramadydembele@yahoo.fr
14.4.3
Unanticipated Problems
All UPs that are also AEs will be reported to the IND Sponsor on the NIH Reportable 
Events Form sent by fax or e-mail attachment no later than 7 calendar days of site 
awareness of the event. 
UPs that are not AEs will also be reported to the IND Sponsor.
14.4.4
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 
IND Sponsor via fax or email within 3 business days from the site awareness of the 
pregnancy. 
Pregnancy outcome data (e.g., delivery outcome, spontaneous, or elective termination of 
the pregnancy) will be reported to the IND Sponsor 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:

Discontinue the study agent

If prior to the 3rd vaccination or within 4 weeks post 3rd or 4th (booster) vaccination, 
withdrawn from the study but continue in follow-up for safety.

If pregnancy occurs more than 4 weeks after the 3rd vaccination, participants will be 
invited to continue in a study on a modified research study schedule with decreased 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 99 of 146
blood draw volumes (Appendix A)

Report to IND Sponsor 

PI will discuss with Sponsor regarding the timing of the pregnancy from last dose 
of PfSPZ Challenge to determine whether treatment is warranted, appropriate 
treatment per standard Malian ministry of health guidelines will be employed if 
needed

Report to FMPOS Ethics Committee as an informational item

Report to Data and Safety Monitoring Board (DSMB), Sponsor Medical Monitor, 
and Site Medical Monitor

Advice research subject to notify the obstetrician of study product exposure 
(infectious SPZ, chloroquine, PYR, artemether, lumefantrine).
14.5 Reporting Procedures to the NIH IRB and FMPOS EC
14.5.1
Reporting to the NIH IRB
UPs, non-compliance and other reportable events will be reported to the NIH IRB as 
per Policy 801. 
14.5.2
Reporting to the FMPOS EC
Events reported to the NIH IRB will simultaneously be reported to the FMPOS EC. In 
addition, SAEs, related or not related to the research will be reported to the FMPOS EC  
within 72 hours of investigator awareness, regardless of expectedness. 
14.6 Follow-up of Adverse Events and Serious Adverse Events
AEs that occur following enrollment of the subject (or AEs related to study procedures after 
signing the informed consent and before enrollment) 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 electronic record and the SERF. 
SAEs that occur after the study follow-up period that are reported to and assessed by the 
Investigator to be possibly, probably, or definitely related must be reported to the IND 
Sponsor as described above.
14.7 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. Sanaria Inc. will also report any 
relevant safety findings, including AEs that are also UPs, to other clinical trial sites where 
related Sanaria products are being studied.
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, using data provided by the PI. 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 100 of 146
14.8 Pausing Criteria for Entire Study Population
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 PfSPZ-CVac (chloroquine) or PfSPZ-
CVac (pyrimethamine) components. 
14.8.1 Parasitemia and Malaria Symptoms 
LMIV has conducted two clinical trials of PfSPZ-CVac (pyrimethamine) immunization in 
malaria naïve participants (NIAID protocol #15-I-0169 and #17-I-0067) in which patent 
parasitemia (positive blood smear) or subpatent parasitemia (qPCR positive) was not detected 
in any PYR participant during immunization. Subjects enrolled in the chloroquine arms did 
have episodes of both patent and subpatent parasitemia at the expected times and showing the 
expected densities, and no subjects were re-treated or withdrawn from the study due to these 
expected episodes of transient parasitemia. 
During the PYR pilot (Arms 1a, 2a, 5a, 6a), the participants will be monitored closely with 
sBS in real time from 7-12 days after vaccination. A positive blood smear or a diagnosis of 
clinical malaria per Malian ministry of health guidelines (presence of symptoms + positive 
malaria diagnostic test) will result in treatment with standard care, artemether/lumefantrine 
regimen. A positive blood smear or diagnosis of clinical malaria will result in pausing of the 
individual participant in the pilot phase only. A positive malaria blood smear during PfSPZ-
CVac immunization pilot phase will therefore not necessarily provide a pause for the study as a 
whole, nor will it be reported as an UP. However, if a positive blood smear occurs, the 
subject’s information, including results and any additional clinical or laboratory information 
will be sent with a Notification Report to the IND Sponsor for review. 
During the main study and booster phase, in the vaccinated PYR Arms (Arms 1b/5b, 
2b/6b), the participants will be monitored closely clinically daily from 7-9 days after 
vaccination. Blood smears and malaria qPCRs will be collected but will be analyzed 
retrospectively. If a participant is symptomatic, the blood smear will be read and reported in 
real time. A diagnosis of clinical malaria per Malian ministry of health guidelines (presence of 
symptoms + positive malaria diagnostic test) will result in treatment with standard care. 
Diagnosis of clinical malaria will not result in pausing of the individual participant in the 
main study phase, but the information will be stored and considered during the analysis at the 
end of the study. A positive malaria blood smear during PfSPZ-CVac immunization will 
therefore not necessarily provide a pause for the study as a whole, nor will it be reported as an 
UP. 
The study may pause for further review during the pilot if patent parasitemia is detected in any 
subjects in Arms 5a or 6a as outlined in Section 3.2 and Figure 7.
In summary, individual participants in PYR arms will be treated per standard care in the pilot 
phase if any of the following occurs:
1. A positive blood smear
2. Diagnosis of clinical malaria
3. At the PI’s discretion (for example, if non-compliance with PYR ingestion is suspected, 
or if concerning signs or symptoms develop) 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 101 of 146
For vaccinated chloroquine only arms (Arms 3b), participants receiving chloroquine may 
present with patent parasitemia as has been seen in other PfSPZ-CVac (chloroquine) and CPS 
with chloroquine studies during the immunization phase, including positive blood smears. In 
previous studies (NIAID protocol #15-I-0169, #17-I-0067), most participants in chloroquine 
only arm did not develop positive blood smears although they did have subpatent parasitemia 
as documented by positive LMIV qPCR assay. However, in the proposed study, due to higher 
doses of PfSPZ Challenge, a positive blood smear may occur. Therefore, for Arm 3, a positive 
blood smear during PfSPZ-CVac (chloroquine) immunization will not necessarily result in 
pausing for an individual participant or the study as a whole, nor will it be reported as an UP. 
Individual subjects in the chloroquine arms will be re-treated with another antimalarial 
medication (artemether/lumefantrine) and withdrawn if any of the following occurs:
1. A positive blood smear outside of the range of expected peak parasitemia (on or after 
day 12 post PfSPZ Challenge) regardless of symptoms (maximum expected peak 
parasitemia is 7-9 days post PfSPZ Challenge). 
2. A subject with a positive blood smear at any time point and Grade 3 or greater 
symptoms (NOT laboratory values) lasting longer than 48 hours despite adequate 
symptomatic management (e.g. non-steroidal anti-inflammatory drugs) 
3. At the PI’s discretion (for example, if non-compliance with chloroquine ingestion is 
suspected, or if concerning signs or symptoms develop) 
If a positive blood smear occurs during the pilot phase (Arm 3a), the subject’s information, 
including blood smear results and any additional clinical or laboratory information will be sent 
with a Notification Report to the IND Sponsor for review. The Sponsor may additionally 
notify the Chair of the DSMB for evaluation and consequent further review by the DSMB for 
determination if the subject’s patent parasitemia is clinically significant and warrants re-
treatment. The DSMB and the IND Sponsor may recommend close clinical follow-up but no 
immediate re-treatment, or may determine that the findings warrant re-treatment with a back-
up antimalarial regimen. 
The following criteria will be used to define unacceptable parasitemia results and will result 
in an immediate pause of further administration of any PfSPZ Challenge to an entire study 
group pending review by the DSMB to determine if the study should be terminated, 
modified, or continued:

Two (in the pilot)/ four (in the main study) or more subjects experience a positive 
blood smear at any time point associated with Grade 3 symptom(s) (NOT laboratory 
values) lasting longer than 48 hours despite adequate symptomatic management (e.g. 
non-steroidal anti-inflammatory drugs) that is determined to be possibly, probably or 
definitely related to PfSPZ Challenge  
OR

Four or more subjects total (pilot and main study) experience positive blood smears 
outside of the expected peak parasitemia (on or after day 12 post PfSPZ Challenge) in 
chloroquine arms that is determined to be possibly, probably or definitely related to 
PfSPZ Challenge


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 102 of 146
In addition to considering the optimal clinical decisions for the subject, the Sponsor and the 
DSMB will determine if the event impacts the remainder of the study participants and whether 
any protocol procedures should be modified.
14.8.2 Reactogenicity
If a study product (PfSPZ Challenge, chloroquine, PYR) is considered unacceptably 
reactogenic (as described in the following criteria), the study will be paused. 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 IRB.
The following criteria will be used to define unacceptable reactogenicity and will result in 
an immediate pause of further administration of any component of the investigational 
product to an entire study group pending review by the DSMB to determine if the study 
should be terminated, modified, or continued:

One or more subjects experience an SAE that is determined to be possibly, probably 
or definitely related to PfSPZ Challenge, chloroquine, PYR, or

One or more subjects experience a hypersensitivity reaction that is probably 
or definitely related to PfSPZ Challenge or

Any serious clinical illness occurs that is not explained by a diagnosis that is 
unrelated or unlikely related to study product or

Two (Pilot study)/Four (Main study) or more subjects experiencing the same 
solicited AE determined to be possibly, probably or definitely related that persists at 
Grade 3 or higher for >48 hours with adequate symptoms management during the 
PfSPZ-CVac immunization phase of the study or

Two (Pilot study)/Four (Main study) or more subjects experiencing a similar 
unsolicited Grade 3 event or a Grade 4 event that is determined to be possibly, 
probably or definitely related to the study product or

Any safety issue that the study PI or IND Sponsor determines should pause 
the study
The IRB, FMPOS EC, 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, 
IRB, FMPOS EC, the IND Sponsor, the FDA, and other regulatory authorities may also result 
in suspension of further administration of immunizations at the clinical site (in case of the 
DSMB, a recommendation of suspension to the Sponsor). The FDA, other regulatory 
authorities, and the study Sponsor(s) retain the authority to suspend additional enrollment and 
administration of immunizations for the entire study as applicable.
14.8.3 Reporting of Study Pausing
If a pausing 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 IND Sponsor by fax or email.


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 103 of 146
The site investigator will inform the LMIV PI, the ISM, and the local IRB that a pausing rule 
has been met according to their requirements. The LMIV PI will notify the NIH IRB. The IND 
Sponsor will notify the DSMB as well as all sites conducting Sanaria-sponsored studies, or 
studies using Sanaria related products that the study has been paused.
14.8.4 Resumption of a Paused Study
The IND Sponsor, in collaboration with the PI and the DSMB will determine if it is safe to 
resume the study. The IND Sponsor will notify the PI of this decision. The conditions for 
resumption of the study will be defined in this notification. The site investigator will notify 
their local IRB(s) of the study pause and of the decision to resume the study. The NIH IRB(s) 
will also be notified. 
14.9 Pausing Criteria for a Subject or Group
The decision to pause administration of the study agent(s) for a single participant or for all 
participants in a specific group requires discontinuation of study agent administrated for the 
study participant(s) or group until a decision is made whether or not to continue study agent 
administration. 
The pausing criteria for a single participant or for the subjects in a specific arm in this study 
include: 

A subject experiences an SAE or ≥ two Grade 3 or greater AEs that are unexpected (as 
determined by the IND Sponsor) and lasting longer than 48 hours despite adequate 
symptomatic management (e.g. non-steroidal anti-inflammatory drugs) that is 
determined to be possibly, probably or definitely related to a study agent; or

Any safety issue that the Site Investigator determines should pause administration of 
the study agent to a single participant or to all participants in a specific group. 
The IND Sponsor, in collaboration with the PI, may also pause for an individual participant or 
entire group if a safety concern is identified during routine aggregate data analysis.
14.9.1 Reporting of Pausing for a Participant or Group
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 1 business day to the IND Sponsor and LMIV PI. 
The PIs will notify the ISM, and the local IRB (NIH IRB, FMPOS EC). The IND Sponsor will 
report this to the DSMB.
14.9.2 Resumption of a Paused Study
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.
14.10 Withdrawal Criteria for an Individual Participant
A subject will not be considered to have completed the trial if any of the following reasons 
apply:


