Conditions: Malaria
Keywords: Malaria Parasitemia Immune Responses Sporozoites
Allocation: NON_RANDOMIZED | Intervention model: PARALLEL
Enrollment: 55
Healthy volunteers: yes

Brief summary:
Background:

People get malaria from bites from infected mosquitos. Researchers are studying a vaccine strategy. They will give people malaria parasites by injecting them with live infectious malaria parasites with antimalarial medications and then see if this strategy prevents malaria infection while off antimalarial medications.

Objective:

To see if combining a high dose of live, infectious malaria parasites (known as Sanaria PfSPZ Challenge) and two FDA approved drugs that kill malaria parasites (pyrimethamine \[PYR\] OR chloroquine \[CQ\]) is safe and can provide people protection against malaria.

Eligibility:

Healthy adults ages 18-50 who:

* are not pregnant or breastfeeding or planning on becoming pregnant while in the study
* are not infected with HIV, Hepatitis B or Hepatitis C
* have reliable early morning access to the NIH Clinical Center
* are able to come to the outpatient clinic frequently, sometimes daily
* have not been diagnosed with malaria within the past 10 years

Design:

* Participants will be screened with medical history and physical exam. They will have heart, blood, and urine tests.
* Participants will have blood drawn for tests at most visits.
* Participants will keep track of their temperature and symptoms during some sections of the study.
* Participants will join one part of the study.

Part 1 is one month:

* Participants will get the parasites by an injection into a vein on day 1 and receive antimalarial medications.
* They will have daily visits on days 7-14
* They will take another antimalarial at visits on days 15-17.
* The final visit will be on day 29.

Part 2 is seven months:

* For the first 3 months, participants will get the parasite injection into a vein for 3 injections in total. Each injection will occur once per month while taking an antimalarial drug.
* They will have daily visits on days 7-14 after the first injection, and on days 7-11 after the second and third injection.
* They will have a final (fourth) injection around month 6 without any antimalarial medication.
* After this fourth injection, participants may have up to 21 daily visits from day 7 after injection until end of study.

Part 3 is one month:

* Participants will get the parasites by injection into a vein on day 1 without antimalarial medications.
* They will have visits almost every day starting day 7 from injection.
* They will take an antimalarial medication when they are diagnosed with malaria
* They will return for final end of study visit on days 27-29.

Detailed description:
Human studies have shown that immunization by the bite Plasmodium falciparum (Pf) sporozoite(SPZ)-infected mosquitoes under drug coverage with chloroquine, an approach called chemoprophylaxis with sporozoites (CPS) or infection treatment vaccination (ITV), can provide high level, long term protection against homologous controlled human malaria infection (CHMI). The Sanaria PfSPZ chemoprophylaxis vaccination (PfSPZ CVac) approach duplicates this with an injectable SPZ regimen. In both approaches, whether mosquitoes or syringes are used for SPZ administration, when chloroquine is used as the chemoprophylactic agent, transient, limited, asexual erythrocytic stage is seen in the majority of participants. Thus the question remains whether the significant protective efficacy seen can be achieved with pre-erythrocytic (sporozoite/liver stage) exposure only.

Previously, we performed a phase 1 study to investigate the safety, tolerability, immunogenicity, and protective efficacy of Sanaria PfSPZ CVac with chloroquine (sporozoites, liver, and blood stage) or pyrimethamine with chloroquine (sporozoites and liver stage only) to further describe stage specific sterile protection (NIAID protocol #15-I-0169). In this study, we demonstrated that pyrimethamine is safe to administer, well tolerated, and can prevent subpatent and patent parasitemia (qPCR and blood smear negative) 100% of the time during Sanaria PfSPZ CVac. The study also duplicated the results previously reported with Sanaria PfSPZ CVac with chloroquine in terms of safety profile and protective efficacy against homologous CHMI. Although a combination of Sanaria PfSPZ- CVac with pyrimethamine and PfSPZ Challenge at 51,200 PfSPZ did not provide a significant protection level against homologous CHMI, we demonstrated that some subjects did develop protective immunity without any evidence of blood stage exposure during PfSPZ-CVac.

Building on these results and taking the lessons learned from other pre-erythrocytic vaccine studies and models that have shown the importance of reaching a minimal antigen threshold required for the development of sterile immunity, this proposed study will assess the safety, tolerability, immunogenicity, and protective efficacy of increasing the dose of Sanaria PfSPZ Challenge sporozoites while receiving the same, or, if needed to successfully prevent parasitemia, a higher dose of pyrimethamine. Unlike the first study, however, pyrimethamine will be administered by itself as the partner drug, and will not be co-administered with chloroquine. The efficacy of PfSPZ-CVac with pyrimethamine will be assessed against CHMI with homologous parasites (Arm 2a) and CHMI with heterologous parasites (Arm 2b). Additionally, we will explore the impact of increasing the dose of Sanaria PfSPZ Challenge sporozoites while receiving chloroquine alone prophylaxis. The efficacy of PfSPZ-CVac with chloroquine will be assessed only against CHMI with heterologous parasites (Arm 3), as protection against homologous parasites has now been shown in two separate trials. It will thus be possible to compare the efficacy of the two partner drugs against heterologous CHMI. The results of this study will contribute to understanding the targets and mechanisms of immunity against Pf malaria infection and how the degree of exposure to the parasite (pre-erythrocytic or erythrocytic stage only or both) impacts these responses and subsequent protective efficacy.

Arms:
arm 1: Pilot Phase - 1 injection of 5x10\^4 sporozoites of PfSPZ Challenge (NF54) with 50mg of pyrimethamine dosed on days 2 and 3 after injection arm 2: Pilot Phase - 1 injection of 1x10\^5 sporozoites of PfSPZ Challenge (NF54) with 50mg of pyrimethamine dosed on days 2 and 3 after injection arm 3: Pilot Phase - 1 injection of 2x10\^5 sporozoites of PfSPZ Challenge (NF54) with 50mg of pyrimethamine dosed on days 2 and 3 after injection arm 4: Pilot Phase - 1 injection of 1x10\^5 sporozoites of PfSPZ Challenge (NF54) with 1000mg of chloroquine (loading dose) 2 days prior to injections and 500mg (maintenance dose) 5 days post injection arm 5: Pilot Phase - 1 injection of 2x10\^5 sporozoites of PfSPZ Challenge (NF54) with 1000mg of chloroquine (loading dose) 2 days prior to injections and 500mg (maintenance dose) 5 days post injection arm 6: Main Phase - 3 monthly doses of 2x10\^5 sporozoites of PfSPZ Challenge + pyrimethamine on days 2 and 3 post injection + homologous controlled human malaria infection (CHMI) with 3.2x10\^3 PfSPZ Challenge (NF54) at 12 weeks after third injection arm 7: Main Phase - 3 monthly doses of 2x10\^5 sporozoites of PfSPZ Challenge + pyrimethamine on days 2 and 3 post injection + heterologous CHMI with 3.2x10\^3 PfSPZ Challenge (7G8) at 12 weeks after third injection arm 8: Main Phase - 3 monthly doses of 2x10\^5 sporozoites of PfSPZ Challenge + weekly chloroquine (1000mg given 2 days prior to injections and 500mg given 5 days post injection and weekly thereafter until 5 days post third injection) + heterologous controlled human malaria infection (CHMI) with 3.2x10\^3 PfSPZ Challenge (7G8) at 12 weeks after third injection arm 9: Main Phase - Infectivity Control with one dose of 3.2x10\^3 sporozoites of PfSPZ Challenge (NF54) (homologous) controlled human malaria infection (CHMI) arm 10: Main Phase - Infectivity Control with 3.2x10\^3 sporozoites of PfSPZ Challenge 7G8 (heterologous) controlled human malaria infection (CHMI)

Interventions: intervention 1: Chloroquine Phosphate intervention 2: Pyrimethamine intervention 3: Sanaria PfSPZ Challenge
Intervention descriptions: intervention 1: 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). Chloroquine phosphate 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. intervention 2: 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. January2014) intervention 3: Sanaria Inc has manufactured two strains of Sanaria PfSPZ Challenge: NF54 and 7G8. The Sanaria 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, pyrimethamine, atovaquone, artesunate, but not mefloquine. Sanaria PfSPZ Challenge (7G8) is known to be susceptible to mefloquine, atovaquone, artemether and artesunate but not to chloroquine or pyrimethamine.

Protocol document:
Prot_SAP_000.pdf: 
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Sanaria® PfSPZ Challenge with Pyrimethamine Chemoprophylaxis (PfSPZ-CVac 
Approach): Phase 1 Dose Escalation Trial to Determine Safety and Development of 
Protective Efficacy after Exposure to Only Pre-erythrocytic Stages of Plasmodium 
falciparum  
 
 
 
 
                                     NIAID Protocol Number:  17-I-0067  
Project Assurance:  FWA #00005897 
 
Multi-institution:  No 
 
Principal Investigator: 
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: 10.0 
November 12, 2019 
  
 
 
 
 
CONFIDENTIAL 
 
 
 
 
 
 
 


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Team Roster 
Principal Investigator: 
 
Patrick Duffy, MD (LMIV/NIAID/NIH) 
 
 
 
 
 
Building 29B 
Room 4NN06 
29 Lincoln Drive 
Bethesda, MD 20892 
 
 
 
 
 
Patrick.Duffy@nih.gov 
Office: (301) 761-5089 
Fax: (301) 480-1962 
 
 
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 
 
Associate Investigators: 
 
Sara Healy, MD, MPH  
 
 
 
 
 
Jacquelyn K. Lane PA-C 
 
 
 
 
 
David Cook, MD 
 
 
 
 
 
 
 
 
 
 
 
Biostatistician: 
 
 
Martha C. Nason PhD 
BRB/NIH 
5601 Fishers Lane 
Room 4C40 
Rockville, MD 20852  
 
 
(240) 669-5251 
 
 
 
Clinical Trial Site:  
 
NIH Clinical Center 
10 Center Drive 
Bethesda, MD 20892 USA 
 
 
Institutional Review Board   
NIH IRB 


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Immunology Laboratories: 
LMIV/NIAID/NIH 
5640 Fishers Lane, Twinbrook 1 
Rockville, Maryland 20852 USA 
 
Antigen Discovery Inc 
Irvine, CA 92618, USA  
 
Protein Potential LLC 
9800 Medical Center Drive 
Rockville, MD 20850 USA 
 
Sanaria, Inc. 
9800 Medical Center Drive 
Rockville, MD 20850 USA 
 
VRC/NIAID/NIH 
40 Convent Drive 
Bldg. 40 
Bethesda, MD 20892 USA 
 
Malaria Infection Laboratory: 
NIH Clinical Center 
10 Center Drive 
Bethesda, MD 20892 USA 
 
 
 
LMIV/NIAID/NIH 
12441 Parklawn Drive, Twinbrook 2 
Rockville, MD 20852 USA 
 
University of Washington 
Department of Laboratory Medicine 
Research and Training Building 
300 9th Avenue, Room 725 
Seattle, WA 98104 USA 
 
University of Maryland 
685 W. Baltimore Street, HSF I, Rm. 480  
Baltimore, MD 21201 USA 
 
Clinical Laboratory: 
NIH Clinical Center 
10 Center Drive 
Bethesda, MD 20892 USA 
 
  Drug Assay Laboratories:  
NMS Labs  
3701 Welsh Road  
Willow Grove, PA 19090 USA 
 


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Military Malaria Research Program 
Division of Experimental Therapeutics 
Walter Reed Army Institute of Research 
Silver Springs, MD USA  
 
Collaborating Laboratories: 
NIH Intramural Sequencing Center 
National Human Genome Research Institute 
5625 Fishers Lane 
Room 5S-16C MSC9400 
Bethesda, MD 20892 
USA  
 
NIH Research Technologies Branch 
Genomic Technologies Section 
Building 50, Room 5509 
50 South Drive, MSC 8005 
Bethesda, MD 20892 
USA  
 
CHI (Center for Human Immunology, Autoimmunity 
and Inflammation) 
NHLBI (National Heart, Lung and Blood Institute) 
Building 10 - Magnuson CC, 7N110a 
10 Center Dr 
Bethesda, MD 20892 USA 
 
 


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Table of Contents 
Team Roster ......................................................................................................................... 2 
Table of Contents ................................................................................................................. 5 
List of Abbreviations .......................................................................................................... 13 
PROTOCOL SUMMARY.................................................................................................. 16 
Précis.................................................................................................................................. 23 
 
Introduction and Rationale ........................................................................................... 25 
1.1 
Malaria Epidemiology .......................................................................................... 25 
1.2 
Malaria Life Cycle and Vaccine Strategies........................................................... 25 
1.3 
Rationale for Chemoprophylaxis Vaccination (CVac) with SPZ Regimens .......... 26 
1.4 
Previous Human Experience ................................................................................. 28 
 
Whole SPZ Vaccination Models.................................................................... 28 
1.4.1.1 
Whole SPZ Vaccination Models – Pre-erythrocytic Stage Exposure ..... 31 
 