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 104 of 146
1. Research terminated by Sponsor or Investigator – applies to the situation where the 
entire study is terminated by the Sponsor or Investigator, or other regulatory authority 
for any reason.
2. Withdrawal of consent – applies to a subject who withdraws consent to participate in 
the study for any reason.
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 study products (other than Grade 3 local reactions lasting <72 
hours, systemic reactions lasting <24 hours or those that occur 7-10 days after PfSPZ 
Challenge and assessed to be related to induced malaria lasting <48 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. HIV/Hepatitis Infection – If a subject acquires HIV or hepatitis infections during the 
course of the study (either by self reporting and confirmed by study team or by repeat 
testing for clinical evaluation), then they meet exclusion criteria for the study. The 
participant will be removed from further scheduled research procedures but will 
continue to be followed for safety. The event will be reported as an SAE as outlined 
in Section 14.
6. Pregnancy – If the subject becomes pregnant during the course of the study, they will 
be withdrawn for safety purposes if the pregnancy happens prior to the third 
vaccination or within 4 weeks of receipt of third or fourth (booster) vaccination. If the 
pregnancy happens more than 4 weeks after the third or fourth (booster) vaccination, 
the participant will be invited to continue with study participation following a 
modified study schedule. Pregnant participants will be followed for the course of the 
pregnancy until pregnancy outcomes are known. 
7.   Inability to tolerate study product – inability to tolerate any component of the study 
product individually or in combination (chloroquine, PYR, PfSPZ Challenge).
8.   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 database. Any subject who has 
received at least 1 dose of PfSPZ Challenge will be strongly encouraged to remain in the 
safety evaluation for the duration of the study (followed at least one more visit post last PfSPZ 
Challenge). In particular, rigorous follow-up for the development of malaria infection 
including the institution of presumptive treatment may be required in order to assure the 
subject’s safety. The subject’s data will be included in the safety and immunogenicity 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 105 of 146
analysis. If a subject fails to complete all planned PfSPZ Challenge administration 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.
14.11 Replacement 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 
will be replaced. 
14.12  Unblinding for the Study
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 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 SOPs and 
submitted to the study statistician for formal unblinding. 
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 one 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. 
For subjects who do not wish to participate in the booster phase, scheduled unblinding will 
occur on or about Study Day 238 at the completion of the subject’s final visit as already 
outlined in Appendix A. For subjects who do enroll in the booster phase, scheduled unblinding 
will occur on or about study day 569.  All primary and secondary endpoints will have been 
completed at this time for the arm of the study in which the participant is enrolled. Individuals 
directly continuing AE assessment or performing assays for exploratory endpoints will remain 
blinded to individual randomization until assessment or assays are completed.
As noted in Section 1.5.1.3, group unblinding for statistical analysis of blood smear results 
from the first year of the study (prior to the booster dose) will occur approximately 4 months 
following receipt of the booster vaccine dose, as long as 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 the 
pyrimethamine comparator arms on an accelerated vaccination schedule in a future planned 
trial.  
14.13
Safety Oversight
14.13.1 Independent Safety Monitor (ISM) in Mali
An independent safety monitor 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 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 106 of 146
monitoring and recommend appropriate action regarding AEs and other safety issues. The ISM 
is an expert in the field of oversight of clinical trials conducted in Mali and internal medicine, 
specifically in the population under study in Mali. The ISM does not have direct involvement 
in the conduct of the study and does not have other interests with any collaborating 
pharmaceutical firms or their competitors. 
All serious adverse events, all UPs, and all FDA IND Safety Reports will be reported by the PI 
to the ISM prior to or at the same time they are submitted to the IRB or IND Sponsor. The ISM 
will be notified immediately if any pausing rule is met and the ISM will provide 
recommendation for continuation, modification, or termination of the study. The PI will also 
notify the medical monitor if intentional or unintentional unblinding occurs. The ISM will be a 
voting member of the DSMB. 
14.13.2 Data and Safety Monitoring Board (DSMB)
As agreed with Office of Clinical Research Policy and Regulatory Operations (OCRPRO), for 
this study a DSMB chartered by the IND Sponsor, Sanaria Inc. will be used instead of the 
NIAID Intramural DSMB. The DSMB will review the study data to evaluate the safety, 
efficacy, study progress, and conduct of the study, these reviews to be conducted during 
scheduled and ad hoc meetings hosted by the IND Sponsor. The six scheduled meetings are: 
prior to initiation, in the first year; between the pilot and main phase, between the first and the 
second vaccination in the main phase, after completion of main phase during the malaria 
follow up period. In the subsequent year of the study for the CQ arms, additional meetings will 
be scheduled between the first and second vaccination and at close-out, as outlined in Figure 7 
and Figure 9. In addition, the DSMB will review data from the main phase of the study prior 
to the administration of the booster vaccination. The PI will generate safety reports for review 
by the IND Sponsor for all but the first meeting; these reports will be submitted to the DSMB 
prior to the meeting for DSMB review. The board will additionally convene on an ad hoc basis 
as needed, and will issue recommendations concerning continuation, modification, or 
termination of the study.  
All SAEs and UPs will be reported by the Sponsor to the DSMB. The Sponsor will also notify 
the DSMB at the time pausing criteria are met and obtain a recommendation concerning 
continuation, modification, or termination of the study. The Sponsor will initially consult with 
the DSMB chair with regard to these events, to obtain recommendations whether an ad hoc 
meeting should be called.
The PI will submit written DSMB summary reports with recommendations to the IRB(s).
15 Clinical Monitoring
15.1 Site Monitoring Plan
As per International Council for Harmonisation of Technical Requirements for 
Pharmaceuticals for Human Use- Good Clinical Practice (ICH-GCP) guideline 5.18 and FDA 
21 CFR 312.50, clinical protocols are required to be adequately monitored by the study 
Sponsor. OCRPRO will provide oversight and monitor the compliance of this trial. 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 site to 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 107 of 146
monitor aspects of the trial in accordance with appropriate regulations. The objectives of a 
monitoring visit will be:

to verify the prompt reporting of all monitored data points, and prompt 
reporting of all SAEs

to check the existence of signed informed consent documents and 
documentation of the ICF process for each monitored subject

to compare individual subject’s records (e.g. CRFs, electronic data) to the source
documents (supporting data, laboratory specimen records, clinical notes)

to ensure the investigators are in compliance with the protocol
The monitors will also inspect the clinical site’s regulatory files to ensure that applicable 
regulatory requirements (FDA, Office for Human Research Protections [OHRP]) and ICH 
guidelines are being obeyed. During the monitoring visits, the PI and/or designated study 
staff will be available to discuss the study. The site PI will provide direct access and allow 
the study monitors, LMIV, the IND Sponsor, and regulatory authorities to access all study-
related documents.
A specific monitoring plan will be discussed with the PI, study staff, and IND Sponsor prior 
to enrollment. The plan will outline the frequency of monitoring visits based on such factors 
as study enrollment, data collection status and regulatory obligations.
Quality control procedures will be implemented beginning with the data entry system, and 
data quality control checks that will be run on the database will be generated. Any missing 
data or data anomalies will be communicated to the site for clarification/resolution.
16 Statistical Considerations 
16.1 General Pilot Study
PfSPZ-CVac (pyrimethamine) groups: The pilot study will investigate whether 75 mg of PYR 
administered either concurrently (day 0) with PfSPZ injection or on day 2&3 post injection 
will prevent breakthrough parasitemia in all subjects following DVI of 4.0x105 PfSPZ. The 
pilot may also explore whether administration of 75mg of PYR on either day 0 or on day 2&3 
with 3.0x105 PfSPZ may prevent parasitemia if there is breakthrough with higher dose of 
PfSPZ. 
For the PfSPZ-CVac (chloroquine) groups: The pilot study will investigate whether the 4.0x105 
dose of PfSPZ Challenge is safe and well tolerated in malaria-exposed adults or whether 
NSAIDS are needed to improve tolerability.
16.2 General Main Study and Booster Study
The main phase and booster phase is designed to investigate the safety profile of the selected 
PfSPZ Challenge regimen with concurrent chemoprophylaxis with PYR or chloroquine in 
healthy adults. As well, we wish to estimate protective efficacy against natural malaria 
transmission, starting two weeks after the third vaccination. We will explore whether the 
timing of the PYR and dose of PfSPZ Challenge can be adjusted to occur more quickly after 
PfSPZ Challenge without impacting the protective efficacy. 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 108 of 146
16.3 Primary Endpoints:
Safety
Pilot Phase

Incidence of positive sBS occurring after PfSPZ-CVac immunization 
starting on day 7 post DVI (Arms 1a, 2a, 5a, 6a).

Incidence and severity of local and systemic grade 3 signs or symptoms 
lasting more than 48 hours despite adequate management and serious 
adverse events (SAEs) occurring after PfSPZ-CVac DVI (Arm 3a)
Main Phase and Booster Phase

Incidence and severity of local and systemic adverse events (AEs) and 
serious adverse events (SAEs) occurring after PfSPZ-CVac 
immunization. (Arms 1b/5b, 2b/6b, 4a, 4b)

Incidence of clinical malaria diagnosis occurring after PfSPZ-CVac 
immunization as defined by the occurrence of grade 3 signs or symptoms 
lasting more than 48 hours despite adequate management. (Arm 3b, 4c)
Protective Efficacy

P. falciparum blood stage infection defined as detection of at least 2 P. 
falciparum parasites by microscopic examination of 0.5 µL of blood starting 2 
weeks after the 3rd vaccination for approximately 6 months and then 2 weeks 
after the 4th vaccination for approximately 6 months for the booster phase. (Arm 
1b/5b, 2b/6b, 3b, 4)
Pyrimethamine regimen efficacy and Immunogenicity

P. falciparum blood stage infection defined as detection of P. falciparum parasites 
by qPCR following PfSPZ Challenge. (Arms 1, 2, 5, 6).)

Humoral immune responses after PfSPZ-CVac regimens by assessing antibodies 
to PfSPZ, Pf asexual erythrocytic stages (AES), and specific Pf sporozoite, liver 
and blood-stage antigens such as CSP, MSP-1, AMA-1 in subjects with no 
patent and/or subpatent parasitemia and those with patent and/or subpatent 
parasitemia, among drug groups. (All Arms)

Cellular immune responses after PfSPZ-CVac regimens to PfSPZ, PfAES, and 
specific Pf sporozoite, liver and blood-stage antigens, such as CSP, MSP-1, 
AMA-1, in subjects with no patent and/or subpatent parasitemia and those with 
patent and/or subpatent parasitemia, among drug groups. (All Arms) 

Cellular and humoral responses that correlate with protective efficacy


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 109 of 146

Prolonged prepatent period and/or reduced density and duration of parasitemia 
in those developing parasitemia during PfSPZ-CVac immunization (Arms 1, 2, 
3, 5, 6)

Comparison of γδ T cells before and after PfSPZ-CVac immunization and 
malaria infection during transmission season using ex vivo whole blood or in 
vitro staining. (All Arms)
16.3 Analysis
16.3.1 Analysis of Primary Endpoints
Safety in each arm will be summarized descriptively, using proportions and exact 95% 
confidence intervals.
16.3.2 Analysis of Secondary Endpoints
The protective efficacy of each PYR experimental arm (Arms 1b and 2b) in the main phase 
will be assessed against the first two  control arms, pooled (Arms 4a and 4b) using a Holm’s 
adjustment based on an overall one-sided type 1 error rate of .025.  
The protective efficacy of the chloroquine experimental arm (Arm 3b) in the main phase will 
be assessed against the final control arm (Arms 4c) using an overall one-sided type 1 error rate 
of .025. This will not be adjusted for multiple comparisons, as it will be be considered a 
separate study for all practical purposes.
The infection rates over time will be compared using log-rank tests. Fisher’s Exact Tests will 
also be conducted, and both will be reported. 
The same efficacy analyses will be performed at the end of the main phase and the end of the 
booster phase. 
16.3.3 Analysis of Exploratory Endpoints
Appropriate methods will be used in exploratory data analysis. All findings will be 
considered hypothesis generating, rather than evidence of any given pre-specified 
hypothesis. 
16.4 Sample Size and Power Calculations
16.4.1 Power calculations for primary objective (safety): Pilot Phase
Table 17 describes the probability of breakthrough parasitemia, infection, or an adverse event 
during the pilot phase. For a group of four participants, there is a 76% chance of observing at 
least 1 event if the true rate of such an event is 30% or more; the probability of observing at 
least 1 event among four participants is 94% if this same true rate of 50%. If we see one event 
among 4, we will be 95% confident that the true rate will be between 1% and 81%. 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 110 of 146
Table 17. Probability of observing 0, 1 and 2 or more break through parasitemia or 
adverse event among arms of size 4, for different true event rates
True event rate (%)
Pr(0/4)
Pr(1/4)
Pr(2+/4)
1
0.96
0.04
<0.01
5
0.81
0.17
0.01
10
0.66
0.29
0.05
20
0.41
0.41
0.18
30
0.24
0.41
0.435
50
0.06
0.25
0.69
16.4.2 Power calculations for Main Phase and Booster Phase
16.4.2.1
Power calculations for primary objective (safety)
The goal of the safety evaluation for this study is to identify safety concerns associated with 
product administration. The ability of the study to detect serious adverse events (SAEs) can be 
expressed by the true event rate above which at least 1 SAE would likely be observed and the 
true event rate below which no events would likely be observed. Specifically, for Arms 1b and 
3b (n=90), there is a 90% chance of observing at least 1 event if the true rate of such an event 
is 2.5% or more, and a 90% chance of observing at least 2 events if the true rate is at least 
4.5%. For Arm 2b, (n=60), there is a 90% chance of observing at least 1 event if the true rate of 
such an event is 4% or more, and a 90% chance of observing at least 2 events if the true rate is 
at least 6.3%. 
Probabilities of observing 0,1, and 2 or more events among Arms of size 90 and 60 are 
presented in 
Table 18, for a range of possible true adverse event rates. These calculations provide a more 
complete picture of the sensitivity of this study design to identify potential safety problems 
with the vaccine. 
Based on data from existing studies, we expect the number of participants in the booster phase 
to be similar to that in the main phase. The above sample size considerations should 
approximately apply to the booster phase.
Table 18. Probability of observing 0, 1 and 2 or more events, among arms of size 90 or 60, 
for different true event rates
True event rate (%)
Pr(0/90)
Pr(1/90)
Pr(2+/90)
Pr(0/60)
Pr(1/60)
Pr(2+/60)
1
0.40
0.77
0.23
0.55
0.33
0.12
3
0.06
0.24
0.76
0.16
0.30
0.54
5
0.01
0.06
0.94
0.05
0.15
0.80
10
<0.01
<0.01
>0.99
<0.01
0.01
0.99