Sanaria® PfSPZ Challenge (NF54) ................................................................ 32 
1.4.2.1 
Live (non-attenuated) PfSPZ (NF54 Sanaria® PfSPZ Challenge) Used for 
Controlled Human Malaria Infection (CHMI).......................................................... 32 
1.4.2.2 
CHMI using Intravenous Inoculation of NF54 Sanaria® PfSPZ Challenge 
in Malaria Naïve Subjects in Germany ..................................................................... 33 
1.4.2.3 
CHMI using NF54 Sanaria® PfSPZ Challenge by DVI .......................... 36 
 
Using Sanaria® PfSPZ Challenge (NF54) for CVac ...................................... 37 
1.4.3.1 
NF54 Sanaria® PfSPZ Challenge CVac under Chloroquine Prophylaxis 37 
1.4.3.2 
Sanaria® PfSPZ-CVac (NF54) using Chloroquine and Pyrimethamine 
Prophylaxis .............................................................................................................. 42 
1.4.3.2.1 CVac period........................................................................................ 42 
1.4.3.2.2 Homologous CHMI ............................................................................ 45 
1.4.3.2.3 Protective Efficacy Against Homologous CHMI ................................ 46 
 
Sanaria® PfSPZ Challenge (7G8) .................................................................. 47 
1.5 
Clinical Trial Plan ................................................................................................ 51 
 
Study Objectives ......................................................................................................... 51 
2.1 
Primary Objective................................................................................................. 51 
2.2 
Secondary Objective ............................................................................................ 52 
2.3 
Exploratory Objectives ......................................................................................... 52 


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Study Design ............................................................................................................... 52 
3.1 
Overall Design ..................................................................................................... 52 
3.2 
Pilot Study............................................................................................................ 52 
 
Pilot Study Outcomes .................................................................................... 58 
3.3 
Main Study ........................................................................................................... 60 
 
Preliminary results of the first cohort of the main phase ............................... 62 
 
Rationale for staggered enrollment ............................................................... 65 
 
Rationale for stopping the study prior to full enrollment............................... 67 
 
Description of Investigational Products and Plan ........................................................ 69 
4.1 
Chloroquine Phosphate ........................................................................................ 69 
4.2 
Pyrimethamine ..................................................................................................... 69 
4.3 
Sanaria® PfSPZ Challenge ................................................................................... 70 
4.4 
Description of Intervention .................................................................................. 70 
4.5 
Presumptive Antimalarial Treatment .................................................................... 74 
 
Study Endpoints .......................................................................................................... 74 
 
Primary Endpoints ......................................................................................... 74 
 
Secondary Endpoints ..................................................................................... 74 
 
Exploratory Endpoints .................................................................................. 74 
5.2 
Sample Size and Estimated Duration of Study...................................................... 75 
 
Study Population ......................................................................................................... 75 
6.1 
Description of Population and Site ....................................................................... 75 
6.2 
Recruitment .......................................................................................................... 75 
6.3 
Participation of NIH Staff .................................................................................... 76 
6.4 
Inclusion Criteria .................................................................................................. 76 
6.5 
Exclusion Criteria ................................................................................................. 77 
 
Study Agents ............................................................................................................... 79 
7.1 
Sanaria® PfSPZ Challenge ................................................................................... 79 
 
Sanaria® PfSPZ Challenge (NF54) ................................................................ 79 
 
Sanaria® PfSPZ Challenge (7G8) .................................................................. 80 
 
Phosphate buffered saline and human serum albumin diluent ....................... 80 
 
Storage and Handling of Sanaria® PfSPZ Challenge ..................................... 81 


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7.2 
Malarone®, Pyrimethamine and Chloroquine ....................................................... 81 
 
Malarone®, Pyrimethamine and Chloroquine Packaging and Labeling ......... 81 
 
Malarone®, Pyrimethamine and Chloroquine Accountability ........................ 81 
7.2.2.1 
Receipt ................................................................................................... 81 
7.2.2.2 
Preparation and Administration ............................................................. 81 
7.2.2.3 
Storage and Handling ............................................................................. 82 
7.2.2.4 
Return of Study Product ......................................................................... 82 
 
Study Procedures......................................................................................................... 82 
8.1 
Screening ............................................................................................................. 82 
8.2 
Malaria Comprehension Exam ............................................................................. 83 
8.3 
Assignment to Groups .......................................................................................... 83 
8.4 
Study Schedule ..................................................................................................... 83 
8.5 
Large Volume Blood Draw ................................................................................ 109 
8.6 
Symptom Memory Enhancement Card (Diary) ................................................... 109 
8.7 
Photographs of Rash or Injection Site Reactions ................................................ 110 
8.8 
Indications for Deferral of Sanaria® PfSPZ Challenge ....................................... 110 
8.9 
Treatments that Could Potentially Interfere with Vaccine-induced Immunity ..... 110 
8.10 Clinical Laboratory Testing ................................................................................ 111 
8.11 Electrocardiograms ............................................................................................ 111 
8.12 Malaria Blood Smears ........................................................................................ 111 
8.13 qPCR (Retrospective LMIV and Real Time NIH) .............................................. 112 
8.14 Drug Assays (Chloroquine, Desethylchloroquine, and Pyrimethamine) ............. 113 
8.15 Immunology Laboratories .................................................................................. 114 
 
Research Use, Storage, and Tracking of Specimens and Data .................................... 115 
 Retention of Specimens for Future Use ..................................................................... 116 
 Data Sharing Plan...................................................................................................... 116 
 Assessment of Safety ................................................................................................ 117 
12.1 Documenting, Recording, and Reporting Adverse Events.................................. 117 
12.2 Definitions ......................................................................................................... 117 
12.3 Investigator Assessment of Adverse Events ....................................................... 119 
 
Adverse Event Definitions........................................................................... 121 


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Severity ....................................................................................................... 123 
 
Causality ..................................................................................................... 124 
12.4 Investigator Reporting Responsibilities to the Sponsor ...................................... 125 
 
Adverse Events ........................................................................................... 125 
 
Serious Adverse Events............................................................................... 125 
 
Unanticipated Problems .............................................................................. 126 
 
Pregnancy ................................................................................................... 126 
12.5 Reporting Procedures to the IRB ........................................................................ 126 
12.6 Follow-up of Adverse Events and Serious Adverse Events ................................ 127 
12.7 Sponsor’s Reporting Responsibilities ................................................................. 127 
12.8 Pausing Criteria for Entire Study Population ..................................................... 127 
 
Parasitemia and Malaria Symptoms ............................................................ 127 
 
Reactogenicity ............................................................................................ 130 
 
Reporting of Study Halting ......................................................................... 130 
 
Resumption of a Halted Study..................................................................... 131 
12.9 Pausing Criteria for a Subject or Group ............................................................. 131 
 
Reporting of Pausing for a Participant or Group ......................................... 131 
 
Resumption of a Paused Study .................................................................... 131 
12.10 Withdrawal Criteria for an Individual Participant .............................................. 131 
12.11 Replacement of Withdrawn Subjects.................................................................. 132 
12.12 Safety Oversight ................................................................................................. 133 
 Safety Monitoring Committee (SMC) ......................................................... 133 
 Clinical Monitoring ................................................................................................... 133 
13.1 Site Monitoring Plan .......................................................................................... 133 
 Statistical Considerations ........................................................................................... 134 
14.1 General Pilot Study ............................................................................................ 134 
14.2 General Main Study ........................................................................................... 134 
14.3 Primary Objectives ............................................................................................. 134 
14.4 Secondary Objective ........................................................................................... 134 
14.5 Exploratory Objectives ....................................................................................... 136 
14.6 Sample Size and Power Calculations .................................................................. 136 


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Power calculations for primary objective (safety): Pilot Phase ................... 136 
 
Power calculations for Main Phase ............................................................. 137 
14.6.2.1 
Power calculations for primary objective (safety) ................................ 137 
14.6.2.2 
Power calculations for secondary objective (efficacy) ......................... 139 
14.7 Randomization ................................................................................................... 139 
 Human Subject Protections and Ethical Obligations .................................................. 139 
15.1 Institutional Review Board ................................................................................. 139 
15.2 Informed Consent Process .................................................................................. 139 
15.3 Justification for Exclusion of Children ............................................................... 140 
15.4 Subject Confidentiality ....................................................................................... 140 
15.5 Risks .................................................................................................................. 140 
 
Venipuncture ............................................................................................... 140 
 
Sanaria® PfSPZ Challenge via DVI ............................................................ 140 
 
Malaria Symptoms after Administration of PfSPZ-CVac ........................... 141 
 
Malaria Symptoms and Infection after PfSPZ CHMI .................................. 142 
15.5.4.1 
Cardiac Abnormalities .......................................................................... 142 
15.5.4.2 
Pregnancy ............................................................................................. 143 
 
Medications used in the study ..................................................................... 143 
15.5.5.1 
Chloroquine Phosphate......................................................................... 143 
15.5.5.2 
Pyrimethamine ..................................................................................... 144 
15.5.5.3 
Malarone® (Atovaquone/proguanil) .................................................... 145 
15.5.5.4 
Ibuprofen ............................................................................................. 145 
15.6 Risk to the Community ....................................................................................... 145 
15.7 Benefits .............................................................................................................. 146 
15.8 Compensation ..................................................................................................... 146 
 Data Handling and Record Keeping ........................................................................... 148 
16.1 Source Documentation ....................................................................................... 148 
16.2 Retention of Study Records ................................................................................ 148 
16.3 Protocol Revisions .............................................................................................. 148 
Appendix A: Clinical Evaluation and Laboratory Procedures .......................................... 149 
Appendix B:  Cardiovascular Risk Assessment ................................................................ 156 


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Appendix C:  Toxicity Table ............................................................................................ 158 
References ........................................................................................................................ 164 
 
 
 


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List of Figures 
 
Figure 1: Mean Parasite Density Following P. falciparum Experimental Infection ............ 29 
Figure 2: Mean Parasite Density in Control Group and Vaccinees Following P falciparum 
Challenge ..................................................................................................................... 30 
Figure 3: Parasite Density after Re-challenge with P. falciparum ...................................... 30 
Figure 4: Frequency of adverse events in all 4 groups after Sanaria® PfSPZ Challenge DVI
 ..................................................................................................................................... 38 
Figure 5: Kaplan Meier curve comparing vaccinated groups and infective control ........... 47 
Figure 6: Pyrimethamine Pilot Study Up-Down Schema ................................................... 54 
Figure 7: Ideal Pyrimethamine Pilot Study Pathway Schema ............................................ 55 
Figure 8:Chloroquine Pilot Flow Chart ............................................................................. 57 
Figure 9: Chloroquine Pilot Ideal Pathway ........................................................................ 58 
Figure 10: Main Study Schema ......................................................................................... 62 
Figure 11: LMIV qPCR results during vaccination phase ................................................. 64 
Figure 12: NHANES I Classification for Women ............................................................. 156 
Figure 13: NHANES I Classification for Men .................................................................. 157 
 
 
List of Tables 
Table 1: Protection against blood stage versus sporozoites challenge after chloroquine 
CVac ............................................................................................................................ 31 
Table 2: CVac-PYR may lead to protective immunity following homologous CHMI ....... 32 
Table 3: Infection rate and pre-patent period ..................................................................... 33 
Table 4: Number of all Adverse Events for Groups 1-5 ..................................................... 34 
Table 5: Number of erythocytic stage adverse events and clinically significant laboratory 
abnormalities (Day 6 to Day 21) .................................................................................. 35 
Table 6:Summary of study design and infectivity outcomes .............................................. 36 
Table 7: Summary of PfSPZ-CVac doses and number of qPCR positive subjects per CVac
 ..................................................................................................................................... 38 
Table 8: Summary of PfSPZ-CVac doses and mean parasite density per CVac................. 39 
Table 9: List of AEs post PfSPZ-CVac with weekly 51,200 Sanaria® PfSPZ Challenge DVI 
under chloroquine in NCT02773979 ............................................................................ 42 
Table 10: Summary of Adverse Events during Vaccination Period ................................... 44 
Table 11: Summary of Reported Symptoms (Reactogenicity and AEs) in Arms 1-4 during 
CHMI ........................................................................................................................... 46 
Table 12: Results of CHMI#1 by exposure to the bites of PfSPZ (7G8)-infected 
mosquitoes in the WRAIR study described above ....................................................... 48 
Table 13: Results of CHMI#2 by exposure to the bites of PfSPZ (7G8)-infected 
mosquitoes in the WRAIR study described above. ...................................................... 49 
Table 14: Results of CHMI by exposure to the bites of PfSPZ (3D7)-infected mosquitoes 