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 111 of 146
An alternative way of describing the statistical properties of the study design is in terms of the 
95% confidence interval for the true rate of an adverse event based on the observed data. 
Error! Reference source not found.Table 19 shows the 2-sided 95% confidence intervals for 
the probability of an event based on a particular observed rate. If none of the 60 participants in 
an arm in the main study experience a safety event, the 95% 2-sided upper confidence bound 
for the true rate of such events in the total vaccinated population is 0.06. 
Table 19. Two-sided 95% confidence intervals based on observing a particular rate of 
safety endpoints for arms of size 90 or 60
Observed event rate
95% Confidence interval (%)
0/60
(0.00, 0.06)
1/60
(<0.01, 0.09)
2/60
(<0.01, 0.11)
0/90
(0.00, 0.04)
1/90
(<0.01, 0.06)
2/90
(<0.01, 0.08)
16.4.2.2
Power calculations for secondary objective (efficacy)
Each experimental arm will be compared to the control arm using a Holm’s adjustment for 
multiple comparisons. 
Table 20. Sample size calculations for various plausible true event rates among both control 
and vaccinated subjects below gives the sample size needed for 80% power to detect a 
difference using a Fisher’s Exact Test or a logrank test and a Bonferroni adjusted alpha of 
.05/2=.025. Table 21 gives the power for comparisons of 81/group, allowing 10% loss to 
follow up. We will have good power to detect a 50% VE for the comparisons of either Arm 1b 
or 3b to control (Arm 4). Since the actual analysis will use a Holm’s adjustment, this 
calculation is conservative. For a given comparison we will have at least 90% power to detect a 
difference if the true infection rate among the controls is approximately 70% and VE, defined 
here as 1- the probability of infection in the treatment group divided by the probability of 
infection in the controls, is at least 50%.
Table 20. Sample size calculations for various plausible true event rates among both 
control and vaccinated subjects
True infection Rate in
Number needed per group
(no LTFU)
Controls
Vaccinated
VE
Fisher’s Exact Test
Log-rank test 
(Freedman)
.3
.4
123
110
.5
.25
.5
77
75


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 112 of 146
.2
.6
52
52
.36
.4
87
83
.3
.5
56
55
.6
.24
.6
39
37
Table 21: Power to detect a difference in efficacy with 81/group (after 10% loss to follow 
up)
True Infection 
Rate, controls
True Infection Rate, 
experimental arm
Vaccine 
Efficacy (VE)
Power
81/group
Power
54/group
.6
.3
50%
95%
84%
.5
.25
50%
86%
69%
16.5 Randomization
Participants in the pilot arms will be randomized into either Arm 1a, 2a or 3a. If additional 
arms are to be enrolled in the pilot phase, these additional arms (Arms 5a and 6a) will not be 
randomized unless enrollment into Arms 5a and 6a occurs prior to completion of Arms 1a, 2a or 3a 
9 (study day 13). If enrollment occurs early, the participants will be randomized into Arms 5a 
and 6a. They will however be enrolled sequentially as they become eligible to enroll. Subjects 
will be randomized into enrollment into Arms 1b/5b, 2b/6b, 4a, and 4b in the first year. 
Subjects will be randomized into Arms 3b, and 4c in a subsequent year. The pilot phase of the 
study will be open-label, but the main part of the study will be blinded in regard to PfSPZ 
Challenge or NS administration: subjects and investigators will not be aware of the injection 
being given, whether Arm 1b/5b or 4b; Arm 2b/6b or 4b; or Arm 3b or 4c). The subjects and 
investigators will be aware of the medication assignments, PYR or chloroquine.
17 Human Subject Protections and Ethical Obligations
This research will be conducted in compliance with the protocol, GCP, and all applicable 
regulatory requirements.
17.1 Institutional Review Board
A copy of the protocol, informed consent forms, and other information to be completed by 
subjects, such as questionnaires, 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 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 113 of 146
throughout the duration of the study. The investigators will notify the reviewing IRBs of 
protocol deviations and SAEs as specified in the relevant sections of the protocol.
17.2 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. 
The informed consent will be translated into French and administered orally in the native 
dialect in the case of potential subjects who cannot read. 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. A copy of the informed consent 
document will be given to the subjects for their records. The researcher will document the 
signing of the consent form in the subject’s medical record. 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. 
17.2.1 Mali Site Community Permission and Individual Informed Consent Process
17.2.1.1 Community Permission
Community permission will be obtained from village elders, family heads, and other 
community members after explanation and discussion of the study.(Diallo, Doumbo et al. 
2005) The community permission process goes through the following steps:

Study investigators/personnel explain the study to village leaders, including the 
village chief, family heads, women association, and elders.

The village leaders then discuss the study with family heads and community members 
and relay any additional questions or concerns they may have to study personnel.

The study and the informed consent process are explained in detail to heads of 
families by study investigators/personnel.  
At the time of community permission, the need for both husband and wife to agree to avoid 
pregnancy for the specified period if a wife chooses to volunteer will also be addressed.
The individual informed consent process and 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.


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 114 of 146
17.2.1.2 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.
17.3 Justification for Exclusion of Children
This study will not enroll children, since safety of the new chloroquine and PYR regimen has 
not yet been established in adults.
17.4 Justification for the Exclusion of Pregnant Women
This study will not enroll pregnant and/or breastfeeding women since the effects of PfSPZ 
Challenge on the developing human fetus are unknown. In addition, for the duration of the 
study, this study will not enroll pregnant women as artemether/lumefantrine are pregnancy 
category C drugs. The safety and efficacy of artemether/lumefantrine in the treatment of acute, 
uncomplicated malaria in pregnant women have not been established. Artemether/lumefantrine 
should only be used during pregnancy if the potential benefit justifies the potential risk to the 
fetus. Given all subjects in the main study will receive artemether/lumefantrine regardless of 
the presence of malaria parasites in their blood, the risk of drug is not justified in pregnant 
women. However, if a woman becomes pregnant after enrollment she may continue in the 
study for safety follow up or may be invited to continue in the study with modified research 
procedures if pregnancy happens after the third vaccination. No additional investigational 
products will be administered.


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 115 of 146
17.5 Subject Confidentiality
All records will be kept confidential to the extent provided by federal, state and local law. The 
study monitors and other authorized representatives of the Sponsor may inspect all documents 
and records required to be maintained by the Investigator, including but not limited to, 
medical records. Records will be kept locked and 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 
OHRP, Sanaria Inc., or the Sponsor’s designee.
17.6 Risks
Risks to the subjects are associated with venipuncture, malaria infection, PfSPZ Challenge, 
chloroquine, PYR, and large volume blood drawing. For large blood draws, no more than 
80 mL of blood will be drawn at a single time point and the total amount of blood drawn is 
not to exceed 10.5 mL/Kg or 550 mL whichever is smaller in a 56 day period (as defined as 
the amount of blood allowed to be drawn under the M95-9: guidelines of blood drawn for 
research purposes in the NIH Clinical Center).
These study risks are outlined below:
17.6.1 Venipuncture
Risks occasionally associated with venipuncture include pain, bruising, bleeding and 
infection at the site of venipuncture, lightheadedness, and rarely, syncope.
17.6.2 PfSPZ Challenge administered by DVI
Excluding the malaria symptoms caused by the parasitemia that may follow the 
injection of non-attenuated PfSPZ (see next section), PfSPZ Challenge as well as other 
PfSPZ-based products have not caused adverse signs or symptoms, apparently due to 
the low reactogenicity of purified SPZ, and the asymptomatic nature of liver stage 
infection. Following the parenteral injection of any vaccine, possible local reactions 
include transient pain, swelling, erythema, induration, limitation of limb movement, 
lymphadenopathy, or pruritus at the injection site. Administration of a vaccine may 
also cause systemic reactions such as fever, chills, headache, fatigue, malaise, myalgia, 
and joint pain, with some reactions moderate or severe. However, with the exception 
of mild and transient local (site of administration) reactions, these ARs remain 
theoretical with respect to PfSPZ-based products, which appear remarkably well 
tolerated. Similarly, no laboratory abnormalities have been clearly related to the 
administration of PfSPZ, although in some studies a transient drop in peripheral 
lymphocyte counts has been documented, likely resulting from margination, diapedesis 
into interstitial tissues and possibly migration of mononuclear cells to central organs 
such as the liver, in response to the antigenic stimulus. 
In Equatorial Guinea two serious adverse events possibly related to PfSPZ Vaccine have been 
described. One volunteer who received PfSPZ Vaccine had a miscarriage at 10 weeks after 
getting pregnant while participating in a PfSPZ Vaccine trial. On vaccination day, the subject 
had a negative urine pregnancy test. She was started on contraceptive measures on the same 
day she received the vaccine and was advised to additionally use a barrier method.


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 116 of 146
Miscarriages frequently occur without known causes and although it is unlikely the vaccine 
caused the miscarriage, the temporal relationship meant this was a possibility. All women of 
child-bearing age are required to take birth control measures as specified within the protocol 
and/or consent form to avoid getting pregnant while participating in trials of PfSPZ-based 
products. In this protocol, volunteers have to use two forms of contraception and must have 
started a hormonal method at least 21 days prior to receipt of first PfSPZ Challenge injection. 
Another volunteer, a 15-year-old boy who received PfSPZ Vaccine, had a generalized seizure 
3 ½ hours after receiving his third dose. The boy fully recovered from the seizure. The EEG 
showed that he was predisposed to having seizures. It is unlikely the vaccine
caused the seizure, but like all vaccines, PfSPZ Vaccine causes an immune response in the 
body which may increase the chance that those individuals predisposed to seizures 
experience a seizure.
As with any infusion, 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.
Subjects may be asked to defer routine immunization (such as influenza) until 14 – 28 days 
following vaccination. 
17.6.3 Malaria Symptoms during immunization with PfSPZ-CVac (chloroquine)
Research subjects immunized with PfSPZ-CVac (chloroquine) may experience the usual, well-
known side effects of chloroquine, which are discussed in a following section (see below). 
Other than the side effects of chloroquine, the adverse event profile associated with PfSPZ, as 
discussed in the preceding section, is benign. However, this changes after six days, due to the 
exiting of the parasites from the liver into the blood. In malaria naïve participants, there 
follows a 3 to 4-day period of transient parasitemia that occurs 6-10 days after injection, 
reaching maximum density on day 7, 8 or 9. Parasitemia then falls quickly, as the parasites are 
exposed to toxic levels of chloroquine, and are rapidly killed. In the Tübingen TÜCHMI-002 
trial, the highest density recorded after administering 5.12x104 PfSPZ of PfSPZ Challenge by 
DVI was 36.4 parasites/μL. The geometric mean density in all 9 volunteers after the first dose 
of PfSPZ Challenge was 15.7 parasites/μL. These densities are high enough to cause symptoms 
in some malaria-naïve individuals, since the symptom threshold is typically crossed at densities 
of 5-15 parasites/μL, although others may not experience symptoms until parasite densities 
exceed 50 parasites/μL. The individual with a density of 36.4 parasites/μL in the Tübingen trial 
did experience transient grade 2 (moderate) symptoms of malaria, which rapidly resolved 
without intervention. In the second PfSPZ-CVac trial that we are conducting at the NIH 
(NIAID protocol #17-I-0067), a 4-fold higher dose of PfSPZ was be used, and signs and 
symptoms of malaria were common, including Grade 3 signs and symptoms, and there were 
also several subjects with positive thick blood smears. The symptoms quickly resolved and 
could be prevented almost entirely by presumptive treatment with ibuprofen or another 
NSAID. Parasite densities fall precipitously during this period, and when highly sensitive 
quantitative polymerase chain reaction (qPCR) is used for detection, parasite densities fall were 
very low by day 10 ( 0.01 parasite/µL) or were undetectable. Any residual low readings on 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 117 of 146
qPCR appear to represent detection of nucleic acid from non-viable parasites, since 
recrudescent infections have not occurred in these individuals. 
Given the fact that parasitemia occurs during PfSPZ-CVac (chloroquine) immunization, 
administering the partner drug is a critical aspect of the procedure for maintaining the safety of 
the study subjects. Chloroquine is an appropriate partner drug due to the high efficacy of 
PfSPZ-CVac when this partner drug is used (see the description of VE earlier in the protocol), 
and because the NF54 parasite strain is highly sensitive to this drug. The 50% inhibitory 
concentrations (IC50s) for the PfSPZ Challenge master cell bank (MCB) and WCB are 8.6-8.7 
ng/mL, and this cannot change (i.e., resistance cannot evolve) because each lot of PfSPZ 
Challenge manufactured by Sanaria is derived from the same WCB. When these IC50s are 
compared to the lowest CQ level ever measured to date in a trial of PfSPZ-CVac (chloroquine), 
32 ng/mL, it can be seen that there is a large safety margin. CQ levels are generally much 
higher than this, ranging upward to 110 ng/mL. Safety is assured by the fact that CQ is 
metabolized to desethyl-CQ, which also has parasiticidal activity against asexual blood stages. 
It is thus not surprising that, with one exception (see below), no recrudescent infections have 
occurred in any research subject during PfSPZ-CVac immunization.
To assure the proper administration of CQ to all volunteers, directly observed treatment (DOT) 
will be done, including oral examination to assure that the tablets have been swallowed. 
Equally important to assure safety is to monitor for signs and symptoms of parasitemia after 
immunization, especially days 7-10 after each injection of PfSPZ Challenge as outlined in 
Appendix A. If signs or symptoms likely caused by parasitemia occur, they will be followed 
closely. Thick blood smears will be collected but read in real time if the participant is 
symptomatic. If needed (see discussion earlier in the protocol), re-treatment will be instituted 
with another antimalarial drug or drug combination if clinically indicated. 
Interestingly, despite using the same dose of PfSPZ Challenge that induced symptomatic 
parasitemia in these studies, malaria-related side effects did not occur at all in the two trials 
where PfSPZ-CVac (chloroquine) has been tested in malaria exposed adults in Africa, one 
performed in Mali and one in Equatorial Guinea (discussed in detail earlier in the protocol). 
This is almost certainly due to naturally acquired immunity, which has developed in these 
individuals after years of exposure to repeated malaria infections. Because the current study 
will be done in the same population – malaria-exposed African adults – we do not expect 
significant ARs, even using a dose of PfSPZ Challenge (4.0x105 PfSPZ) that is nearly twice as 
high as that studied in the prior trial in Mali (2.048x105 PfSPZ).
No SAEs deemed related to PfSPZ Challenge have occurred in any trials of PfSPZ-CVac. 
However, two noteworthy AEs have occurred. The first occurred in the first PfSPZ-CVac 
(chloroquine trial), conducted in Holland, where PfSPZ Challenge was administered by ID 
injection. Several hours after the fourth chloroquine dose, one participant experienced 
transitory urticaria at multiple sites of the body lasting for 3 days (corresponding to days 5–8 
after PfSPZ Challenge injection). The subject did not receive any treatment for the urticaria, 
continued in the study, received two more injections with PfSPZ Challenge and underwent 
CHMI, but did not develop recurrent urticaria or any other indication of an allergic reaction. 
The etiology of the urticaria was unclear. The second noteworthy adverse event was an SAE 
that occurred 3 days after immunization #3 in the 15-I-0169 trialat NIH. A 23-year-old 
participant in a group receiving the antimalarial drugs PYR and CQ presented with the acute 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 118 of 146
onset of nausea, vomiting, headache, tinnitus, mental “fogginess” and confusion worsening 
over the course of a week. The participant was hospitalized for 4 days to facilitate evaluation 
and management, and treated with IV acyclovir for presumed herpes simplex virus 
encephalitis. Cerebrospinal fluid was not obtained, because the volunteer had a Chiari 
malformation noted on brain MRI scan. Symptoms improved over time with the main 
symptom of “fogginess” fully resolving in 11 days. Convalescent viral titers were unrevealing. 
Final diagnosis was encephalopathy of unknown etiology deemed possibly related to 
chloroquine, which has been reported to cause a similar clinical picture.
In addition to these two AEs, there was an unanticipated event that placed a study subject at 
risk and was reported to the SMC, the IRBs and the US and German regulatory agencies. This 
occurred during the second part of the TÜCHMI-002 trial, during follow-up after the first 
immunization. The research subject experienced parasitemia documented by PCR on day 7 
after PfSPZ Challenge injection, with the parasite burden peaking on day 8 at a density by 
qPCR (15.7 parasites/µL) that was typical for the study. Parasitemia then fell progressively on 
days 9 and 10, consistent with natural cycling of blood stage parasite growth and/or partial 
killing by CQ. However, there was increasing parasitemia on day 11 by qPCR, and the subject 
was found to be thick blood smear positive on day 13, and experienced mild symptoms of 
malaria (headache, sweating), suggesting that sufficient CQ was either not ingested or not 
absorbed. Parasitemia peaked on day 13 (1271.2 parasites/µL). The volunteer was treated with 
atovaquone / proguanil on days 13 to 15 and parasitemia cleared promptly. This case is 
discussed in more detail earlier in the protocol. 
In summary, we do not expect the study subjects to experience signs and symptoms of malaria 
due to naturally acquired immunity, but because 4.0x105 PfSPZ is the largest dose of PfSPZ 
Challenge yet administered to humans, the study subjects will be followed closely during the 
period of transient parasitemia, including symptom checks and blood smears, and retrospective 
measurement of parasite densities using qPCR.
Immunization with PfSPZ-CVac (pyrimethamine) should not lead to parasitemia. However, 
this could occur during this trial, as the optimal doses of PfSPZ and PYR are worked out. For 
this reason, the study subjects in the PYR arms will also be monitored closely starting day 6 
after injection of PfSPZ Challenge, with sBS performed daily (qPCRs performed 
retrospectively) from 6-12 days post injection after first vaccination and 7-9 days post injection 
after subsequent vaccinations to permit early detection of any breakthrough parasitemia. If 
parasitemia is detected and clinical malaria is diagnosed per Malian ministry of health 
guidelines, they will be treated immediately. Otherwise, participants in the pilot phase will be 
treated for malaria at the end of their study participation.
17.6.4  Medications used in the study
The antimalarial medications administered in the study (PYR, chloroquine, 
artemether/lumefantrine) are generally well tolerated in the dosing regimens to be used, 
although common side effects (e.g., nausea, vomiting, diarrhea, headaches, sleep 
disturbance) are anticipated to occur in some subjects. Potential subjects with known 
sensitivity or contraindications to the antimalarials administered in this study are excluded 
from participation. Subjects in the study will be closely monitored for adverse drug reactions 
and where possible, attempts will be made to minimize anticipated side effects (e.g. dosing 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 119 of 146
of medications with food). Specific toxicities for PYR, chloroquine and 
artemether/lumefantrine are noted below and further outlined in the package insert for each 
drug.
17.6.4.1 Chloroquine Phosphate (CQ)
The most commonly reported side effects of chloroquine dosing include gastrointestinal 
disturbance, headache, dizziness, blurred vision, insomnia, tinnitus, and pruritus, but generally 
these effects do not require discontinuation of the drug. High doses of chloroquine, as used to 
treat rheumatic diseases (>3. 5 mg/kg/day over 1 year or more), have been associated with 
retinopathy, although generally requires a cumulative dose exceeding 100 grams (Salako 
1984) and thus is extremely unlikely at doses used for routine weekly malaria prophylaxis 
especially for the limited duration in this study. Chloroquine is reported to exacerbate 
psoriasis; therefore, subjects with a history of psoriasis are excluded. Rarely, chloroquine may 
cause seizures or life-threatening allergic reactions. If these or other ARs known to be 
associated with chloroquine occur, even if they are serious (leading to hospitalization), this 
will not mandate pausing the study. Rather, chloroquine will be immediately discontinued in 
the affected study subject, any further treatment needed will be given, and no further PfSPZ 
Challenge injections will be administered.
In a recent study recently completed by LMIV here at the NIH (NIAID protocol # 15-I-0169) 
using a combination of the live malaria parasites, PYR and chloroquine, one volunteer 
developed a serious adverse event (SAE) after the third injection. The symptoms included 
confusion, dizziness, headache, and nausea requiring a short hospital stay to find out the cause 
of the volunteer’s symptoms. The volunteer was treated with an antiviral medication and 
recovered quickly and fully. It was suspected that the volunteer either had a virus infection of 
the brain or had possibly developed a rare side effect of chloroquine. Chloroquine is a safe 
FDA approved medication but there is very low risk of neurological side effects. Volunteers 
who take chloroquine will be asked about these types of symptoms frequently and at all clinic 
visits.
In an ongoing LMIV PfSPZ-CVac study at NIH (NIAID protocol # 17-I-0067) in which some 
participants are receiving chloroquine, one participant presented with acute change in mental 
status. This was later diagnosed to be due to acute anticholinergic poisoning after ingesting seeds 
of a Datura stramonium plant for recreational purposes. This episode was deemed to also be 
possibly related to chloroquine due to timing as it is unclear whether concurrent consumption with 
chloroquine may potentiate the effects of this plant. 
For complete chloroquine safety information, including less commonly reported side effects, 
please refer to the Package Insert for chloroquine that is provided.
17.6.4.2 Pyrimethamine
The most commonly reported side effects of PYR dosing include vomiting and anorexia but 
generally these effects do not require discontinuation of the drug and can be minimized by 
giving the medication with food. These sides effects may increase with PYR dose but usually 
disappears promptly upon reduction of dosage. Prolonged and higher doses of PYR, as used 
to treat toxoplasmosis, may produce megaloblastic anemia, leukopenia, thrombocytopenia, 
pancytopenia, atrophic glossitis, hematuria, and disorders of cardiac rhythms though this is 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 120 of 146
extremely unlikely at doses used for this study. Hematologic effects, however, may also occur 
at low doses in certain individuals, thus hematological parameters will be closely 
monitored. 
PYR has occasionally been associated with severe hypersensitivity reactions (such as 
Stevens-Johnson syndrome, toxic epidermal necrolysis, erythema multiforme, and 
anaphylaxis), and hyperphenylalaninemia, can occur particularly when PYR is administered 
concomitantly with a sulfonamide. The approved dosage of PYR for treatment of 
toxoplasmosis can be higher than utilized for malaria treatment or chemoprophylaxis (75-100 
mg per day) and given for prolonged periods of time (weeks to months), usually in 
conjunction with sulfadoxine and folinic acid. Chronic high doses used in treatment of 
toxoplasmosis may produce megaloblastic anemia, leukopenia, thrombocytopenia, 
pancytopenia, atrophic glossitis, hematuria, and disorders of cardiac rhythm. 
For complete PYR safety information, including less commonly reported side effects, please 
refer to the Package Insert for PYR that is provided.
17.6.4.3 Artemether/Lumefantrine
For artemether/lumefantrine the most commonly reported side effects in adults are: headache, 
anorexia, dizziness, asthenia, arthralgia and myalgia. Generally, these effects do not require 
discontinuation of the drug.
Artemether/lumefantrine 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 artemether/lumefantrine due to AE is rare (0.2%) in 
adults. Rare but serious hypersensitivity reactions (urticarial and angioedema) and skin 
reactions (bullous eruption) have been reported post marketing. 
Artemether/lumefantrine is a Category C pregnancy drug. Thus, all female participants will 
undergo pregnancy testing prior to receipt of the investigational dose of 
artemether/lumefantrine at the start of the study. Also, per the package insert, 
artemether/lumefantrine 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 artemether/lumefantrine safety information, including less commonly reported 
side effects, please refer to the Package Insert for Coartem® that is provided.
17.6.4.4 Ibuprofen 
Another medication that may be used in the study is ibuprofen or an equivalent nonsteroidal 
anti-inflammatory drug (NSAIDs). These medications pose very low risk in general and are 
usually available over the counter. Specific toxicities of ibuprofen are outlined below and 
further details are available in the provided package insert.
Ibuprofen likewise is not a part of the investigational product/regimen, but will be provided 
to participants in the chloroquine arm (Arm 4) if required to mitigate typical symptoms of 
malaria infection such as headache, myalgia, fever and chills. The most commonly reported 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 121 of 146
side effects of ibuprofen include abdominal discomfort, heartburn, nausea, skin rash, 
dizziness, and tinnitus (ringing in the ears). Less frequent side effects include headache, 
nervousness, itching, edema (fluid retention), constipation, diarrhea, decreased appetite, 
flatulence, and vomiting. Ibuprofen has rarely been associated with abnormal hepatic 
function tests, decreased Cr clearance, leukopenia, thrombocytopenia, allergic rhinitis, 
anaphylaxis, aseptic meningitis, blurred vision, cardiac arrhythmia, confusion, 
conjunctivitis, cystitis, depression, drowsiness, duodenal ulcer, erythema multiforme, gastric 
ulcers, gastritis, gastrointestinal hemorrhage, hallucinations, hearing loss, hematuria, 
hypertension, insomnia, jaundice, palpitations, pancreatitis, peripheral neuropathy, Stevens-
Johnson syndrome, tachycardia, and urticaria.
Note: If an equivalent NSAID is provided, the subject will be informed of the possible side 
effects. 
17.7 Risk to the Community 
Malaria is not contagious and cannot be spread by person-to-person contact. Vaccination for 
this study is designed to occur during the dry season in Mali when malaria transmission and the 
presence of appropriate mosquito vectors in the community to transmit the parasite to others is 
low. 
Benefits: Subjects will not receive any direct benefit from participation in this study. The effect 
of the vaccine on long term anti-malarial immunity is unknown at this time. It is hoped that 
information gained in this study will contribute to the development of a safe and effective 
malaria vaccine.
17.8 Compensation
Subjects will be given in kind (such as rice and/or millet) or cash equivalent, that can be given 
in multiple installments, to compensate for the time taken to come to the study clinic for study-
related visits (Table 22). Preferred compensation will be decided in consultation with village 
elders, but case-by-case exceptions to receive the cash equivalent may be acceptable. The Mali 
EC recommends compensating the study subject for their time lost for study procedures. The 
amount is equivalent to USD $6 for each scheduled visit with blood drawn and equivalent to 
USD $3 for each scheduled visit without blood drawn. For unscheduled visits that request 
research labs to be drawn at that time, such as positive blood smear visits during the malaria 
transmission season, subjects will be compensated USD $6. 
Table 22: Compensation during the study
Study Group(s)
Study Activity
US Dollar Equivalent in Rice 
or Millet Dispensed (Local 
Currency [CFA]
All Arms (Pilot,  
Main, & Booster)
Screening & Enrollment
$18 – $24 = 9,684 – 12,912F
CFA
All Arms (Pilot 
& Main)
Completion of Vaccination #1 & follow up
$60-$66 = 32,280F – 35,508F  
CFA
Main Arms
Completion of Vaccination #2 & follow up
$57-63 = 30,666-33,894F CFA