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in the WRAIR study described above. ......................................................................... 50 
Table 15: Summary of AEs by severity and frequency observed in pyrimethamine pilot 
study ............................................................................................................................ 59 
Table 16: Summary of AEs by severity and frequency observed in chloroquine pilot study
 ..................................................................................................................................... 59 
Table 17: Results of CHMI for the first Cohort ................................................................. 65 
Table 18: Number of participants in each Arm during first, second and third cohort ........ 67 
Table 19: Combined Results of CHMI for the first and Second Cohort ............................. 68 
Table 20: Chemoprophylaxis regimen and assignment ..................................................... 73 
Table 21: Solicited Adverse Events ................................................................................. 123 
Table 22: Definitions for Severity of AE Grading ........................................................... 124 
Table 23: Decision Rule: ................................................................................................. 136 
Table 24: Probability of observing 0 and 2 or more break through parasitemia, among arms 
of size 2 and 4, for different true event rates .............................................................. 137 
Table 25: Probability of observing 0 events and 2 or more events, among arms of size 17 
and 10, for different true event rates .......................................................................... 138 
Table 26: Two-sided 95% confidence intervals based on observing a particular rate of 
safety endpoints for arms of size 17 and 10 ............................................................... 138 
Table 27: Compensation Table ........................................................................................ 146 
 
 


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List of Abbreviations 
 
AE 
Adverse event 
AES 
Asexual erythrocytic stages 
ALT 
Alanine transaminase 
ANC 
Absolute neutrophil count 
AP 
Alkaline phosphatase 
AST 
Aspartate aminotransferase 
β-hCG 
β human choriogonadotropin 
CBC 
Complete blood count 
CFR 
Code of Federal Regulations 
cGMP 
Current Good Manufacturing Practices 
CHD 
Coronary heart disease 
CHI 
Center for human Immunology, Autoimmunity and Inflammation 
CHMI 
Controlled human malaria infection 
CoA 
Certificate of Analysis 
CPS 
Chemoprophylaxis with sporozoites 
CQ 
Chloroquine Phosphate 
CRF 
Case report form 
CRIMSON 
Clinical Research Information Management System of the NIAID 
CRP  
CSP 
C reactive protein 
Circumsporozoite protein 
CVac 
Chemoprophylaxis Vaccination 
DHHS 
Department of Health and Human Services 
DSMB 
Data Safety Monitoring Board 
DVI 
Direct Venous Inoculation 
EC 
Ethics Committee 
ECG 
Electrocardiogram 
ELISA 
Enzyme-linked immunosorbent assay 
ELISPOT 
Enzyme-linked immunosorbent spot assay 
FDA 
Food and Drug Administration 
FWA 
Federal Wide Assurance 
GAP 
Genetically attenuated parasites 
GCP 
Good Clinical Practice 
HBsAg 
Hepatitis B surface antigen 
HCV 
Hepatitis C virus 
HIV 
Human immunodeficiency virus 
HRP2 
histidine-rich protein 2 
HSA 
Human serum albumin 
IB 
Investigator’s Brochure 
ICS 
Intracellular cytokine staining 
ID 
Intradermal 
IFA 
Immunofluorescence assay 


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IHI 
Ifakara Health Institute 
IM 
Intramuscular 
IND 
Investigational New Drug 
IRB 
Institutional Review Board 
ISI 
Inhibition of sporozoite invasion 
ITV 
Infection treatment vaccine 
IV 
Intravenous 
LDH  
LMIV 
Lactate dehydrogenase 
Laboratory of Malaria Immunology and Vaccinology (of NIAID) 
LMVR 
Laboratory of Malaria Vector Research 
N 
Number (typically refers to subjects or participants) 
NASBA 
nucleic acid sequence–based amplification 
NHLBI 
National Heart, Lung and Blood Institute 
NIAID 
National Institute of Allergy and Infectious Diseases, NIH 
NIH 
National Institutes of Health 
NOCI 
New Onset of Chronic Illness 
OCRPRO 
Office of Clinical Research Policy and Regulatory Operations  
OHRP 
Office for Human Research Protections 
OHSR 
Office of Human Subjects Research 
PATH 
Program for Appropriate Technology in Health 
PBS 
Phosphate-buffered saline 
PI 
Principal Investigator 
Pf 
Plasmodium falciparum 
Pf7G8 
Plasmodium falciparum, 7G8 strain 
PfNF54 
Plasmodium falciparum, NF54 strain 
PfAES 
Plasmodium falciparum asexual erythrocytic stages 
PfSPZ 
Plasmodium falciparum sporozoites 
PfSPZ-CVac 
Sanaria® PfSPZ Challenge Chemoprophylaxis vaccination 
pRBC 
Packed Red Blood Cells 
PYR 
Pyrimethamine 
qPCR 
Quantitative polymerase chain reaction 
Real time qPCR 
Real time quantitative polymerase chain reaction (qPCR assay 
performed the same day) 
RUNMC 
Radboud University Nijmegen Medical Center 
Sanaria® PfSPZ 
Challenge  
Aseptic, purified, cryopreserved Plasmodium falciparum  
sporozoites used for controlled human malaria infection (CHMI) 
SAE 
Serious adverse event/serious adverse experience 
SOP 
Standard Operating Procedure 
SPZ 
Sporozoites 
SUSARS 
Serious and unexpected suspected adverse reactions 
UP 
Unanticipated Problem 
WBC 
White blood cell 
WHO 
World Health Organization 


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WRAIR 
Walter Reed Army Institute of Research 


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PROTOCOL SUMMARY 
Full Title: 
Sanaria® PfSPZ Challenge with Pyrimethamine 
Chemoprophylaxis (PfSPZ-CVac Approach): Phase 1 
Dose Escalation Trial to Determine Safety and 
Development of Protective Efficacy after Exposure to 
Only Pre-erythrocytic Stages of Plasmodium 
falciparum  
  
Short Title: 
Dose Escalation PfSPZ-CVac  
Clinical Phase: 
1 
IND Sponsor: 
Sanaria (product); OCRPRO (clinical activities) 
 
Conducted by: 
Laboratory of Malaria Immunology and Vaccinology 
(LMIV) 
 
Principal Investigators: 
Patrick Duffy, MD (LMIV/NIAID/NIH) 
 
Site 
National Institute of Health (NIH), Bethesda, Maryland, 
USA 
 
Sample Size: 
N=67 to 71 
 
Accrual Ceiling: 
230 
 
Study Population: 
Healthy malaria-naïve US adults, 18-50 years of age 
 
Accrual Period: 
Approximately June 2017 to June 2020 
 
Study Design: 
This is a phase 1 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 (Sanaria® PfSPZ Challenge (NF54)), 
combined with either pyrimethamine or chloroquine 
drug dosing, known as PfSPZ chemoprophylaxis 
vaccination (CVac). This study will also investigate the 
protective efficacy of pre-erythrocytic stage only 
parasite exposure (pyrimethamine arm; Arm 2a) against 
homologous (same strain as that used for vaccination, Pf 
NF54) controlled human malaria infection (CHMI;) and 
that of pre-erythrocytic stage only parasite exposure 
(pyrimethamine, Arm 2b) AND pre-erythrocytic and 
erythrocytic stage parasite exposure (chloroquine arm; 
Arm 3) against heterologous (different strain from that 
used during vaccination Pf 7G8) CHMI. 


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In brief: 
x Arm 1 is a pilot arm to determine dose of 
sporozoites and pyrimethamine for Arm 2 
x Arm 5 is a pilot arm to determine a safe dose of 
sporozoites under chloroquine prophylaxis for 
Arm 3 
x Arm 2 and 3 are main study arms under 
pyrimethamine only (Arm 2) and chloroquine 
only prophylaxis (Arm 3) 
x Arm 4 is the infectivity control arm for Arm 2 
and 3 during CHMI 
The study is to occur in two parts, pilot study and main 
study. The pilot study will be divided into two, first an 
adaptive pilot study (n=8-12) to assess safety, 
tolerability and identify a PfSPZ-CVac pyrimethamine 
regimen that maximizes the PfSPZ Challenge dose 
while still preventing subpatent parasitemia. Second, a 
pilot study (n=6) to assess safety and tolerability of 
PfSPZ-CVac chloroquine with increasing dose of PfSPZ 
Challenge dose.  The regimens selected in the pilot 
study will then be followed by the main study which is 
designed to assess safety, tolerability, immunogenicity, 
and protective efficacy of PfSPZ-CVac with 
pyrimethamine (against homologous and heterologous 
CHMI) and PfSPZ-CVac with chloroquine (against 
heterologous CHMI).  
 
In the first pilot study (Arm 1), subjects will undergo 
PfSPZ-CVac with one exposure of Sanaria® PfSPZ 
Challenge (NF54) with pyrimethamine dosing on days 
2, 3 post PfSPZ Challenge. The goal of the pilot study is 
to progressively increase the dose of PfSPZ Challenge 
(50,000; 100,000; 200,000) while evaluating safety and 
tolerability of these increasing PfSPZ doses. The dose of 
pyrimethamine may also be increased as needed if there 
is detectable subpatent parasitemia (via qPCR) in the 
preceding arm. Subjects will be treated with standard 
treatment doses of Malarone® at the end of their 
enrollment. Subjects from the pilot study will not join 
the main study nor undergo CHMI.  
In the second pilot study (Arm 5), subjects will undergo 
PfSPZ-CVac with one exposure of Sanaria® PfSPZ 
Challenge (NF54) with chloroquine, loading dose 


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administered 2 days before PfSPZ Challenge and 
maintenance dose administered 5 days post PfSPZ 
Challenge. The goal of the pilot study is to progressively 
increase the dose of PfSPZ Challenge (100,000; then 
200,000) while evaluating safety and tolerability of 
these increasing PfSPZ doses. Subjects will be treated 
with standard treatment doses of Malarone® at the end 
of their enrollment. Subjects from the pilot study will 
not join the main study nor undergo CHMI.  
 
In the main study, subjects will be enrolled in Arm 2a 
(pyrimethamine only; homologous CHMI n=17), Arm 
2b (pyrimethamine only; heterologous CHMI n=10) or 
Arm 3 (chloroquine only; heterologous CHMI n=10). 
Subjects in the main study will receive three exposures 
of Sanaria® PfSPZ Challenge (NF54), separated by 4 
weeks, with pyrimethamine or chloroquine coverage 
(PfSPZ-CVac). Arm 2a and 2b will use the optimal 
doses of PfSPZ-CVac with pyrimethamine as 
determined in the pilot study. Arm 3 will use the optimal 
dose of PfSPZ-CVac with chloroquine as determined in 
the pilot study. The goal is for Arm 2a, 2b and 3 to use 
the same highest number of PfSPZ Challenge if this is 
determined to be safe and tolerable in the pilot study. 
  
Subjects in the main study together with non-immunized 
infectivity controls (Arms 4a and 4b) will then undergo 
CHMI. Arm 2a and Arm 4a will undergo CHMI using 
Sanaria® PfSPZ Challenge NF54 whereas Arm 2b, 3 and 
Arm 4b will undergo CHMI using Sanaria® PfSPZ 
Challenge 7G8. 
Enrollment in the study was divided into three cohorts. 
High significant level of protection against heterologous 
CHMI was observed after enrollment of cohorts 1&2. 
As a result, the study will not complete enrollment in 
Arms 2a and 4a that were planned to undergo 
homologous CHMI (see details in section 3.3.2). 
 
Study Duration: 
Start Date: Approximately June 2017 
End Date: Approximately June 2020 
Study subjects will be enrolled for a total of  
approximately 1 (pilot/controls) to 7 (main) months 
  
Study Agent/ 
Intervention Description: 
 
Pyrimethamine: Arms 1, 2a and 2b will  receive orally two 
to three single strength pills (for a total of 50-75 mg 
pyrimethamine per day. The dose of pyrimethamine  
will be determined in the pilot study, Arm 1) dosed 2 


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and 3 days post Sanaria® PfSPZ Challenge (NF54) 
during the PfSPZ-CVac immunization phase. Arms 3 
(chloroquine arm) and 4 (infectivity controls) will not 
receive pyrimethamine. 
 
Chloroquine: Arm 3 and 5 will receive a loading dose of 
approximately 1000 mg chloroquine (600 mg base) 2 days 
prior to Sanaria® PfSPZ Challenge (NF54). Arm 5 will 
receive a second and last dose of chloroquine (500 mg 
salt, 300 mg base) 5 days post Sanaria® PfSPZ 
Challenge. After loading dose, Arm 3 will receive 500 
mg (300 mg base) weekly continuously with the last 
dose administered 5 days post 3rd Sanaria® PfSPZ 
Challenge injection (NF54). A total of 10 doses of 
chloroquine will be given to Arm 3. Arms 1, 2, and 4 
will not receive chloroquine. 
 
 
Sanaria® PfSPZ Challenge during CVac   
Arms 1: 1 dose of 50,000 or 100,000 or 200,000 
aseptic, purified, vialed, cryopreserved, fully infectious 
Sanaria® PfSPZ Challenge (NF54) will be administered 
by DVI to Arms 1. The first day of administration of 
Sanaria® PfSPZ Challenge (NF54) is Study Day 1 for 
each Arm.  
Arms 5: 1 dose of 100,000 or 200,000 aseptic, purified, 
vialed, cryopreserved, fully infectious Sanaria® PfSPZ 
Challenge (NF54) will be administered by DVI to Arms 
5. The first day of administration of Sanaria® PfSPZ 
Challenge (NF54) is Study Day 1 for each Arm  
Arm 2: 3 doses of 200,000 aseptic, purified, vialed, 
cryopreserved, fully infectious Sanaria® PfSPZ 
Challenge (NF54) will be administered by DVI four 
weeks apart. The first day of administration of Sanaria® 
PfSPZ Challenge is Study Day 1 thus Sanaria® PfSPZ 
Challenge will be administered on Study Days 1, 29 and 
57.   
Arms 3: 3 doses of 200,000 aseptic, purified, vialed, 
cryopreserved, fully infectious Sanaria® PfSPZ 
Challenge (NF54) will be administered by DVI four 
weeks apart. The first day of administration of Sanaria® 
PfSPZ Challenge is Study Day 1 thus Sanaria® PfSPZ 
Challenge will be administered on Study Days 1, 29 and 
57.   
 