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 122 of 146
*Assuming currency exchange rate of USD $1 = 538 CFA
18  Data Handling and Record Keeping
18.1 Data Capture and Management
In Mali, the study data will be collected on paper CRFs and then put into a study specific 
DataFax electronic database or entered directly into the DataFax electronic database. Data from 
CRFs will be collected directly from subjects during study visits. Paper or electronic CRFs will 
be used as source. Any type of corrections to paper CRFs must be initialed and dated by the 
person making the correction. Any type of corrections to electronic CRFs will be tracked 
electronically with time, date, individual making the correction, and what was changed. All 
CRFs should be reviewed by the Investigator or designee and signed.
Data entry will be performed by authorized individuals. Corrections to the electronic data 
systems will be tracked electronically (password protected or through an audit trail) with time, 
date, individual making the correction, and what was changed. The investigator is responsible 
for assuring that the data collected are complete, accurate, and recorded in a timely manner.
18.2 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.
18.3 Retention of Study Records
The investigator is responsible for retaining all essential documents listed in the ICH GCP 
Guideline. Study records will be maintained by the PI for a minimum of three 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 and Sanaria Inc. 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 or Sanaria Inc.
Main Arms
Completion of Vaccination #3 & follow up
$45 = 24,210F CFA
Main Arms
Natural Human Malaria Infection (NHMI)
$72 = 38,736F CFA
Booster Phase
Completion of Vaccination #4 & follow up
$75 = 30,666F CFA
Booster Phase
Natural Human Malaria Infection (NHMI)
$72 = 38,736F CFA


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 123 of 146
18.4 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 for review and then 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.


Mali PfSPZ-CVac (pyrimethamine) 
V8.0 03 January 2022      Page 124 of 146
Appendix A: Clinical Evaluation and Laboratory Procedures
Please note: dates of subsequent vaccinations are determined based on the dates of preceding vaccinations rather than on a correlation between Study Day and calendar 
date.
 


Mali PfSPZ-CVac (pyrimethamine) 
V8.0 03 January 2022      Page 125 of 146
 


Mali PfSPZ-CVac (pyrimethamine) 
V8.0 03 January 2022      Page 126 of 146
 


Mali PfSPZ-CVac (pyrimethamine) 
V8.0 03 January 2022      Page 127 of 146
 


Mali PfSPZ-CVac (pyrimethamine) 
V8.0 03 January 2022      Page 128 of 146
Clinic visits 
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Unscheduled Visit 
Study Day (Main 
Study)
29
30
31
32
34
36
37
38
41
43
44
45
48
55
N/A
Days post-PfSPZ Cvac
0
1
2
3
5
7
8
9
12
14
15
16
19
26
N/A
Visit windows (days)
+/-7
0
0
0
+/-1
0
0
0
+/-1
+/-1
+/-1
+/-1
+/-2
+/-5
N/A
PfSPZ CVac #2
Clinic  F/U
Arm 2b, 6b, 4b Only: 
PYR dose
Clinic f/u
Arm 3b, 4c Only: CQ 
dose
 Clinic    F/U; Pre-pat
Clinic F/U;  Pre-pat
Clinic F/U;   Pre-pat
Arm 3b, 4c Only: CQ 
dose
AL dose2
AL dose
AL dose
Arm 3b, 4c Only: CQ 
dose
Clinic f/u 
Unscheduled Visit 
/Positive BS
Complete medical history/ physical
Informed consent
Malaria Comprehension Exam
Pre-test/Post-test HIV counseling
Pregnancy Prevention Counseling
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Interim clinical evaluation
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
AE/SAE assessment
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Conmed review
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
CQ dosing (Arm 3b,4c ONLY)
X
X
X
X
PYR Dose (Arms 2b, 6b, 4b 
ONLY)
X
X
PYR Dose (Arms 1b, 5b, 4a 
ONLY)
X
Artemether/Lumefantrine TX 
Dose (All Arms)
(X)
(X)
(X)
X
X
X
(X)
PfSPZ (Arms 1b, 2b, 3b, 5b, 6b 
ONLY)5
X
LABORATORY PROCEDURES
Designated Laboratory
Tube Type
CBC with differential
EDTA
2
2
2
ALT, Cr
SST
3
3
3
Urine/Serum Pregnancy Test 
(females only)
Urine Container or 
Lithium Hep
X
X
Retrospective Research qPCR
EDTA
0.5
0.5
0.5
0.5
0.5
Retrospective Peripheral Blood Smear1
EDTA 
0.5
0.5
0.5
0.5
0.5
Humoral Assays 
SST
4
Cellular Assays 
NaHep
20
20
Transcriptional Assays 
PAXgene/ Nucleic Acid 
Stabilizer
1
1
1
Ex vivo
EDTA 
1
1
1
1
1
1
0
0
7
0
28
1
1
0
6
0
0
0
26
133.5
133.5
133.5
140.5
140.5
168.5
169.5
170.5
170.5
176.5
176.5
176.5
176.5
202.5
Appendix A: Clinic and Laboratory Procedures For  All  Arms [1b, 2b, 3b, 5b, 6b, 4a, 
4b, 4c] PfSPZ CVac #2
CLINICAL PROCEDURES
LMIV/MRTC
Screening/Safety Labs
Malaria Infection Assays
MRTC CAP/UB/LPD
5 Arms 4a, 4b, 4c will receive Normal Saline injection (not PfSPZ)
2AL dosing to occur AFTER labs collected
1Blood smear will be run Real Time in Symptomatic subjects
Daily total
Study cumulative total
Research Assays


Mali PfSPZ-CVac (pyrimethamine) 
V8.0 03 January 2022      Page 129 of 146
Clinic visits 
32
33
34
35
36
37
38
39
40
Unscheduled Visit 
Study Day (Main Study)
57
58
59
60
62
64
65
66
71
N/A
Days post-PfSPZ Cvac
0
1
2
3
5
7
8
9
14
N/A
Visit windows (days)
+/-5
0
0
0
+/-1
0
0
0
+/-3
N/A
PfSPZ CVac #3
 Clinic F/U
Arm 2b, 6b, 4b Only: PYR dose
Clinic f/u
Arm 3b, 4c Only: CQ dose
Clinic F/U; Pre-pat
Clinic F/U;  Pre-pat
Clinic F/U;     Pre-pat
Clinic f/u
Unscheduled Visit /Positive 
BS
Complete medical history/ 
physical
Informed consent
Malaria Comprehension 
Exam
Pre-test/Post-test HIV 
counseling
Pregnancy Prevention 
Counseling
X
X
X
X
X
X
X
X
X
X
Interim clinical evaluation
X
X
X
X
X
X
X
X
X
X
AE/SAE assessment
X
X
X
X
X
X
X
X
X
X
Conmed review
X
X
X
X
X
X
X
X
X
X
CQ dosing (Arm  3b, 4c 
ONLY)
X
PYR Dose (Arms 2b, 6b, 4b 
ONLY)
X
X
PYR Dose (Arms 1b, 5b, 4a 
ONLY)
X
Artemether/Lumefantrine TX 
Dose
(X)
(X)
(X)
(X)
PfSPZ (Arms 1b, 2b, 3b, 5b, 
6b ONLY)5
X
LABORATORY 
PROCEDURES
Designated Laboratory
Tube Type
CBC with differential
EDTA
2
2
ALT, Cr
SST
3
3
Urine/Serum Pregnancy Test 
(females only)
Urine Container or Lithium 
Hep
X
Retrospective Research qPCR
EDTA
0.5
0.5
0.5
0.5
0.5
0.5
Retrospective Peripheral Blood 
Smear1
EDTA 
0.5
0.5
0.5
0.5
0.5
0.5
Humoral Assays 
SST
4
4
Cellular Assays 
NaHep
20
20
Transcriptional Assays 
PAXgene/ Nucleic Acid 
Stabilizer
1
1
1
Ex vivo
EDTA 
1
1
1
1
1
0
0
7
0
32
1
1
27
203.5
203.5
203.5
210.5
210.5
242.5
243.5
244.5
271.5
5 Arms 4a, 4b, 4c will receive Normal Saline injection (not PfSPZ)
Appendix A: Clinic and Laboratory Procedures For All  Arms [1b, 2b, 3b, 5b, 6b, 4a, 4b, 4c] PfSPZ CVac #3
CLINICAL PROCEDURES
Screening/Safety Labs
MRTC CAP/UB
Malaria Infection Assays
Research Assays
LMIV/MRTC
Daily total
Study cumulative total
1Blood smear will be run Real Time in Symptomatic subjects


Mali PfSPZ-CVac (pyrimethamine) 
V8.0 03 January 2022      Page 130 of 146


Mali PfSPZ-CVac (pyrimethamine) 
V8.0 03 January 2022      Page 131 of 146


Mali PfSPZ-CVac (pyrimethamine) 
V8.0 03 January 2022      Page 132 of 146
Appendix A: Clinic and Laboratory Procedures For All  Arms [1b, 2b, 3b, 4a, 4b, 4c]  Natural Human Malaria Infection (NHMI) Surveillance Post Cvac #4 (Booster)
 
 
Clinic visits 
67
68
69
70
71
72
73
74
75
76
77
78
Unscheduled Visit 
 
 
Study Day (Main Study)
415       
429     
443   
457   
471   
485   
499   
513   
527   
541   
555   
569        
N/A
 
 
Days post Vaccine #4 
(Booster)
28   
41          
56   
70   
84 
98 
112
126 
140 
154 
168 
182 
N/A
 
 
Weeks post Vaccine #4 
(Booster)
4
6
8
10
12
14
16
18
20
22
24
26
 
 
 
Visit windows (days)
+/-3
+/-5
+/-5
+/-5
+/-5
+/-5
+/-5
+/-5
+/-5
+/-5
+/-5
+/-5
N/A
 
 
 
Clinic f/u
Clinic 
f/u
Clinic 
f/u
Clinic 
f/u
Clinic 
f/u
Clinic 
f/u
Clinic 
f/u
Clinic 
f/u
Clinic 
f/u
Clinic 
f/u
Clinic 
f/u
Clinic f/u 
Final 
Unblinding 
visit
Unscheduled Visit 
/ Positive BS
CLINICAL PROCEDURES
Pregnancy Prevention Counseling
 
 
X
 
 
 
 
 
 
 
 
 
 
 
X
Interim clinical evaluation
 
 
X
X
X
X
X
X
X
X
X
X
X
X
X
AE/SAE assessment
 
 
X
X
X
X
X
X
X
X
X
X
X
X
X
Conmed review
 
 
X
X
X
X
X
X
X
X
X
X
X
X
X
Artemether/Lumefantrine TX Dose
 
 
(X)
(X)
(X)
(X)
(X)
(X)
(X)
(X)
(X)
(X)
(X)
(X)
X
LABORATORY PROCEDURES
Designated 
Laboratory
Tube Type
 
 
 
 
 
 
 
 
 
 
 
 
 
Screening/Safety Labs
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CBC with differential
MRTC 
CAP/UB/LPD
EDTA
2
 
2
 
 
2
 
 
2
 
 
 
 
ALT, Cr
 
SST
3
 
3
 
 
3
 
 
3
 
 
 
 
Malaria Infection Assays
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Retrospective Research qPCR
 
EDTA
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Retrospective Peripheral Blood Smear1
 
EDTA 
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Research Assays
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Humoral Assays 
LMIV/MRTC
SST
4
 
 
 
 
 
 
 
 
 
 
4
 
Cellular Assays2 
 
NaHep
30
 
 
 
 
 
 
 
 
 
 
30
 
Transcriptional Assays 
 
PAXgene/ Nucleic Acid 
Stabilizer
1
 
 
 
 
 
 
 
 
 
 
 
 
Parasite Purification (CF11)3
 
EDTA
(4)
 
(4)
 
 
(4)
 
 
(4)
 
 
 
(4)
Ex vivo
 
EDTA
1
 
 
 
 
1
 
 
 
 
 
1
(0.5)
 
Daily total  
42
1
6
1
1
7
1
1
6
1
1
36
 
Study cumulative total  
548.5 
549.5 
555.5 
556.5  
557.5  
564.5 
565.5 
566.5 
572.5 
573.5 
574.5 
610.5 
 
1 Blood smear will be run Real Time in Symptomatic subjects
2 If a woman becomes pregnant 4 weeks after the 3rd vaccination and remains in the study, ONLY 5 mL will be drawn if there are no other contraindications for blood draw.
3  Parasite purification only to be completed if there is a positive blood smear and a sample has not been completed in the previous 28 days


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 133 of 146
Appendix B: Toxicity Table
These tables are modified versions of the FDA Toxicity Grading Scale for healthy adult and 
adolescent subjects enrolled in Preventive Vaccine Clinical Trials to be used to grade adverse 
events.
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
Erythema/Redness *
2.5 – 5 cm
5.1 – 10 cm
> 10 cm
Necrosis or
exfoliative 
dermatitis
Induration/Swelling
**
2.5 – 5 cm and does
not 
interfere 
with 
activity
5.1 – 10 cm 
or
interferes 
with activity
> 10 cm or
prevents 
daily activity
Necrosis
Bruising
2.5 – 5 cm
5.1 – 10 cm
> 10 cm
Necrosis or
exfoliative 
dermatitis
Tenderness
Mild discomfort to 
touch
Discomfort with 
movement
Significant 
discomfort at 
rest
ER visit or 
hospitalization
Pruritus
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
* In addition to grading the measured local reaction at the greatest single diameter, the measurement should be recorded as a 
continuous variable.
** Induration/Swelling should be evaluated and graded using the functional scale as well as the actual measurement


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 134 of 146
Vital Signs *
/Systemic adverse 
events
Mild (Grade 1)
Moderate (Grade 2)
Severe (Grade 3)
Potentially Life-
Threatening
(Grade 4)
Fever (°C) **
(°F) **
38.0 – 38.4
100.4 – 101.1
38.5 – 38.9
101.2 – 102.0
39.0 – 40
102.1 – 104
> 40
> 104
Tachycardia -
beats per 
minute
101 – 115
116 – 130
> 130
ER visit or
hospitalization for 
arrhythmia
Bradycardia -
beats per 
minute***
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
Shortness of breath
Shortness of breath with 
running or doing more 
than ordinary effort**** 
Shortness of breath on 
doing ordinary effort
Shortness of doing less 
than ordinary effort
Shortness of 
breath at rest
Palpitations
No or minimal 
interference with usual 
activities
Greater than minimal 
interference with usual 
activities or with 
associated symptoms
Inability to perform usual
activities
ER visit or 
hospitalization
Photosensitivity/ 
Rash
Localized rash with no 
medical intervention 
indicated
Localized rash with 
medical intervention 
indicated 
Generalized rash with 
medical intervention 
indicated 
ER visit or 
hospitalization
Abdominal Pain
No or minimal 
interference with usual 
activities
Greater than minimal 
interference with usual 
activities
Inability to perform usual
activities
ER visit or 
hospitalization
Anorexia
Transient (< 24 hours) or 
intermittent anorexia with 
no or minimal interference
with oral intake
Persistent anorexia 
resulting in decreased 
oral intake for 24 – 48 
hours
Persistent anorexia 
resulting in minimal oral 
intake for > 48 hours OR
Aggressive rehydration 
indicated (e.g., IV fluids)
Life-threatening 
consequences 
(e.g., 
hypotensive 
shock)
Weight loss
5 to <10% from baseline, 
nutritional supplement not 
required
10 to <20% from 
baseline, nutritional 
supplement required
≥ 20% from baseline, 
invasive intervention for 
nutritional support 
required
Life-threatening 
consequences


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 135 of 146
 
Vital Signs *
/Systemic adverse 
events
Mild (Grade 1)
Moderate (Grade 2)
Severe (Grade 3)
Potentially Life-
Threatening
(Grade 4)
Back Pain
No or minimal 
interference with usual 
activities
Greater than minimal 
interference with usual 
activities
Inability to perform 
usual activities
ER visit or 
hospitalization
Chills/Sweats/
Rigors
No or minimal 
interference with usual 
activities
Greater than minimal 
interference with usual 
activities 
Inability to perform 
usual activities
ER visit or 
hospitalization
Generalized 
Pruritus
Itching causing no or 
minimal interference with 
usual activities
Greater than minimal 
interference with usual 
activities
Inability to perform 
usual activities
ER visit or 
hospitalization
Generalized 
Edema
Localized edema causing 
no or minimal interference
with usual activities
Greater than minimal 
interference with usual 
activities
Inability to perform 
usual activities
ER visit or 
hospitalization
Dizziness/Vertigo
Dizziness/vertigo causing 
minimal interference and 
above baseline
Dizziness/vertigo 
causing greater than 
minimal interference 
with usual social & 
Dizziness/vertigo causing
inability to perform usual
social & functional 
activities 
Disabling dizziness/ 
vertigo causing 
inability to perform 
basic self-care 
Change in Vision
Change in vision causing 
minimal interference and 
above baseline
Difficulty seeing 
causing greater than 
minimal interference 
with usual social & 
functional activities 
Difficulty seeing 
(diplopia, blurriness) 
causing inability to 
perform usual social & 
functional activities 
Disabling change in 
vision (visual field 
loss, blindness) 
causing inability to 
perform basic self-
care functions 
Sleep disorder 
(Insomnia, or 
Difficulty 
Sleeping, 
Drowsiness, etc.)
Difficulty sleeping/     
Sleep disorder causing 
minimal interference and 
above baseline
Sleep disorder/ 
Difficulty sleeping 
causing greater than 
minimal interference 
with usual social & 
functional activities 
Sleep disorder/   
Difficulty sleeping 
causing inability to 
perform usual social & 
functional activities 
Disabling sleep 
disorder/insomnia 
causing inability 
to perform basic 
self-care functions 
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


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 136 of 146
Vital Signs *
/Systemic 
adverse events
Mild (Grade 1)
Moderate (Grade 2)
Severe (Grade 3)
Potentially Life
Threatening
(Grade 4)
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
Fatigue/Malaise
No interference 
with 
activity
Repeated use of non-
narcotic pain reliever 
> 24 hours or
some interference 
with activity
any use of narcotic 
pain reliever or 
prevents daily activity
ER visit or 
hospitalization
Myalgia
No interference 
with 
activity
Repeated use of non-
narcotic pain reliever 
> 24 hours or
some interference 
with activity
any use of narcotic 
pain reliever or 
prevents daily activity
ER visit or 
hospitalization
Arthralgia
No interference 
with 
activity
Repeated use of non-
narcotic pain reliever 
> 24 hours or
some interference 
with activity
any use of narcotic 
pain reliever or 
prevents daily activity
ER visit or 
hospitalization
Muscle 
weakness
No interference 
with 
activity
Greater than minimal 
interference with usual 
activities
Inability to 
perform usual 
activities
ER visit or 
hospitalization
Urticaria
No interference 
with 
activity
Requiring PO or
topical treatment >
24 hours or IV 
medications or 
steroids for ≤24 
hours
Requiring IV 
medication or 
steroids for >24 
hours
ER visit or 
hospitalization
Tinnitus
Ringing in your ears 
causing no or 
minimal interference 
with usual activities
Greater than minimal 
interference with usual 
activities secondary to 
ringing in your ears
Inability to perform 
usual activities 
secondary to ringing in 
your ears.
ER visit or 
hospitalization
Peripheral
Neuropathy
Tingling in your 
hands or feet 
causing no or 
minimal interference 
with usual activities
Tingling in your hands 
or feet causing greater 
than minimal 
interference with usual 
activities
Inability to perform 
usual activities due to 
tingling or pain in your 
hands or feet
ER visit or 
hospitalization


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 137 of 146
     * Subject should be at rest for all vital sign measurements.
** Oral temperature; no recent hot or cold beverages or smoking.
*** 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.
**** Ordinary effort is that of the person himself / herself regards as previous effort of tolerance and usual life style (New York 
Heart Association definition)
Vital Signs *
/Systemic 
adverse 
events
Mild (Grade 
1)
Moderate (Grade 2)
Severe (Grade 
3)
Potentially Life
Threatening
(Grade 4)
Anxiety
No 
interference 
with activity
Repeated use of anti-
anxiety medication  > 24 
hours or
some interference with 
activity
Prevents daily 
activity
ER visit or 
hospitalization
Chest Pain (non- 
musculoskeletal)
Transient (< 24 
hours) or 
intermittent chest 
pain with no or 
minimal 
interference
Persistent chest pain 
resulting in greater than 
minimal interference 
with usual activities
Persistent chest pain 
resulting in inability to 
perform usual activities 
secondary to chest pain
ER visit or 
hospitalization
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


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 138 of 146
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 – 10.3
8.0 – 9.4
6.5 – 7.9
< 6.5 and / or 
requiring 
transfusion
Hemoglobin 
(Female) 
gm/dL 
8.0 – 9.0
7.0 – 7.9
6.0 – 6.9
< 6 and /or 
requiring 
transfusion
WBC Increase – 
103/µL
11.5 – 15.0
15.1 – 20.0
20.1 – 25.0
> 25.0
WBC Decrease - 
103/µL
2.5 – 3.3
1.5 – 2.4
1.0 – 1.4
< 1.0 with fever
Granulocyte or 
Neutrophil 
Decrease - 103/µL
0.8 – 1.0
0.5 – 0.7
< 0.5
< 0.5 with fever
Platelets Decreased 
– 103/µL
100 – 110
70 – 99
25 – 69
< 25
Creatinine (Male) 
µmol/L
124.00 – 
150.99
151.00 – 176.99
177.00 – 
221.00
> 221.00 and 
requires dialysis
Creatinine 
(Female) µmol/L
107.00 – 
132.99
133.00 – 159.99
160.00 – 
215.99
> 216.00 and 
requires dialysis
Liver Function 
Tests –ALT U/L
75.0 – 150.9
151.0 – 300.9
301.0 – 600.0
> 600.0
1The laboratory values provided in the tables serve as guidelines and are dependent upon institutional normal parameters. 
Institutional normal reference ranges should be provided to demonstrate that they are appropriate. 
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. 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 139 of 146
Mali Adult Normals
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 
Granulocyte or 
Neutrophil 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
Blood 
(microscopic) –
red blood cells per 
high power field 
(RBC/HPF)
None or Trace
< 1
1The laboratory values provided in the table are based on Bancoumana, Malian adults (age 18-45 years old)