Sanaria® PfSPZ Challenge for CHMI:  
Arms 2a, 4a: Sanaria® PfSPZ Challenge (NF54; dose: 
3,200 PfSPZ) will be administered by DVI to assess 


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homologous protective efficacy by CHMI 
approximately 13 weeks after 3rd Sanaria® PfSPZ 
Challenge for Arm 2a (Study Day 147). Infectivity 
controls (Arm 4a) join the main study at CHMI. Neither 
Arm 2a nor 4a will  receive any antimalarial 
chemoprophylaxis during CHMI. 
 
Arms 2b, 3, 4b: Sanaria® PfSPZ Challenge (7G8; dose: 
3,200 PfSPZ, if this dose is confirmed by ongoing 
studies) will be administered by DVI to assess 
heterologous protective efficacy by CHMI 
approximately 13 weeks after 3rd Sanaria® PfSPZ 
Challenge (NF54) for Arm 2b and 3 (Study Day 147). 
Infectivity controls (Arm 4b) join the main study at 
CHMI. Neither Arm 2b and 3 nor 4b will receive any 
antimalarial chemoprophylaxis during CHMI. 
 
Primary Objectives: 
 
  Safety 
x To monitor the safety and tolerability of Sanaria® PfSPZ 
Challenge (NF54) via DVI with pyrimethamine (PfSPZ-
CVac pyrimethamine) or chloroquine (PfSPZ-CVac 
chloroquine). (Arms 1, 2, 3, 5) 
         
x To evaluate whether pyrimethamine on days 2, 3 prevents 
patent parasitemia post Sanaria® PfSPZ Challenge 
(NF54) via DVI at increasing PfSPZ Challenge doses 
(maximum of 200,000 PfSPZ).  (Arm 1, 2) 
x To evaluate whether weekly chloroquine prevents clinical 
malaria diagnosis requiring treatment with additional 
antimalaria post Sanaria® PfSPZ Challenge (NF54) via 
DVI at increasing PfSPZ Challenge doses (maximum of 
200,000 PfSPZ).  (Arm 3, 5) 
 
Secondary 
Objectives: 
 
Protective Efficacy 
x To assess the protective efficacy of PfSPZ-CVac 
pyrimethamine against homologous (NF54) CHMI. (Arm 
2a and 4a) 
x To assess the protective efficacy of PfSPZ-CVac 
pyrimethamine or chloroquine against heterologous 
(7G8) CHMI. (Arm 2b, 3 and 4b) 
 


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Exploratory 
Objectives: 
Immunogenicity 
x To assess the humoral and cell mediated immune 
responses to PfSPZ and to known pre-erythrocytic and 
blood stage antigens 
 
x To assess the humoral and cell mediated immune 
responses to novel pre-erythrocytic antigens 
 
x To assess the kinetics of subpatent parasitemia during 
PfSPZ-CVac 
x To describe changes in γδ T cells in malaria naïve 
individuals after CVac and CHMI 
x To explore new diagnostic testing platforms (qPCR, rapid 
diagnostics, ELISA) 
Primary Endpoints: 
Safety 
x Incidence and severity of local and systemic adverse 
events (AEs) and serious adverse events (SAEs) 
occurring after PfSPZ-CVac. (Arms 1, 2, 3, 5) 
x P. falciparum blood stage infection defined as detection 
of P. falciparum parasites by qPCR (real time NIH 
qPCR and sensitive retrospective LMIV qPCR) 
following Sanaria® PfSPZ Challenge. (Arm 1, 2) 
x Incidence of clinical malaria diagnosis occurring after 
PfSPZ-CVac – chloroquine requiring treatment with 
additional antimalaria (Arms 3 and 5) 
 
Secondary 
Endpoints: 
 
Protective Efficacy 
x P. falciparum blood stage infection defined as detection 
of at least 2 P. falciparum parasites by microscopic 
examination of 0.5 μL of blood or one positive real time 
NIH qPCR after homologous PfSPZ CHMI (NF54) via 
DVI. (Arm 2a and 4a) 
x P. falciparum blood stage infection defined as detection 
of at least 2 P. falciparum parasites by microscopic 
examination of 0.5 μL of blood or one positive real time 
NIH qPCR after heterologous PfSPZ CHMI (7G8) via 
DVI. (Arm 2b, 3 and 4b) 
Exploratory 
Endpoints: 
 
x 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, EXP-1, LSA-1, MSP-3, 


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MSP-1, AMA-1 in subjects with no patent and/or 
subpatent parasitemia and those with patent and/or 
subpatent parasitemia, among drug groups. 
 
x Cellular immune responses after PfSPZ-CVac regimen to 
PfSPZ, PfAES, and specific Pf sporozoite, liver and 
blood-stage antigens, such as CSP, EXP-1, LSA-1, MSP-
3, MSP-1, AMA-1, in subjects with no patent and/or 
subpatent parasitemia and those with patent and/or 
subpatent parasitemia, among drug groups. 
 
x Subpatent parasitemia and degree of subpatent 
parasitemia by sensitive retrospective LMIV qPCR 
following Sanaria® PfSPZ-CVac pyrimethamine and 
Sanaria® PfSPZ-CVac chloroquine. 
x Comparison of γδ T cells before and after Sanaria® 
PfSPZ-CVac and CHMI using ex vivo whole blood 
staining 
x Comparison of time to positivity of various malaria 
diagnostic platforms (qPCR, rapid diagnostics, ELISA). 


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Précis 
Human studies have shown that immunization by the bite Plasmodium falciparum (Pf) 
sporozoite(SPZ)-infected mosquitoes under drug coverage with chloroquine, an approach 
called chemoprophylaxis with sporozoites (CPS) or infection treatment vaccination 
(ITV), can provide high level, long term protection against homologous controlled human 
malaria infection (CHMI) (Roestenberg, McCall et al. 2009, Roestenberg, Teirlinck et al. 
2011). The Sanaria® PfSPZ chemoprophylaxis vaccination (PfSPZ-CVac) approach 
duplicates this with an injectable SPZ regimen. In both approaches, whether mosquitoes 
or syringes are used for SPZ administration, when chloroquine is used as the 
chemoprophylactic agent, transient, limited, asexual erythrocytic stage is seen in the 
majority of participants. Thus the question remains whether the significant protective 
efficacy seen can be achieved with pre-erythrocytic (sporozoite/liver stage) exposure 
only.    
Previously, we performed a phase 1 study to investigate the safety, tolerability, 
immunogenicity, and protective efficacy of Sanaria® PfSPZ-CVac with chloroquine 
(sporozoites, liver, and blood stage) or pyrimethamine with chloroquine (sporozoites and 
liver stage only) to further describe stage specific sterile protection (NIAID protocol #15-
I-0169). In this study, we demonstrated that pyrimethamine is safe to administer, well 
tolerated, and can prevent subpatent and patent parasitemia (qPCR and blood smear 
negative) 100% of the time during Sanaria® PfSPZ-CVac. The study also duplicated the 
results previously reported with Sanaria® PfSPZ-CVac with chloroquine in terms of 
safety profile and protective efficacy against homologous CHMI. Although a 
combination of Sanaria® PfSPZ-CVac with pyrimethamine and PfSPZ Challenge at 
51,200 PfSPZ did not provide a significant protection level against homologous CHMI, 
we demonstrated that some subjects did develop protective immunity without any 
evidence of blood stage exposure during PfSPZ-CVac.  
Building on these results and taking the lessons learned from other pre-erythrocytic 
vaccine studies and models that have shown the importance of reaching a minimal 
antigen threshold required for the development of sterile immunity, this proposed study 
will assess the safety, tolerability, immunogenicity, and protective efficacy of increasing 
the dose of Sanaria® PfSPZ Challenge sporozoites while receiving the same, or, if needed 
to successfully prevent parasitemia, a higher dose of pyrimethamine. Unlike the first 
study, however, pyrimethamine will be administered by itself as the partner drug, and 
will not be co-administered with chloroquine. The efficacy of PfSPZ-CVac with 
pyrimethamine will be assessed against CHMI with homologous parasites (Arm 2a) and 
CHMI with heterologous parasites (Arm 2b). Additionally, we will explore the impact of 
increasing the dose of Sanaria® PfSPZ Challenge sporozoites while receiving chloroquine 
alone prophylaxis. The efficacy of PfSPZ-CVac with chloroquine will be assessed only 
against CHMI with heterologous parasites (Arm 3), as protection against homologous 
parasites has now been shown in two separate trials. It will thus be possible to compare 
the efficacy of the two partner drugs against heterologous CHMI. The results of this 
study will contribute to understanding the targets and mechanisms of immunity against Pf 


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malaria infection and how the degree of exposure to the parasite (pre-erythrocytic or 
erythrocytic stage only or both) impacts these responses and subsequent protective 
efficacy.


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 Introduction and Rationale 
1.1 
Malaria Epidemiology 
According to the World Health Organization (WHO), global control efforts have resulted in a 
reduction in the number of deaths since 2000; however 438,000 people are estimated to have 
died due to malaria in 2015, 69% of whom are African children under the age of 5 (WHO 2015). 
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 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 Anopheline 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-
stage, is responsible for the majority of 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 even 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®), in which phase 3 trials were completed throughout Africa in 2015, and 
phase 4 and implementation studies are underway. Subunit vaccines of this type utilize conserved 
antigenic targets to elicit protection against sporozoite migration, hepatocyte infection and 
intrahepatocytic parasite replication.  RTS, S has protected malaria-naïve adults against an 
experimental Pf CHMI and reduced malaria-associated episodes in children living in malaria 
endemic areas, but the level and length of immunity seen is relatively modest (Stoute, Kester et 
al. 1998, Agnandji, Lell et al. 2011, Rts 2015). 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 


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(Campo, Sacarlal et al. 2014). The mechanism by which RTS,S and other SPZ and pre-
erythrocytic vaccine strategies confer protective immunity is still under investigation.  
 
In the absence of more effective subunit vaccines, malaria vaccine development has continued to 
emphasize strategies that increase the feasibility of whole organism vaccination to induce sterile 
immunity. Prior studies using mosquito bites or irradiated SPZ (including Sanaria® PfSPZ 
Vaccine) for vaccination have required a large number of SPZ for the development of 
protective immunity (Hoffman, Goh et al. 2002, Roestenberg, McCall et al. 2009, Roestenberg, 
Teirlinck et al. 2011, Seder, Chang et al. 2013). In 2009, a human study suggested that anti-
infection immunity can be achieved with a much smaller parasite inoculum. 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). This method of immunization by experimental Pf infection with PfSPZ in conjunction 
with antimalarial prophylaxis is referred to as chemoprophylaxis with SPZ (CPS), infection 
treatment vaccine (ITV), and chemoprophylaxis vaccination (PfSPZ-CVac) by different authors. 
We use the term CVac in this protocol to refer to this vaccination concept with PfSPZ Challenge 
by direct venous inoculation (DVI).  
1.3 
Rationale for Chemoprophylaxis Vaccination (CVac) with SPZ Regimens 
In rodent models, sterile protection against malaria can be achieved by the inoculation of intact 
SPZ while treating the animals concomitantly with chloroquine (Beaudoin, Strome et al. 1977, 
Belnoue, Costa et al. 2004) a drug that kills parasites in the asexual blood stage but not in the pre-
erythrocytic stages. Importantly, the protective efficacy of this approach requires fewer SPZ to 
achieve high-level protective efficacy than does the radiation-attenuated SPZ model (Belnoue, 
Costa et al. 2004). Furthermore, as noted above, in humans, fully infectious PfSPZ administered 
by mosquito bites to subjects taking chloroquine chemoprophylaxis also induced protection in 
10/10 volunteers and this protection lasted for at least 28 months in 4 of 6 participants 
(Roestenberg, McCall et al. 2009, Roestenberg, Teirlinck et al. 2011). Although this approach 
requires far fewer mosquito bites, thus fewer SPZ, than the strategy using irradiated mosquitoes, 
it still suffers from the same logistical limitation that providing infectious mosquitoes at a scale 
large enough for mass vaccination campaigns is not practical. And since chloroquine is a blood-
stage schizonticide, the degree to which the protective immune response induced by the 
chloroquine CVac model targets sporozoites, liver, or blood-stage antigens is unclear from the 
study of Roestenberg et al.  
 