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 140 of 146
Appendix C: Cardiovascular Risk Assessment
As part of the eligibility determination, subjects will be screened for cardiac risk based on the 
NHANES I study criteria (Gaziano, Young et al. 2008) and screening electrocardiogram. Results will 
be documented in the subject source documentation and Cardiac Risk Assessment CRF. 
NHANES cardiovascular risk assessment includes the following assessments: 
• Evaluation of risk factors: Calculated BMI [weight (kg)/height (m2)], measured Systolic 
Blood Pressure, smoking status and known diabetes status as reported by the subject on review 
of medical history 
• Evaluation of 5-year cardiovascular risk for females or males: Low, Moderate, High 
Note: subjects under the age of 35 are considered low risk by the NHANES I risk assessment 
Only subjects classified as low risk by the NHANES I criteria (green and blue categories below) 
who have a non-significant ECG, as determined by the study cardiologist, are eligible for the 
study.
NHANES I Classification for Women


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 141 of 146
NHANES I Classification for Men


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 142 of 146
References
Agnandji, S. T., B. Lell, S. S. Soulanoudjingar, J. F. Fernandes, B. P. Abossolo, C. Conzelmann, B. G. 
Methogo, Y. Doucka, A. Flamen, B. Mordmuller, S. Issifou, P. G. Kremsner, J. Sacarlal, P. Aide, M. 
Lanaspa, J. J. Aponte, A. Nhamuave, D. Quelhas, Q. Bassat, S. Mandjate, E. Macete, P. Alonso, S. Abdulla, 
N. Salim, O. Juma, M. Shomari, K. Shubis, F. Machera, A. S. Hamad, R. Minja, A. Mtoro, A. Sykes, S. 
Ahmed, A. M. Urassa, A. M. Ali, G. Mwangoka, M. Tanner, H. Tinto, U. D'Alessandro, H. Sorgho, I. Valea, 
M. C. Tahita, W. Kabore, S. Ouedraogo, Y. Sandrine, R. T. Guiguemde, J. B. Ouedraogo, M. J. Hamel, S. 
Kariuki, C. Odero, M. Oneko, K. Otieno, N. Awino, J. Omoto, J. Williamson, V. Muturi-Kioi, K. F. Laserson, 
L. Slutsker, W. Otieno, L. Otieno, O. Nekoye, S. Gondi, A. Otieno, B. Ogutu, R. Wasuna, V. Owira, D. 
Jones, A. A. Onyango, P. Njuguna, R. Chilengi, P. Akoo, C. Kerubo, J. Gitaka, C. Maingi, T. Lang, A. Olotu, 
B. Tsofa, P. Bejon, N. Peshu, K. Marsh, S. Owusu-Agyei, K. P. Asante, K. Osei-Kwakye, O. Boahen, S. 
Ayamba, K. Kayan, R. Owusu-Ofori, D. Dosoo, I. Asante, G. Adjei, G. Adjei, D. Chandramohan, B. 
Greenwood, J. Lusingu, S. Gesase, A. Malabeja, O. Abdul, H. Kilavo, C. Mahende, E. Liheluka, M. Lemnge, 
T. Theander, C. Drakeley, D. Ansong, T. Agbenyega, S. Adjei, H. O. Boateng, T. Rettig, J. Bawa, J. 
Sylverken, D. Sambian, A. Agyekum, L. Owusu, F. Martinson, I. Hoffman, T. Mvalo, P. Kamthunzi, R. 
Nkomo, A. Msika, A. Jumbe, N. Chome, D. Nyakuipa, J. Chintedza, W. R. Ballou, M. Bruls, J. Cohen, Y. 
Guerra, E. Jongert, D. Lapierre, A. Leach, M. Lievens, O. Ofori-Anyinam, J. Vekemans, T. Carter, D. 
Leboulleux, C. Loucq, A. Radford, B. Savarese, D. Schellenberg, M. Sillman, P. Vansadia and S. C. T. P. Rts 
(2011). "First results of phase 3 trial of RTS,S/AS01 malaria vaccine in African children." N Engl J Med 
365(20): 1863-1875.
Andrews, L., R. F. Andersen, D. Webster, S. Dunachie, R. M. Walther, P. Bejon, A. Hunt-Cooke, G. 
Bergson, F. Sanderson, A. V. Hill and S. C. Gilbert (2005). "Quantitative real-time polymerase chain 
reaction for malaria diagnosis and its use in malaria vaccine clinical trials." Am J Trop Med Hyg 73(1): 
191-198.
Bastiaens, G. J., M. P. van Meer, A. Scholzen, J. M. Obiero, M. Vatanshenassan, T. van Grinsven, B. K. 
Sim, P. F. Billingsley, E. R. James, A. Gunasekera, E. M. Bijker, G. J. van Gemert, M. van de Vegte-Bolmer, 
W. Graumans, C. C. Hermsen, Q. de Mast, A. J. van der Ven, S. L. Hoffman and R. W. Sauerwein (2016). 
"Safety, Immunogenicity, and Protective Efficacy of Intradermal Immunization with Aseptic, Purified, 
Cryopreserved Plasmodium falciparum Sporozoites in Volunteers Under Chloroquine Prophylaxis: A 
Randomized Controlled Trial." Am J Trop Med Hyg 94(3): 663-673.
Bijker, E. M., G. J. Bastiaens, A. C. Teirlinck, G. J. van Gemert, W. Graumans, M. van de Vegte-Bolmer, R. 
Siebelink-Stoter, T. Arens, K. Teelen, W. Nahrendorf, E. J. Remarque, W. Roeffen, A. Jansens, D. 
Zimmerman, M. Vos, B. C. van Schaijk, J. Wiersma, A. J. van der Ven, Q. de Mast, L. van Lieshout, J. J. 
Verweij, C. C. Hermsen, A. Scholzen and R. W. Sauerwein (2013). "Protection against malaria after 
immunization by chloroquine prophylaxis and sporozoites is mediated by preerythrocytic immunity." 
Proc Natl Acad Sci U S A 110(19): 7862-7867.
Burkot, T. R., J. L. Williams and I. Schneider (1984). "Infectivity to mosquitoes of Plasmodium falciparum 
clones grown in vitro from the same isolate." Trans R Soc Trop Med Hyg 78(3): 339-341.
Campo, J. J., J. Sacarlal, J. J. Aponte, P. Aide, A. J. Nhabomba, C. Dobano, P. L. Alonso, J. J. Campo, J. J. 
Aponte, J. Skinner, R. Nakajima, D. M. Molina, L. Liang, J. Sacarlal, P. L. Alonso, P. D. Crompton, P. L. 
Felgner and C. Dobano (2014). "Duration of vaccine efficacy against malaria: 5th year of follow-up in 
children vaccinated with RTS,S/AS02 in Mozambique


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 143 of 146
RTS,S Vaccination Is Associated With Serologic Evidence of Decreased Exposure to Plasmodium 
falciparum Liver- and Blood-Stage Parasites." Vaccine 32(19): 2209-2216.
Chulay, J. D., I. Schneider, T. M. Cosgriff, S. L. Hoffman, W. R. Ballou, I. A. Quakyi, R. Carter, J. H. Trosper 
and W. T. Hockmeyer (1986). "Malaria transmitted to humans by mosquitoes infected from cultured 
Plasmodium falciparum." Am J Trop Med Hyg 35(1): 66-68.
Church, L. W., T. P. Le, J. P. Bryan, D. M. Gordon, R. Edelman, L. Fries, J. R. Davis, D. A. Herrington, D. F. 
Clyde, M. J. Shmuklarsky, I. Schneider, T. W. McGovern, J. D. Chulay, W. R. Ballou and S. L. Hoffman 
(1997). "Clinical manifestations of Plasmodium falciparum malaria experimentally induced by mosquito 
challenge." J Infect Dis 175(4): 915-920.
Delves, M., D. Plouffe, C. Scheurer, S. Meister, S. Wittlin, E. A. Winzeler, R. E. Sinden and D. Leroy (2012). 
"The activities of current antimalarial drugs on the life cycle stages of Plasmodium: a comparative study 
with human and rodent parasites." PLoS Med 9(2): e1001169.
Diallo, D. A., O. K. Doumbo, C. V. Plowe, T. E. Wellems, E. J. Emanuel and S. A. Hurst (2005). "Community 
permission for medical research in developing countries." Clin Infect Dis 41(2): 255-259.
Doolan, D. L., C. Dobano and J. K. Baird (2009). "Acquired immunity to malaria." Clin Microbiol Rev 22(1): 
13-36, Table of Contents.
Epstein, J. E., K. M. Paolino, T. L. Richie, M. Sedegah, A. Singer, A. J. Ruben, S. Chakravarty, A. Stafford, R. 
C. Ruck, A. G. Eappen, T. Li, P. F. Billingsley, A. Manoj, J. C. Silva, K. Moser, R. Nielsen, D. Tosh, S. 
Cicatelli, H. Ganeshan, J. Case, D. Padilla, S. Davidson, L. Garver, E. Saverino, T. Murshedkar, A. 
Gunasekera, P. S. Twomey, S. Reyes, J. E. Moon, E. R. James, N. Kc, M. Li, E. Abot, A. Belmonte, K. Hauns, 
M. Belmonte, J. Huang, C. Vasquez, S. Remich, M. Carrington, Y. Abebe, A. Tillman, B. Hickey, J. Regules, 
E. Villasante, B. K. Sim and S. L. Hoffman (2017). "Protection against Plasmodium falciparum malaria by 
PfSPZ Vaccine." JCI Insight 2(1): e89154.
Epstein, J. E., S. Rao, F. Williams, D. Freilich, T. Luke, M. Sedegah, P. de la Vega, J. Sacci, T. L. Richie and 
S. L. Hoffman (2007). "Safety and clinical outcome of experimental challenge of human volunteers with 
Plasmodium falciparum-infected mosquitoes: an update." J Infect Dis 196(1): 145-154.
Epstein, J. E., K. Tewari, K. E. Lyke, B. K. Sim, P. F. Billingsley, M. B. Laurens, A. Gunasekera, S. 
Chakravarty, E. R. James, M. Sedegah, A. Richman, S. Velmurugan, S. Reyes, M. Li, K. Tucker, A. 
Ahumada, A. J. Ruben, T. Li, R. Stafford, A. G. Eappen, C. Tamminga, J. W. Bennett, C. F. Ockenhouse, J. 
R. Murphy, J. Komisar, N. Thomas, M. Loyevsky, A. Birkett, C. V. Plowe, C. Loucq, R. Edelman, T. L. Richie, 
R. A. Seder and S. L. Hoffman (2011). "Live attenuated malaria vaccine designed to protect through 
hepatic CD8(+) T cell immunity." Science 334(6055): 475-480.
Friesen, J., S. Borrmann and K. Matuschewski (2011). "Induction of antimalaria immunity by 
pyrimethamine prophylaxis during exposure to sporozoites is curtailed by parasite resistance." 
Antimicrob Agents Chemother 55(6): 2760-2767.
Gama, H., A. Ismael, F. Sitoi, A. Matola, H. Barros and N. Lunet (2009). "Factors associated with 
chloroquine-induced pruritus during malaria treatment in Mozambican university students." Gac Sanit 
23(4): 306-310.