The concept that sterile protective immunity to Pf can be induced by parasite exposure limited to 
the pre-erythrocytic stages of the parasite life cycle is supported by experimental infection studies 
in animals and humans.  In rodent malaria models, blood-stage parasites may modulate dendritic 
cell responses and suppress protective CD8+ T cell responses that target parasites infecting 
hepatocytes (Ocana-Morgner, Mota et al. 2003).  Additionally, CVac pyrimethamine regimens 
in mice has been shown to induce sterile protective immunity (Friesen, Borrmann et al. 2011). It 
is important to further define whether sterile protective immunity to Pf can be induced in humans 
by wild-type (non-attenuated) SPZ immunizations when exposure is limited to SPZ and pre-
erythrocytic stages of the parasite life cycle and by a low dose of SPZ inoculum.  


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We performed a human study that explored this question (NIAID protocol #15-I-0169) and 
showed that a CVac regimen using chloroquine weekly in addition to pyrimethamine can prevent 
subpatent parasitemia. Using quantitative reverse transcriptase polymerase chain reaction (qRT-
PCR) we showed that none of the participants in pyrimethamine + chloroquine arms (Arm 1a, 2) 
were exposed to blood stage parasites. Participants in the chloroquine only arm (Arm 3) all had 
detectable subpatent parasitemia after the first CVac, with fewer participants being parasitemic 
after CVac #2 and #3. In addition, this initial study showed that CVac with pyrimethamine can 
lead to protective immunity against homologous CHMI, as 2/9 subjects in the pyrimethamine + 
chloroquine arm (Arm 2) remained uninfected and 1 additional participant was significantly 
delayed. 
 
Other antimalarial drugs with activity against pre-erythrocytic stage parasites that have been 
considered for CVac regimens include primaquine, proguanil, and azithromycin. Proguanil alone 
is not FDA approved for treatment or prophylaxis for malaria. In a recently completed human 
trial, we found that single dose (45 mg) primaquine is insufficient to prevent blood stage 
parasitemia after Pf SPZ exposure (Healy, Duffy et al. in preparation). Azithromycin is currently 
under study for PfSPZ-CVac and is active against liver stage parasites, however it allows non-
infectious merozoites to emerge from the infected hepatocyte and did not prevent subpatent 
parasitemia during PfSPZ-CVac. 
 
Natural infection with wild-type Pf does not normally induce sterile protection against pre-
erythrocytic stage parasites, possibly because only low levels of pre-erythrocytic or liver-stage 
specific cellular immune responses are generated (Plebanski, Aidoo et al. 1997). But unlike 
natural infections with wild-type parasites, radiation-attenuated P. falciparum parasites that arrest 
during liver-stage development can induce sterile protection in humans, but >1000 infectious 
mosquito bites (>105 cumulative sporozoites) are required to achieve this effect (Hoffman 1997, 
Hoffman, Goh et al. 2002). One possible conclusion drawn from this observation is that sterile 
protective immunity requires substantial or extended antigenic exposure.  But this observation 
also highlights the possibility of a broadly effective combination SPZ and liver stage vaccine.  
 
As seen in prior studies using irradiated sporozoites, a high number of sporozoites is required to 
develop protective immunity (Seder, Chang et al. 2013). Although CVac models that lead to 
blood stage exposure require lower number of Pf SPZ (Bijker, Borrmann et al. 2015), the optimal 
number of SPZ during CVac approaches using SPZ and liver stage exposure only has not been 
established. With results from our previous study, it is possible that this number is higher than the 
three episodes of exposure to 51,200 Sanaria® PfSPZ Challenge but less than the 4 PfSPZ 
Vaccine vaccinations of 270,000 irradiated sporozoites required to achieve 73% vaccine efficacy 
(Ishizuka, Lyke et al. 2016).  
 
The study being proposed here aims to increase by up to four fold the number of Sanaria® PfSPZ 
Challenge (NF54) that was used in the prior trial (up to 200,000 PfSPZ). Our study attempts to 
determine the optimal combination of pyrimethamine and dose of PfSPZ Challenge required to 
prevent the development of asexual erythrocytic stages of the malaria parasite and subsequently be 
evaluated for protective immunity. 
 


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We propose to evaluate an optimal regimen of pyrimethamine in combination with an increasing 
number of PfSPZ to determine whether protective efficacy can be achieved in humans only 
exposed to pre-erythrocytic parasite stages. Exploration of stage-specific immune responses 
involved in protective immunity to Pf and discovery of new target antigens is critical for future 
development of malaria vaccine strategies. 
1.4 
Previous Human Experience 
 Whole SPZ Vaccination Models 
 
Experimental infection studies in the early 1970s with radiation attenuated parasites demonstrated 
that induction of sterile protective immunity is achievable by whole SPZ vaccination. However, 
because of the substantial number of infectious mosquito bites (>1000) required to achieve this 
effect, this vaccine approach was logistically prohibitive to implement on a large scale. However, 
evaluation of the response to vaccination in these early studies provided valuable insight on anti-
infection immunity and allowed identification of several candidate immunogens included in later 
vaccine platforms (Hoffman, Goh et al. 2002). 
 
A recent variation on the whole SPZ vaccination concept achieves attenuation of the parasite 
through targeted disruption of genes critical to liver-stage development (Vaughan, Wang et al. 
2010). Vaccination of mice with live, whole SPZ genetically attenuated parasites (GAP) that 
arrest during liver-stage development has been shown to confer durable and complete protection 
against subsequent challenge with wild-type (non-attenuated) sporozoites. A dose or genetic 
disruption required to achieve a sterile protective immune response in a Pf human GAP vaccine 
model is currently unknown, but initial dose-finding studies of the first GAP vaccine in humans 
have recently begun and will initially target a cumulative dose consistent with the irradiated SPZ 
model (Vaughan, Wang et al. 2010). 
 
Recent human studies suggest that anti-infection immunity might be achieved with much smaller 
exposure or SPZ inoculum using wild type (non-attenuated) parasites in a CVac model. 
Roestenberg et al. have reported that human subjects who received a limited number of infected 
mosquito bites while receiving concurrent antimalarial prophylaxis were subsequently protected 
from development of blood-stage parasitemia upon challenge with homologous parasites. 
Subjects in this CVac model were exposed to three monthly episodes of experimental infection 
by the bites of 12-15 Pf infected mosquitoes while taking concurrent standard dose weekly 
suppressive prophylaxis with chloroquine.  Four weeks after stopping chemoprophylaxis, 
subjects underwent subsequent challenge with homologous sporozoites by the bites of five 
infected mosquitoes and were completely protected from development of blood-stage 
parasitemia (Roestenberg, McCall et al. 2009). The total exposure over three episodes was 36 to 
45 infected mosquito bites per subject, implying a one to two log-fold reduction in SPZ load 
required for induction of protective immunity compared to the irradiated SPZ model in humans. 
The degree to which this immune response targeted liver or blood-stage antigens is unknown. 
However, this approach to immunization does not protect against infection by DVI of Pf- 
infected erythrocytes, suggesting that immunity is primarily directed at the liver stages (Bijker, 
Bastiaens et al. 2013).  
 


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In addition, heterologous protection induced by CVac pyrimethamine and CVac chloroquine 
models is being explored. This is an important step in understanding the immune development 
and vaccine design as in endemic areas, people are exposed to multiple strains of Pf. Schats R et 
al. conducted a study (Clinicaltrials.gov Identifier: NCT01660854) in which they challenged 
subjects who had been immunized with NF54 infected mosquitoes (low to high dose) with 
NF135.C10 1 year after initial homologous challenge. Of the 13 subjects who were protected 
against homologous challenge, 2 were protected against this heterologous challenge. Those who 
were not protected against homologous challenge, were also not protected against heterologous 
challenge (Schats, Bijker et al. 2015). As with pre-erythrocytic stage vaccines, such as 
irradiated SPZ based vaccines, a higher number of sporozoites used for vaccination may be 
necessary. A vaccine that can induce protection against a various number of strains will be 
important in the goals to eliminate malaria.  
 
Chloroquine is a blood-stage schizonticide frequently used for prophylaxis (suppressive therapy) 
against Pf malaria infection in non- immune populations with weekly oral dosing (1g [600 mg 
base] loading dose then 500 mg [300 mg base]). It has no activity on SPZ liver or early 
merozoite forms, but when used for prophylaxis it effectively kills intra-erythrocytic parasites 
early in the blood stage of infection preventing development of patent parasitemia and clinical 
disease (Yayon, Vande Waa et al. 1983). Accordingly, subjects in the first chloroquine CVac 
study were completely protected from development of patent parasitemia following each episode 
of experimental infection under chloroquine prophylaxis, although a brief subpatent parasitemia 
was detected by nucleic acid sequence–based amplification (NASBA) in progressively 
decreasing magnitude, duration and number of subjects following each of the three CVac 
infections (Figure 1). 
 
 
Figure 1: Mean Parasite Density Following P. falciparum Experimental Infection 
 
 
The mean parasite density following P. falciparum experimental infection with concurrent chloroquine suppressive prophylaxis, as 
measured by nucleic acid sequence–based amplification (NASBA). The numbers of subjects who had positive results on peripheral 
blood smears for malarial parasites (MPS) and NASBA are shown below the graph. Adapted from (Roestenberg, McCall et al. 2009) 


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Following the challenge with homologous sporozoites in the absence of drug, subjects were 
monitored twice daily from day 6-21 for the development of patent and subpatent parasitemia. All 
subjects in the chloroquine CVac group were protected from development of both patent and 
subpatent parasitemia, whereas control subjects who received drug and uninfected mosquito bites 
during CVac developed a patent infection post-challenge (Figure 2). 
 
Figure 2: Mean Parasite Density in Control Group and Vaccinees Following P falciparum 
Challenge 
 
 
The mean parasite density in control group (open triangles) and vacinees (diamonds) following P. falciparum challenge as determined 
by real-time polymerase-chain-reaction assay. The bars denote standard errors. Adapted from (Roestenberg, McCall et al. 2009). 
 
Six of the previously immunized subjects from this study were rechallenged 28 months after the 
first controlled human malaria infection (CHMI) (Roestenberg, Teirlinck et al. 2011). Five newly 
recruited malaria-naïve subjects who served as infectivity controls developed patent parasitemia 
as previously reported. In contrast, four of the six vaccinees continued to have sterile protection, 
meaning both peripheral blood smear and real-time PCR evaluations remained negative (Figure 
3). In the remaining two immunized subjects, patent parasitemia was delayed to 15 and 18 days 
post-challenge. 
 
Figure 3: Parasite Density after Re-challenge with P. falciparum 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Panel A shows the geometric mean (95% confidence interval) for parasite density in control subjects (n=5). Panel B shows individual 
plots for the six previously immunized subjects (n=6). Numbers of parasites were determined by real-time PCR. Adapted from 
(Roestenberg, Teirlinck et al. 2011) 


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In order to further dissect the role of pre-erythrocytic and erythrocytic stages in the development 
of protective immunity in a CVac model, Bijker EM, et al. as part of the above-described study, 
challenged immunized volunteers with stage specific parasites. Group 1 (n=9) were challenged 
by DVI of Pf infected erythrocytes. Group 2 (n=5) immunized volunteers were challenged by 
infected mosquito bites. Groups 3 and 4 were naïve subjects, infection controls for infected 
mosquito bites and DVI with erythrocytic stages respectively. 100% of volunteers in Group 2 
were protected where as 0% of Group 1 were protected. In addition the group challenged by 
infected erythrocytes had pre-patent period similar to that of naïve controls (see Table 1). This 
study elucidated that the presence of erythrocytic stages during chloroquine CVac model may not 
have a significant role in development of protective immunity and that the developed immunity 
may be stage specific (Bijker, Bastiaens et al. 2013). 
 
Table 1: Protection against blood stage versus sporozoites challenge after chloroquine CVac 
 
^Adapted from Bijker EM, et al. 2013 
 
To further characterize the immune response, Behet, M. et al. demonstrated that vaccinated 
volunteers developed IgG against circumsporozoite protein (CSP) that inhibited SPZ ability to 
glide and traverse in hepatocytes in vitro. In addition, compared to human liver-chimeric mice 
receiving pre-vaccination IgG, mice receiving post vaccination IgG had a 91% decrease pre-
erythrocytic stages when infected with Pf SZP. This study demonstrated that CVac vaccination 
strategy results in development of inhibitory antibodies against CSP that may play a role in the 
development of protective immunity (Behet, Foquet et al. 2014). 
 
In the above studies using chloroquine CVac, immunized volunteers were exposed to both 
erythrocytic and pre-erythrocytic blood stages, hence it is still unclear the role of each stage in 
the development of protective pre-erythrocytic stage immunity in this model.  
1.4.1.1 Whole SPZ Vaccination Models – Pre-erythrocytic Stage Exposure 
In order to dissect the role of various parasite stages to protective immunity, as noted prior, we 
conducted a study in which we used CVac with pyrimethamine to limit parasite exposure to pre-
erythrocytic stages alone (NIAID protocol #15-I-0169). In that study, the main group (Arms 2 
and 3: Arm 2, PYR+CQ, n=12; Arm 3, CQ only, n=6) received 3 immunization with PfSPZ-
CVac (51,200 Sanaria® PfSPZ Challenge by DVI plus 50mg pyrimethamine on 2 and 3 days post 
injection and weekly chloroquine until 5 days after 3rd CVac (Arm 2) or plus weekly chloroquine 
only (Arm 3) before undergoing homologous CHMI with 3,200 Sanaria® PfSPZ Challenge via 
DVI. In Arm 3, 4/5 participants were protected as seen in above study. In Arm 2, 2/9 subjects 
remained uninfected and one subject (seen in red) was delayed which demonstrated that the CVac 


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with pyrimethamine approach may lead to protective immunity, even if the doses of PfSPZ 
Challenge were too low to induce high grade immunity (Table 2) 
 
Table 2: CVac-PYR may lead to protective immunity following homologous CHMI 
Arm 
Number of 
subjects 
challenged 
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. 
 
This proposed study will evaluate whether increasing number of Sanaria® PfSPZ Challenge will 
lead to protective immunity. The results of this study will help to evaluate the stage-specific 
exposure and immune response required for sterile protection following a complete CVac 
immunization series and subsequent homologous CHMI with Sanaria® PfSPZ Challenge. 
 Sanaria® PfSPZ Challenge (NF54) 
To date, several trials have used Sanaria® PfSPZ Challenge produced by Sanaria® Inc. and 
are summarized here briefly: 
1.4.2.1 Live (non-attenuated) PfSPZ (NF54 Sanaria® PfSPZ Challenge) Used for 
Controlled Human Malaria Infection (CHMI) 
This product, Sanaria® PfSPZ Challenge, has now been used for CHMI in 15 different trials 
conducted in the USA, the Netherlands, the United Kingdom, Tanzania, Kenya, Germany, 
Gabon, Mali, and Spain (see below).  Four hundred and fifty two (452) volunteers have received 
456 doses of PfSPZ Challenge administered by the intradermal (ID), intramuscular (IM), 
intravenous (IV) and DVI routes to induce CHMI, and like PfSPZ Vaccine, it has been extremely 
well tolerated. 3,200 PfSPZ by DVI has been established as a gold standard regimen for inducing 
blood stage infection in malaria-naïve volunteers, and has infected 36/36 volunteers to date with 
a pre-patent period of 11.0-11.5 days, equivalent to the pre-patent period of the standard 5 
mosquito bite CHMI (Mordmuller, Supan et al. 2015).  
 
The availability of vialed Sanaria® PfSPZ Challenge has facilitated the broader use of CHMI, 
and therefore, has the potential to accelerate malaria vaccine development. Studies with CHMI in 


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endemic areas - to test the efficacy of new vaccine candidates - are now feasible at sites that do 
not have access to an insectary, enabling the malaria community to test new vaccine candidates 
directly in the target population in Africa. 
 
In addition to ease of administration, the possibility to inject PfSPZ by needle and syringe offers 
further advantages compared to CHMI using infected mosquitoes: 
 
x The number of sporozoites with which volunteers are inoculated can easily be calculated 
and predefined. 
x The impact of variation in infectivity of mosquito-inoculated sporozoites when 
performing parallel clinical trials at multiple sites or sequential clinical trials at the same 
site with different lots of infected mosquitoes will be eliminated. Consequently, 
comparability will be increased substantially. 
x Since challenges do not need to be administered in real time (as with mosquito bites), 
volunteers can be infected over an extended period with different doses, routes, and 
with varying concomitant medications. 
1.4.2.2 CHMI using Intravenous Inoculation of NF54 Sanaria® PfSPZ Challenge in 
Malaria Naïve Subjects in Germany 
The first volunteer received cryopreserved P. falciparum sporozoites by an indwelling intravenous 
catheter of Sanaria® PfSPZ Challenge at the Institute of Tropical Medicine Tübingen, Germany 
(ClinicalTrials.gov Identifier: NCT01624961) (Mordmuller, Supan et al. 2015). In a sequential 
dose-escalation trial 50, 200, 800, and 3,200 Sanaria® PfSPZ Challenge were administered IV until 
a dose that infected 9 out of 9 volunteers was established and, as a secondary endpoint, the 
geometric mean pre-patent period was 12 days or less (Table 3). A control group (Group 1) 
received 2,500 Sanaria® PfSPZ Challenge by intradermal injection to assess infectivity of the 
Sanaria® PfSPZ Challenge lot and to compare parasitological and clinical outcomes between sites.  
The Sanaria® PfSPZ Challenge by DVI dose was escalated to 3,200 PfSPZ. Consequently, 
volunteers were recruited into all five groups as specified in Table 3 below.  
 
Table 3: Infection rate and pre-patent period 
Group 
Route 
PfSPZ 
dose 
Volunteers 
inoculated 
Volunteers 
infected (%) 
Days mean prepatency (ranges) 
1 
ID 
2,500 
6 
4 (67) 
13.6 (12.3 – 15.3) 
2 
IV 
50 
3 
1 (33) 
13.3 (N/A) 
3 
IV 
200 
3 
1(33) 
13.9 (N/A) 
4 
IV 
800 
9 
7 (78) 
11.7 (10.9-12.5) 
5 
IV 
3,200 
9 
9 (100) 
11.2 (10.5-12.5) 
Sanaria® PfSPZ Challenge DVI was well tolerated in all 24 volunteers at all doses. Volunteers 
with parasitemia developed mild or moderate symptoms of malaria and were cured with standard 
antimalarial treatment.  


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In total 179 adverse events (AEs) occurred in 28 of the 30 volunteers: 162 Grade 1 AEs, 16 
Grade 2 AEs and one Grade 3 AE as outlined in Tables 4 and 5 below. No serious AE (SAE) 
occurred. Ten hours after injection one volunteer (800 PfSPZ group) experienced nausea, which 
was considered possibly related to PfSPZ injection. No local AE occurred after ID injection or 
IV of Sanaria® PfSPZ Challenge. Five volunteers had AEs that occurred during the 5 days after 
injection of Sanaria® PfSPZ Challenge, one of which was judged possibly related to the 
inoculation of Sanaria® PfSPZ Challenge. All other AEs occurred from day 5 after injection, and 
were possibly or probably related to the intervention. These AEs were likely to be caused by 
asexual parasite multiplication.  
 
Table 4: Number of all Adverse Events for Groups 1-5 
 
Group 1 
(ID 2500) 
Group 2 
(IV50) 
Group 3 
(IV 200) 
Group 4 
(IV 800) 
Group 5 
(IV 3200) 
Adverse Events 
 
 
 
 
 
Total no. of AEs 
36 
23 
10 
39 
71 
SAEs 
0 
0 
0 
0 
0 
Total solicited AEs 
30 
17 
7 
36 
62 
Grade 1 
22 
16 
6 
35 
58 
Grade 2 
8 
1 
1 
0 
4 
Grade 3 
0 
0 
0 
1 
0 
Total unsolicited AEs 
6 
6 
3 
3 
9 
Grade 1 
6 
6 
3 
3 
9 
Grade 2 & 3 
0 
0 
0 
0 
0 


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Table 5: Number of erythocytic stage adverse events and clinically significant laboratory 
abnormalities (Day 6 to Day 21) 
Post- challenge AEs 
Group 1 
(ID 2500) 
4/6 had 
parasitemia 
Group 2 
(IV 50) 
1/3 had 
parasitemia 
Group 3 
(IV 200) 
1/3 had 
parasitemia 
Group 4 
(IV 800) 
7/9 had 
parasitemia 
Group 5 
(IV 3200) 
9/9 had 
parasitemia 
Abdominal pain 
0 
1 
0 
0 
0 
Anemia 
0 
0 
0 
0 
1 
ALT increase 
0 
1 
0 
0 
0 
AST increase 
0 
1 
0 
0 
0 
Back pain 
0 
1 
0 
0 
0 
Bilirubinuria 
1 
0 
1 
0 
0 
Chills 
2 
1 
0 
0 
0 
Common cold 
0 
0 
0 
0 
1 
Diarrhea 
1 
0 
1 
0 
3 
Dizziness 
0 
0 
0 
0 
1 
Fatigue 
6 
4 
1 
7 
9 
Fever 
3 
1 
1 
2 
6 
Feverish 
0 
0 
0 
0 
1 
Hemoglobinuria 
0 
0 
1 
0 
0 
Headache 
4 
8 
1 
13 
18 
Inflammation of the eye 
0 
0 
0 
0 
1 
Insomnia 
1 
1 
1 
0 
0 
Loss of appetite 
0 
0 
0 
0 
2 
Lymphopenia 
0 
0 
0 
1 
0 
Muscle ache 
1 
0 
0 
0 
0 
Myalgia 
1 
1 
0 
0 
7 
Nausea 
0 
2 
0 
2 
6 
Neck pain 
0 
2 
0 
0 
0 
Neutropenia 
1 
0 
0 
0 
0 
Stomach ache 
1 
1 
0 
0 
0 
Syncope 
1 
0 
0 
0 
0 
Tachycardia 
3 
0 
3 
3 
8 
TOTAL 
29 
22 
10 
28 
64 
 


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1.4.2.3 CHMI using NF54 Sanaria® PfSPZ Challenge by DVI  
A single center, varied dose, open label, controlled trial of Sanaria® PfSPZ Challenge with an 
escalated design in two parts, was conducted in Barcelona, Spain by the Barcelona Center for 
International Health Research (CRESIB) (NCT01771848) (Mordmuller, Supan et al. 2015). Part 
A determined a volume for IM administration of 2,500 Sanaria® PfSPZ Challenge was 10μL. 
Part B was designed to assess the effectiveness of administering different doses of Sanaria® 
PfSPZ Challenge (25,000 and 75,000 Sanaria® PfSPZ Challenge in 10 μL) intramuscularly and 
comparing to DVI administration of 3,200 Sanaria® PfSPZ Challenge, a dose that had been 
proven in a prior study to result in 100% infection. In part B, although 100% of volunteers were 
infected with geometric mean pre-patent period of 11-12 days, a higher number of Sanaria® 
PfSPZ Challenge administered IM was required to achieve a pre-patent period of less than 12 
days that is seen in DVI administration as seen on Table 6.  
 
Table 6:Summary of study design and infectivity outcomes 
 
In total between part A and part B, 588 AEs occurred in 36 of the 36 volunteers: 379 Grade 1 
AEs, 184 Grade 2 AEs and 25 Grade 3 AEs. There were no acute systemic allergic reactions. 
Only one volunteer presented a local AE following the injection. Most AEs recorded following 
challenge were consistent with symptoms associated with clinical malaria. No Grade 3 AEs 
were observed during the pre-erythrocytic phase (between challenge and day 5), nor during the 
erythrocytic phase (from Day 6 until Day 21 or day of treatment).  


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 Using Sanaria® PfSPZ Challenge (NF54) for CVac 
 
As previously described in detail, administration of PfSPZ via mosquito bites under 
chloroquine chemoprophylaxis induced high levels of long lasting protection in man, making it 
the currently most efficient way to induce sterile immunity against P. falciparum 
(Roestenberg, McCall et al. 2009, Roestenberg, Teirlinck et al. 2011). In its current format, 
unfortunately, the requirement of infectious mosquitoes precludes widespread use of this 
approach. However, the advent of vialed, viable sporozoites enabling delivery of PfSPZ via 
needle and syringe could avoid this logistical bottleneck. The previously described trials 
demonstrate that ID, IM, and IV administration of Sanaria® PfSPZ Challenge reliably infects 
both malaria naïve and malaria exposed individuals. We and others therefore consider further 
clinical studies exploring the usefulness of Sanaria® PfSPZ Challenge in the context of CVac 
highly warranted. 
1.4.3.1 NF54 Sanaria® PfSPZ Challenge CVac under Chloroquine Prophylaxis  
A randomized, placebo controlled, double-blinded, Sanaria® PfSPZ Challenge dose finding trial 
for CVac studies has been completed in Germany (NCT02115516) to identify the Sanaria® 
PfSPZ Challenge dose needed to replicate the results see in Roestenberg et al. In stage A, three 
groups (n=9 in each) received three CVac administrations 4 weeks apart by DVI with 3,200, 
12,800 or 51,200 Sanaria® PfSPZ Challenge while under chloroquine prophylaxis for a total of 
10 doses, starting with loading dose 2 days before the first Sanaria® PfSPZ Challenge and last 
dose given 5 days after the third Sanaria® PfSPZ Challenge DVI.  A fourth group (n=5) 
received placebo injections. Nine to ten weeks after 3rd Sanaria® PfSPZ Challenge results show 
that upon homologous CHMI with 3,200 Sanaria® PfSPZ Challenge via DVI, 3/9 (3,200 
PfSPZ-CVac), 6/9 (12,800 PfSPZ-CVac) and 9/9 (51,200 PfSPZ-CVac) of the subjects 
respectively were protected (Mordmuller, Surat et al. 2017). These results highlight the 
importance of higher antigen load required for vaccination efficacy, but significantly lower 
PfSPZ amounts required than needed to obtain 100% protection previously seen by Sanaria® 
PfSPZ Vaccine (irradiated P.falciparum sporozoites) (Seder, Chang et al. 2013).  
 