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 144 of 146
Guindo, M. A., J. P. Shott, R. Saye, M. L. Diakite, S. Sanogo, M. B. Dembele, S. Keita, M. C. Nagel, R. D. 
Ellis, J. A. Aebig, D. A. Diallo and O. K. Doumbo (2012). "Promoting good clinical laboratory practices and 
laboratory accreditation to support clinical trials in sub-Saharan Africa." The American journal of tropical 
medicine and hygiene 86(4): 573-579.
Haeusler, I. L., X. H. S. Chan, P. J. Guerin and N. J. White (2018). "The arrhythmogenic cardiotoxicity of 
the quinoline and structurally related antimalarial drugs: a systematic review." BMC Med 16(1): 200.
Hermsen, C. C., D. S. Telgt, E. H. Linders, L. A. van de Locht, W. M. Eling, E. J. Mensink and R. W. 
Sauerwein (2001). "Detection of Plasmodium falciparum malaria parasites in vivo by real-time 
quantitative PCR." Mol Biochem Parasitol 118(2): 247-251.
Hoffman, S. L., P. F. Billingsley, E. James, A. Richman, M. Loyevsky, T. Li, S. Chakravarty, A. Gunasekera, 
R. Chattopadhyay, M. Li, R. Stafford, A. Ahumada, J. E. Epstein, M. Sedegah, S. Reyes, T. L. Richie, K. E. 
Lyke, R. Edelman, M. B. Laurens, C. V. Plowe and B. K. Sim (2010). "Development of a metabolically 
active, non-replicating sporozoite vaccine to prevent Plasmodium falciparum malaria." Hum Vaccin 6(1): 
97-106.
Hoffman, S. L., L. M. Goh, T. C. Luke, I. Schneider, T. P. Le, D. L. Doolan, J. Sacci, P. de la Vega, M. Dowler, 
C. Paul, D. M. Gordon, J. A. Stoute, L. W. Church, M. Sedegah, D. G. Heppner, W. R. Ballou and T. L. 
Richie (2002). "Protection of humans against malaria by immunization with radiation-attenuated 
Plasmodium falciparum sporozoites." J Infect Dis 185(8): 1155-1164.
Laurens, M. B., C. J. Duncan, J. E. Epstein, A. V. Hill, J. L. Komisar, K. E. Lyke, C. F. Ockenhouse, T. L. 
Richie, M. Roestenberg, R. W. Sauerwein, M. D. Spring, A. K. Talley, V. S. Moorthy and I. Consensus 
Group on Design of Clinical Trials of Controlled Human Malaria (2012). "A consultation on the 
optimization of controlled human malaria infection by mosquito bite for evaluation of candidate malaria 
vaccines." Vaccine 30(36): 5302-5304.
Mordmuller, B., G. Surat, H. Lagler, S. Chakravarty, A. S. Ishizuka, A. Lalremruata, M. Gmeiner, J. J. 
Campo, M. Esen, A. J. Ruben, J. Held, C. L. Calle, J. B. Mengue, T. Gebru, J. Ibanez, M. Sulyok, E. R. James, 
P. F. Billingsley, K. C. Natasha, A. Manoj, T. Murshedkar, A. Gunasekera, A. G. Eappen, T. Li, R. E. 
Stafford, M. Li, P. L. Felgner, R. A. Seder, T. L. Richie, B. K. Sim, S. L. Hoffman and P. G. Kremsner (2017). 
"Sterile protection against human malaria by chemoattenuated PfSPZ vaccine." Nature.
Most, H., I. M. London, C. A. Kane, P. H. Lavietes, E. F. Schroeder and J. M. Hayman, Jr. (1984). 
"Landmark article July 20, 1946: Chloroquine for treatment of acute attacks of vivax malaria. By Harry 
Most, Irving M. London, Charles A. Kane, Paul H. Lavietes, Edmund F. Schroeder and Joseph M. Hayman, 
Jr." JAMA 251(18): 2415-2419.
Ocana-Morgner, C., M. M. Mota and A. Rodriguez (2003). "Malaria blood stage suppression of liver stage 
immunity by dendritic cells." J Exp Med 197(2): 143-151.
Organization, W. H. (April 2013 (rev. January 2014)). WHO policy brief for the implementation of 
intermittent preventive treatment of malaria in pregnancy using sulfadoxine-pyrimethamine (IPTp-SP).
Polhemus, M. E., S. A. Remich, B. R. Ogutu, J. N. Waitumbi, L. Otieno, S. Apollo, J. F. Cummings, K. E. 
Kester, C. F. Ockenhouse, A. Stewart, O. Ofori-Anyinam, I. Ramboer, C. P. Cahill, M. Lievens, M. C. 
Dubois, M. A. Demoitie, A. Leach, J. Cohen, W. R. Ballou and D. G. Heppner, Jr. (2009). "Evaluation of 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 145 of 146
RTS,S/AS02A and RTS,S/AS01B in adults in a high malaria transmission area." PLoS One 4(7): e6465.
Richie, T. L., P. F. Billingsley, B. K. Sim, E. R. James, S. Chakravarty, J. E. Epstein, K. E. Lyke, B. Mordmuller, 
P. Alonso, P. E. Duffy, O. K. Doumbo, R. W. Sauerwein, M. Tanner, S. Abdulla, P. G. Kremsner, R. A. Seder 
and S. L. Hoffman (2015). "Progress with Plasmodium falciparum sporozoite (PfSPZ)-based malaria 
vaccines." Vaccine 33(52): 7452-7461.
Roestenberg, M., E. M. Bijker, B. K. Sim, P. F. Billingsley, E. R. James, G. J. Bastiaens, A. C. Teirlinck, A. 
Scholzen, K. Teelen, T. Arens, A. J. van der Ven, A. Gunasekera, S. Chakravarty, S. Velmurugan, C. C. 
Hermsen, R. W. Sauerwein and S. L. Hoffman (2013). "Controlled human malaria infections by 
intradermal injection of cryopreserved Plasmodium falciparum sporozoites." Am J Trop Med Hyg 88(1): 
5-13.
Roestenberg, M., M. McCall, J. Hopman, J. Wiersma, A. J. Luty, G. J. van Gemert, M. van de Vegte-
Bolmer, B. van Schaijk, K. Teelen, T. Arens, L. Spaarman, Q. de Mast, W. Roeffen, G. Snounou, L. Renia, 
A. van der Ven, C. C. Hermsen and R. Sauerwein (2009). "Protection against a malaria challenge by 
sporozoite inoculation." N Engl J Med 361(5): 468-477.
Roestenberg, M., A. C. Teirlinck, M. B. B. McCall, K. Teelen, K. N. Makamdop, J. Wiersma, T. Arens, P. 
Beckers, G. van Gemert, M. van de Vegte-Bolmer, A. J. A. M. van der Ven, A. J. F. Luty, C. C. Hermsen and 
R. W. Sauerwein (2011). "Long-term protection against malaria after experimental sporozoite 
inoculation: an open-label follow-up study." The Lancet 377(9779): 1770-1776.
RTS, S. C. T. P. (2015). "Efficacy and safety of RTS,S/AS01 malaria vaccine with or without a booster dose 
in infants and children in Africa: final results of a phase 3, individually randomised, controlled trial." 
Lancet.
Salako, L. A. (1984). "Toxicity and side-effects of antimalarials in Africa: a critical review." Bull World 
Health Organ 62 Suppl: 63-68.
Schneider, P., G. Schoone, H. Schallig, D. Verhage, D. Telgt, W. Eling and R. Sauerwein (2004). 
"Quantification of Plasmodium falciparum gametocytes in differential stages of development by 
quantitative nucleic acid sequence-based amplification." Mol Biochem Parasitol 137(1): 35-41.
Schofield, L. and I. Mueller (2006). "Clinical immunity to malaria." Curr Mol Med 6(2): 205-221.
Seder, R. A., L. J. Chang, M. E. Enama, K. L. Zephir, U. N. Sarwar, I. J. Gordon, L. A. Holman, E. R. James, P. 
F. Billingsley, A. Gunasekera, A. Richman, S. Chakravarty, A. Manoj, S. Velmurugan, M. Li, A. J. Ruben, T. 
Li, A. G. Eappen, R. E. Stafford, S. H. Plummer, C. S. Hendel, L. Novik, P. J. Costner, F. H. Mendoza, J. G. 
Saunders, M. C. Nason, J. H. Richardson, J. Murphy, S. A. Davidson, T. L. Richie, M. Sedegah, A. 
Sutamihardja, G. A. Fahle, K. E. Lyke, M. B. Laurens, M. Roederer, K. Tewari, J. E. Epstein, B. K. Sim, J. E. 
Ledgerwood, B. S. Graham, S. L. Hoffman and V. R. C. S. Team (2013). "Protection against malaria by 
intravenous immunization with a nonreplicating sporozoite vaccine." Science 341(6152): 1359-1365.
Sissoko, M. S., S. A. Healy, A. Katile, F. Omaswa, I. Zaidi, E. E. Gabriel, B. Kamate, Y. Samake, M. A. 
Guindo, A. Dolo, A. Niangaly, K. Niare, A. Zeguime, K. Sissoko, H. Diallo, I. Thera, K. Ding, M. P. Fay, E. M. 
O'Connell, T. B. Nutman, S. Wong-Madden, T. Murshedkar, A. J. Ruben, M. Li, Y. Abebe, A. Manoj, A. 
Gunasekera, S. Chakravarty, B. K. L. Sim, P. F. Billingsley, E. R. James, M. Walther, T. L. Richie, S. L. 
Hoffman, O. Doumbo and P. E. Duffy (2017). "Safety and efficacy of PfSPZ Vaccine against Plasmodium 


Mali PfSPZ-CVac (pyrimethamine) V8.0 03 January 2022      Page 146 of 146
falciparum via direct venous inoculation in healthy malaria-exposed adults in Mali: a randomised, 
double-blind phase 1 trial." Lancet Infect Dis 17(5): 498-509.
Stoute, J. A., K. E. Kester, U. Krzych, B. T. Wellde, T. Hall, K. White, G. Glenn, C. F. Ockenhouse, N. 
Garcon, R. Schwenk, D. E. Lanar, P. Sun, P. Momin, R. A. Wirtz, C. Golenda, M. Slaoui, G. Wortmann, C. 
Holland, M. Dowler, J. Cohen, W. R. Ballou, J. A. Stoute, M. Slaoui, D. G. Heppner, P. Momin, K. E. Kester, 
P. Desmons, B. T. Wellde, N. Garcon, U. Krzych and M. Marchand (1998). "Long-term efficacy and 
immune responses following immunization with the RTS,S malaria vaccine
A preliminary evaluation of a recombinant circumsporozoite protein vaccine against Plasmodium 
falciparum malaria. RTS,S Malaria Vaccine Evaluation Group." J Infect Dis 178(4): 1139-1144.
Traebert, M. and B. Dumotier (2005). "Antimalarial drugs: QT prolongation and cardiac arrhythmias." 
Expert Opin Drug Saf 4(3): 421-431.
Walther, M., S. Dunachie, S. Keating, J. M. Vuola, T. Berthoud, A. Schmidt, C. Maier, L. Andrews, R. F. 
Andersen, S. Gilbert, I. Poulton, D. Webster, F. Dubovsky, E. Tierney, P. Sarpotdar, S. Correa, A. 
Huntcooke, G. Butcher, J. Williams, R. E. Sinden, G. B. Thornton and A. V. Hill (2005). "Safety, 
immunogenicity and efficacy of a pre-erythrocytic malaria candidate vaccine, ICC-1132 formulated in 
Seppic ISA 720." Vaccine 23(7): 857-864.
Walther, M., F. M. Thompson, S. Dunachie, S. Keating, S. Todryk, T. Berthoud, L. Andrews, R. F. 
Andersen, A. Moore, S. C. Gilbert, I. Poulton, F. Dubovsky, E. Tierney, S. Correa, A. Huntcooke, G. 
Butcher, J. Williams, R. E. Sinden and A. V. Hill (2006). "Safety, immunogenicity, and efficacy of prime-
boost immunization with recombinant poxvirus FP9 and modified vaccinia virus Ankara encoding the 
full-length Plasmodium falciparum circumsporozoite protein." Infect Immun 74(5): 2706-2716.
Wellems, T. E. (2002). "Plasmodium chloroquine resistance and the search for a replacement 
antimalarial drug." Science 298(5591): 124-126.
WHO. (2013). "Seasonal malaria chemoprevention with sulfadoxine-pyrimethamine plus amodiaquine in 
children: A field guide." from https://www.who.int/malaria/publications/atoz/9789241504737/en/.
WHO (2014). WHO policy brief for the implementation of intermittent preventive treatment of malaria 
in pregnancy using sulfadoxine-pyrimethamine (IPTp-SP). Geneva, Switzerland, World Health 
Organization.
WHO (2018). World Health Organization Malaria Report 2018, World Health Organization.
Yayon, A., J. A. Vande Waa, M. Yayon, T. G. Geary and J. B. Jensen (1983). "Stage-dependent effects of 
chloroquine on Plasmodium falciparum in vitro." J Protozool 30(4): 642-647.
Yoeli, M. (1965). "Studies on Plasmodium Berghei in Nature and under Experimental Conditions." Trans 
R Soc Trop Med Hyg 59: 255-276.