Safety data from this study after unblinding show that adverse event (AE) between the four 
treatment groups were not significantly different in the frequencies of total adverse events [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; abnormal laboratory tests (sodium, 
potassium glucose, bilirubin, creatinine, LDH, ALT, AST, white blood count differential 
component count including neutrophils, lymphocytes, monocytes, basophils, eosinophils, 
erythrocyte count, hemoglobin, hematocrit], as shown in Figure 4.  There were also no 
significant differences between the three injections of Sanaria® PfSPZ Challenge (data not 
shown) in any of the groups. 


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Figure 4: Frequency of adverse events in all 4 groups after Sanaria® PfSPZ Challenge DVI 
 
Parasitemia was seen by qPCR in all PfSPZ-immunized volunteers after the first Sanaria® 
PfSPZ Challenge (except one of the group receiving 3200 PfSPZ), and in a decreasing number 
of volunteers after the second and third Sanaria® PfSPZ Challenge (Table 7).  Parasitemia 
typically occurred during the six to ten day interval after PfSPZ Challenge administration, 
peaking on days 7 or 8.   
Table 7: Summary of PfSPZ-CVac doses and number of qPCR positive subjects per CVac 
PfSPZ Challenge dose per 
immunization 
Number Subjects Positive by qPCR
 
Immunization 1 
Immunization 2 
Immunization 3 
3,200  
8 / 8* 
9 / 9 
6 / 9 
12,800 
9 / 9 
8 / 9 
7 / 9 
51,200 
9/ 9 
8 / 9 
3 / 9 
* Technical difficulties precluded assessment of one volunteer’s sample 
The density of parasites increased with increasing doses of PfSPZ Challenge, approximately 
doubling with each four-fold increase in PfSPZ dose.  The geometric mean parasite densities 
(and 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 8). 
0
1
2
3
4
5
6
7
8
0
3200
12800
51200
Grade1
Grade2
Grade3
Figure 4.  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. 
 


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Table 8: Summary of PfSPZ-CVac doses and mean parasite density per CVac 
 
Geometric Mean Parasite Density (range)* 
Dose  
Immunization 1 
Immunization 2 
Immunization 3 
3,200 
462 (228-989)  
78 (2-1,236) 
210 (31-2,170) 
12, 800 
711 (120-1,891) 
195 (13-762) 
57 (14-417) 
51, 200 
15,652 (5,146-
36,404) 
555 (63-3,089) 
42 (11-126) 
* 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 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 one 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 of the groups. 
 
Thus, in summary, there was no clear indication that immunization with up to 51,200 PfSPZ of 
PfSPZ Challenge caused any excess of abnormal signs, symptoms or laboratory values relative to 
normal saline placebo, with the possible exception of one volunteer in the 51,200 PfSPZ group. 
In stage B of the study, three doses of 51,200 PfSPZ are being administered to 19 volunteers 
divided into three cohorts, and concurrently three doses of normal saline are being administered 
to 6 volunteers in blinded fashion (two per cohort).  The timelines for the regimens were 
condensed relative to Stage A: 9 volunteers are receiving doses of PfSPZ Challenge on days 0, 
14 and 28, and 9 volunteers are received doses of PfSPZ Challenge on days 0, 5 and 10.  Half of 
the 10 volunteers on the four-week schedule are received 2g extended release azithromycin 
(Zithromax UNO, Pfizer) in addition to chloroquine with the first dose of PfSPZ Challenge.  AE 
profiles were similar to those seen in Stage A. 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.  
 
However, there was one unanticipated problem.  One of the volunteers receiving the 0, 14 and 28 
day regimen of Sanaria® PfSPZ Challenge experienced parasitemia by PCR on day 7, peaking 
on day 8 and then falling progressively on days 9 and 10, which is typical pattern previously 
seen.  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 


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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. 
 
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 Sanaria® PfSPZ Challenge in the past.  
 
As a result of this unanticipated event (inadequate levels of chloroquine), the IND Sponsor 
(Sanaria, Inc.) is recommending that chloroquine levels should be collected after chloroquine 
administration and prior to the anticipated peaking of parasitemia post PfSPZ Challenge, and this 
recommendation is followed in this protocol (see Section 8.14).   
 
Although CVac-chloroquine with 51,200 NF54 Sanaria® PfSPZ Challenge in three successive 4-
weekly vaccinations leads to 80-100% protective efficacy against homologous CHMI, it is 
likely that a higher dose of NF54 Sanaria® 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 NF54 Sanaria® PfSPZ Challenge DVI under chloroquine cover in healthy malaria-naïve 
adults in the United States (Clinicaltrials.gov Identifier: NCT02773979) is currently being 
conducted by Seattle Group Health. The trial has 3 groups with increasing PfSPZ Challenge 
doses from 51,200, 102,400 and 204,800 PfSPZ (NF54). Each group has 12 subjects randomized 
3:1 (PfSPZ Challenge: normal saline) and receives 3 CVac vaccinations with a 7 day interval 
between each vaccination (vaccinations were done at 4 week intervals in our prior study NIAID 
protocol # 15-I-0169). Enrollment of successive groups for dose escalation is staggered by 3 
week intervals, with each higher dose group being enrolled after the lower dose group has 
received all three CVac vaccinations. Currently, the initial group receiving 51,200 PfSPZ 
Challenge has completed the vaccination phase. Unexpectedly, subjects experienced a higher 
frequency and severity of AEs (Table 9) in comparison to other PfSPZ-CVac chloroquine 
studies with 51,200 PfSPZ Challenge dose given at 4 weeks interval [NIAID protocol # 15-I-
0169, Table 10; (Mordmuller, Surat et al. 2017)] or at 5 or 14 day intervals (Mordmuller, Surat 
et al. 2017).All severe AEs, which included high grade fever, severe malaise, severe myalgia, 


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severe arthralgia and severe chills, happened approximately 8 days after receiving an injection 
with PfSPZ Challenge, and coincided with periods of transient parasitemia. Investigations are 
ongoing to understand this phenomenon. One of the theories is that there was exaggerated 
immune response due to the administration of a large number of sporozoites in the presence of 
blood stream parasites (parasitemia peaked on roughly the seventh and eighth days after each 
injection of PfSPZ Challenge). Since in this proposed study the vaccinations are at 4 week 
intervals, subjects will already have cleared any blood stage parasitemia by the time of the next 
injection. Thus we do not expect to see the same frequency and severity of AEs as the Seattle 
study.  However, as a precaution, we propose to add a PfSPZ Challenge dose escalation pilot 
study to determine safety and tolerability of 100,000 and 200,000 PfSPZ Challenge under 
chloroquine prophylaxis before proceeding to the main study. Safety data from the pilot study 
will be used to determine which PfSPZ Challenge dose will be used in Arm 3 of the main study. 
 
 


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Table 9: List of AEs post PfSPZ-CVac with weekly 51,200 Sanaria® PfSPZ Challenge DVI 
under chloroquine in NCT02773979  
 
 Note: X (X) X% refers to Absolute number of AEs (Number of subjects with AEs) Percent of subjects with AEs 
* vaccination happened on days 3, 10 and 17 
** 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 CVac #1 
**** Malaria symptoms are expected around 7 days and beyond post injection corresponding to parasitemia 
1.4.3.2 Sanaria® PfSPZ-CVac (NF54) using Chloroquine and Pyrimethamine 
Prophylaxis 
As briefly summarized in Section 1.4.1.1, an open label, single center, phase 1 study was 
undertaken at the National Institutes of Health (NIH) Clinical Center starting in 2015 (NIAID 
protocol #15-I-0169, NCT02511054). The Arm 1a pilot regimen (n=2; dosing of 50 mg 
pyrimethamine  on days 2, 3 post 51,200 PfSPZ Challenge via DVI while under weekly 
chloroquine chemoprophylaxis) successfully prevented patent and subpatent parasitemia 
(negative by blood smear and qPCR) in 2 of the 2 subjects enrolled following first vaccination 
(CVac #1), thus no further pilot regimens were explored. Arm 1a subjects completed 3 
immunization with PfSPZ-CVac, 28 days apart, and underwent homologous CHMI, 
approximately 6 months later, with the main group. 
 
The main group (Arms 2 [pyrimethamine + chloroquine, n=12]; Arm 3 [chloroquine only, n=6]) 
received 3 immunizations with PfSPZ-CVac, 28 days apart. Following approximately 12 weeks 
of drug washout, subjects then underwent homologous CHMI in June 2016. 
To determine if limited exposure to the parasite, pre-erythrocytic stages only (pyrimethamine + 
chloroquine) results in sterile protection against homologous CHMI with 3,200 PfSPZ Challenge 
administered via DVI was conducted. In total 21 subjects were challenged (Arms 1a [n=2], 2 
[n=9] and 3 [n=5] as well as Arm 4 [infectivity controls, n=5]).   
1.4.3.2.1 CVac period 
Overall vaccinations were well tolerated. The majority of AE’s reported, 180/210 (86%) were 
mild (Grade 1) and mostly transient (laboratory abnormalities, insomnia, dizziness, nausea, 
diarrhea, headache, myalgia, fatigue) (Table 10). 
There were a few reported Grade 2 AE’s, 27/210 (13%), that all resolved; most commonly, 
diarrhea, headache, hemoglobin decrease, insomnia and fatigue. In total, three (3) Grade 3 AEs 
were reported: sleep disorder and acute gastroenteritis (unlikely related to any of the study 
products; both occurred in the same individual) and encephalopathy NOS (which was also 
reported as a SAE) that was determined to be possibly related to chloroquine (Table 10). All 
Grade 2 and 3 AEs and the SAE resolved without complication.  
The following SAE was reported following CVac #3. A 23 year old previously healthy female 
who was enrolled in Arm 2 (pyrimethamine + chloroquine) presented with the acute onset of 
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


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nausea, vomiting, headache, tinnitus, fogginess, and confusion worsening over the course of a 
week concerning for encephalitis versus encephalopathy of unknown etiology. During the 
ensuing evaluation, she was diagnosed with an incidental Chiari I malformation with syrinx on 
magnetic resonance imaging. She was hospitalized for 4 days to facilitate prompt clinical 
evaluation and management. She was treated with intravenous acyclovir for presumed herpes 
simplex virus encephalitis while she underwent further evaluation. She was evaluated by NIH 
neurology, neurosurgery, and otolaryngology consulting services during this time. Unfortunately, 
cerebrospinal fluid could not be obtained due to the presence of a Chiari malformation however 
the rest of her work up which included negative malaria diagnostic testing (NIH diagnostic qPCR 
and blood smears), blood and urine cultures, urine toxicology, viral, rickettsial, and autoimmune 
causes of encephalitis were all unrevealing. She completed a 14 days course of intravenous 
acyclovir. Her symptoms improved over time with the main symptom of ‘fogginess’ fully 
resolving in 11 days. A grade 1 headache lingered and finally fully resolved in approximately 40 
days from presentation. Due to the SAE, she was withdrawn from the study and treated with 
Malarone® per protocol. Convalescent viral titers were completed approximately 4 months post 
presentation but were also unrevealing of a diagnosis. Final diagnosis was encephalopathy of 
unknown etiology that may have been possibly related to one of the study products (chloroquine) 
or another etiology unrelated to study participation (such as viral).  
 


PfSPZ-CVac PYR #2 
                Version 10.0    Nov. 12, 2019      Page 44 of 169 
 
 
Table 10: Summary of Adverse Events during Vaccination Period 
 
Note: X (X) X% refers to Absolute number of AEs (Number of subjects with AEs) Percent of subjects with AEs 
 
 
CVac 1
CVac 2
CVac 3
Total
CVac 1
CVac 2
CVac 3
Total
CVac 1
CVac 2
CVac 3
Total
N= 2
N= 2
N= 2
N= 2
N= 12
N= 12
N= 11
N=12
N= 6
N= 5
N= 5
N= 6
Total # AEs
12 (2) 100%
1 (1) 50%
6 (2) 100%
19 (2) 100%
61 (12) 100%
23 (10) 83%
41 (9) 82%
125 (12) 100%
35 (6) 100%
14 (4) 80%
17 (5) 100%
66 (6) 100%
Local Reactogenicity
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
1 (1) 8%
0 (0) 0%
0 (0) 0%
1 (1) 8%
1 (1) 17%
0 (0) 0%
0 (0) 0%
1 (1) 17%
Systemic Reactogenicity
5 (2) 100%
0 (0) 0%
2 (2) 100%
7 (2) 100%
33 (11) 92%
9 (6) 50%
14 (6)55%
56 (12) 100%
18 (6) 100%
10 (4) 80%
5 (3) 60%
33 (6) 100%
Laboratory Abnormalities
1 (1) 50%
1 (1) 50%
1 (1) 50%
3 (1) 50%
5 (4) 33%
4 (3) 25%
4 (2) 18%
13 (8) 67%
3 (2) 33%
1 (1) 20%
3 (1) 20%
7 (2) 33%
Unsolicited AEs
6 (2) 100%
0 (0) 0%
3 (2) 100%
9 (2) 100%
23 (10) 83%
10 (5) 42%
23 (7) 64%
56 (11) 92%
13 (5) 83%
3 (1) 20%
9 (5) 100%
25 (5) 83%
Grade 1      
12 (2) 100%
1 (1) 50%
6 (2) 100%
19 (2) 100%
53 (12) 100%
19 (9) 75%
33 (8) 73%
105 (12) 100%
30 (6) 100%
12 (3) 60%
14 (5) 100%
56 (6) 100%
Related to PfSPZ Challenge
2 (2) 100%
0 (0) 0%
2 (1) 50%
4 (2) 100%
20 (9) 75%
5 (4) 33%
4 (2) 18%
29 (9) 75%
5 (4) 67%
1 (1) 20%
4 (4) 80%
10 (4) 67%
Related to Induced Malaria
1 (1) 50%
0 (0) 0%
0 (0) 0%
1 (1) 50%
6 (4) 33%
1 (1) 8%
7 (3) 27%
14 (4) 33%
4 (3) 50%
1 (1) 20%
1 (1) 20%
6 (3) 50%
Related to Chloroquine
4 (1) 50%
1 (1) 50%
1 (1) 50%
6 (2) 100%
13 (8) 67%
2 (2) 17%
14 (2) 18%
29 (8) 67%
4 (4) 67%
1 (1) 20%
1 (1) 20%
6 (4) 67%
Related to Pyrimethamine
3 (1) 50%
1 (1) 50%
0 (0) 0%
4 (1) 50%
11 (4) 33%
4 (3) 25%
7 (1) 9%
22 (4) 33%
N/A
N/A
N/A
N/A
Grade 2     
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
8 (7) 58%
4 (4) 33%
7 (6) 55%
19 (11) 92%
3 (3) 50%
2 (2) 40%
3 (2) 40%
8 (5) 83%
Related to PfSPZ Challenge
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
1 (1) 8%
1 (1) 8%
1 (1) 9%
3 (3) 25%
1 (1) 17%
0 (0) 0%
0 (0) 0%
1 (1) 17%
Related to Induced Malaria
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
1 (1) 8%
0 (0) 0%
1 (1) 8%
1 (1) 17%
1 (1) 20%
1 (1) 20%
3 (1) 17%
Related to Chloroquine
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
2 (2) 17%
1 (1) 8%
1 (1) 9%
4 (2) 17%
1 (1) 17%
0 (0) 0%
0 (0) 0%
1 (1) 17%
Related to Pyrimethamine
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
N/A
N/A
N/A
N/A
Grade 3     
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
1 (1) 9%
1 (1) 8%
2 (1) 17%
0 (0) 0%
0 (0) 0%
2 (1) 17%
Related to PfSPZ Challenge
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
1 (1) 9%
1 (1) 8%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Related to Induced Malaria
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Related to Chloroquine
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
1 (1) 9%
1 (1) 8%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Related to Pyrimethamine
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
N/A
N/A
N/A
N/A
Grade 4     
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Related to PfSPZ Challenge
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Related to Induced Malaria
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Related to Chloroquine
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Related to Pyrimethamine
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
N/A
N/A
N/A
N/A
Severity and Relationship
Arm 3 (CQ only)
Arm 1a (PYR/CQ)
Arm 2 (PYR/CQ)
Classification


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1.4.3.2.2 Homologous CHMI 
A total of 21 subjects underwent CHMI with 3,200 PfSPZ Challenge (NF54) administered by DVI.  
In this study, two positive NIH Malaria Genus Species 4-plex real time polymerase chain reaction 
(NIH malaria real time qPCR) on separate days or a single peripheral thick blood smear were used 
for diagnosis and determination of treatment.  
The majority of symptoms reported were solicited and expected as part of a malaria diagnosis with 
70/103 (69%) being Grade 1 and most commonly reported were headache, fatigue, myalgia, and 
arthralgia. Twenty- one (21) of the 103 symptoms reported (20%) were Grade 2 which included 
headache, fatigue and myalgia. There were few Grade 3 AEs 12/103 (12%) of which the majority 
were transient laboratory abnormalities seen following the diagnosis of malaria and included 
decreases in white blood cell count, neutrophil count, lymphocyte count, and platelet count (Table 
11); one (1) subject experienced Grade 3 increase in alanine transferase (ALT) and aspartate 
transferase (AST).  Most subjects had mild malaria symptoms following diagnosis of malaria, 
however five (5) subjects (3 vaccinees and 2 controls) developed the final diagnosis of Grade 3 
malaria due to either reported symptoms and/or laboratory abnormalities that made Grade 3 
criteria, though these were often transiently Grade 3, reported after diagnosis and initiation of 
treatment, and all resolved without complication. However, one subject in Arm 1a developed 
Grade 3 symptoms (Grade 3 due to not being able to go to work that day) prior to the second 
positive PCR result being received and was treated per the investigator’s discretion that morning 
based off symptoms and reported previous single positive NIH malaria real time PCR. Both the 
NIH malaria real time PCR and blood smear in this subject were reported positive a few hours later 
that day.  
Of those subjects who had a reported positive blood smear during the study, the majority were 
reported after PCR diagnosis had been made, either at the timing of start of antimalarial treatment 
or day 1 post antimalarial treatment was initiated. Majority of subjects developed symptoms after 
starting treatment for malaria that required management with non-steroidal anti-inflammatory 
drugs (NSAIDs) and anti-nausea medications. None of the subjects required hospitalization or 
significant medical management for symptoms or treatment of malaria. All AEs fully resolved 
without complication. 
 
 


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Table 11: Summary of Reported Symptoms (Reactogenicity and AEs) in Arms 1-4 during CHMI 
 
Note: X (X) X% refers to absolute number of AEs (Number of subjects with AEs) Percent of 
subjects with AEs 
In this study, two positive NIH malaria real time qPCR (limit of detection ~500 parasites/mL) on 
separate days or a single peripheral thick blood smear were used for diagnosis and clinical decisions 
to treat the subjects. Although, the first NIH malaria real time qPCR detected parasitemia 
approximately 2 days prior to positive blood smears and before participants developed any 
symptoms, the second positive NIH malaria real time qPCR coincided with increased symptoms in 
most participants. Other studies, and our experience here, have shown that higher parasite number 
during CHMI is directly related to symptoms. Although most prior CHMI studies have used thick 
blood smears for malaria diagnosis, recently, in order to decrease adverse events related to clinical 
malaria, using qPCR has been suggested as a more sensitive measure (Walk, Schats et al. 2016). 
1.4.3.2.3 Protective Efficacy Against Homologous CHMI 
Final efficacy results by time to positivity are presented (as previously seen in Table 2 and 
below in Figure 5). Briefly 2/2 subjects in pilot Arm 1a (pyrimethamine + chloroquine; 6 months 
post last PfSPZ-CVac to CHMI) were infected. In Arm 2 (pyrimethamine + chloroquine); 7/9 
subjects were infected (1 additional subject was delayed, as seen in Table 2); 1/9 had negative 
PCR until day 16 post CHMI when the subject was withdrawn from study (note hash mark on 
survival curve at day 16). In Arm 3 (chloroquine only), 1/5 subjects was infected and in the 
infectivity control group, Arm 4, all 5/5 subjects were infected (Table 2 and Figure 5). Arm 3 
(chloroquine only) was significantly different in time to positivity and the binary endpoint from 
the infectivity controls however Arm 2 (pyrimethamine + chloroquine) was not significantly 
different in either endpoint when compared to the controls. 
Arm 1a
Arm 2
Arm 3
Arm 4
Total
N= 2
N= 9
N= 5
N= 5
N= 21
Total # AEs
12(2)100%
36(9)100%
23 (5)100%
32(5)100%
103 (21) 100%
Local Reactogenicity
0 (0) 0%
0 (0) 0%
0 (0) 0%
1 (1) 20%
1  (1) 5%
Systemic Reactogenicity
5 (2) 100%
7 (6) 67%
10 (4) 80%
16 (5) 100%
38 (17) 81%
Laboratory Abnormalities
4 (2) 100%
20 (6) 67%
7 (2) 40%
11 (3) 60%
42 (13) 62%
Unsolicited AEs
3 (1) 50%
9 (4) 44%
6 (3) 60%
4 (2) 40%
22 (10) 48%
Grade 1      
9 (2) 100%
22 (9) 100%
22 (5) 100%
17 (5) 100%
70 (21) 100%
Related to PfSPZ Challenge
2 (1) 50%
0 (0) 0%
5 (2) 40%
4 (1) 20%
11 (4) 19%
Related to Induced Malaria
4 (1) 50%
18 (6) 67%
6 (3) 60%
8 (4) 80%
36 (14) 67%
Grade 2     
1 (1) 50%
10 (5) 56%
0 (0) 0%
10 (3) 60%
21 (9) 43%
Related to PfSPZ Challenge
0 (0) 0%
1 (1) 11%
0 (0) 0%
3 (2) 40%
4 (3) 14%
Related to Induced Malaria
1 (1) 50%
5 (4) 44%
0 (0) 0%
4 (2) 40%
10 (7) 33%
Grade 3     
2 (1) 50%
4 (1) 11%
1 (1) 20%
5 (2) 40%
12 (5) 24%
Related to PfSPZ Challenge
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Related to Induced Malaria
2 (1) 50%
4 (1) 11%
1 (1) 20%
5 (2) 40%
12 (5) 24%
Grade 4     
Related to PfSPZ Challenge
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
Related to Induced Malaria
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
0 (0) 0%
CHMI 
Classification
Severity and Relationship


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Figure 5: Kaplan Meier curve comparing vaccinated groups and infective control 
 
Arm 1a- Pilot with pyrimethamine + chloroquine; Arm 2 – pyrimethamine + chloroquine; Arm 3 – 
chloroquine only, Arm 4 – infective controls 
 Sanaria® PfSPZ Challenge (7G8)  
Pf 7G8 infected mosquitoes have been used in a number of CHMI studies with the number of 
mosquitoes required to achieve 100% infectivity rate and safety profile similar to that observed 
in Pf NF54 infected mosquitoes (Egan, Hoffman et al. 1993, Heppner, Gordon et al. 1996, 
Epstein, Rao et al. 2007). Recently, Pf7G8-infected mosquitoes (created from same Working 
Cell Bank (WCB, BMF 15326; used to produce 7G8 PfSPZ Challenge) were used to infect a 
total of 10 control volunteers in a Sanaria-sponsored clinical trial of PfSPZ Vaccine under IND 
14826 (Clinicaltrials.gov Identifier: NCT02215707). The five mosquito bite CHMI that is 
standard for Pf NF54 and the Pf 3D7 clone of Pf NF54 was found to be optimal for Pf 7G8, 
resulting in 100% infection rates, comparable pre-patent periods, similar parasite densities and 
analogous clinical effects (Table 12, 13 and 14 below). Hence these data indicate that the in vivo 
biological profile of Pf 7G8 is highly similar to that of Pf NF54, and also to that of Pf 3D7.  
 
 
 
 
 
 
 
 
 
 


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Table 12: Results of CHMI#1 by exposure to the bites of PfSPZ (7G8)-infected mosquitoes in 
the WRAIR study described above 
 
 
 


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Table 13: Results of CHMI#2 by exposure to the bites of PfSPZ (7G8)-infected mosquitoes in 
the WRAIR study described above. 
 
 
 
 
 
 
 
 
 


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Table 14: Results of CHMI by exposure to the bites of PfSPZ (3D7)-infected mosquitoes in the 
WRAIR study described above. 
 
An initial study to assess the dose of 7G8 Sanaria® PfSPZ Challenge DVI required to achieve 100% 
infectivity was initiated in a MAVACHE study in Tubingen, Germany (Clinicaltrials.gov Identifier: 
NCT02704533). The study is being done in two phases. In phase 1, dose optimization in which 
malaria naïve subjects received 800, 1600 and 3,200 7G8 Sanaria® PfSPZ Challenge DVI (n=3 in 
each group). In phase 2, regimen verification will be done. Per verbal report from the study team, 
3,200 7G8 Sanaria® PfSPZ Challenge DVI (similar to NF54 Sanaria® PfSPZ Challenge DVI) 
resulted in 100% infection (Hoffmann SL, personal communication).  
 
Another study was recently completed at University of Maryland in Baltimore, MD 
(Clinicaltrials.gov Identifier: NCT02780154) to assess the safety and reactogenicity of 7G8 and 
NF54 Sanaria® PfSPZ Challenge DVI. In this trial, the dose of 7G8 Sanaria® PfSPZ Challenge DVI 
will be escalated through 800, 1600, 3,200 (n=9 in each group) and 4,800 (n=3) PfSPZ. Per verbal 
report 3,200 7G8 Sanaria® PfSPZ Challenge DVI resulted in 8/9 infected subjects (92% infection) 
(Richie T, personal communication). Safety data from both clinical trials will be available prior to 
CHMI with 7G8 Sanaria® PfSPZ Challenge DVI in this proposed study.  


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1.5 
Clinical Trial Plan 
The study proposed here by Sanaria and LMIV will assess PfSPZ-CVac using an increased 
number of Sanaria® PfSPZ Challenge (NF54) and pyrimethamine in attempt to determine the dose 
of Sanaria® PfSPZ Challenge required for development of protective immunity with this regimen.  
 
The design of the proposed clinical trial  entailed 2 phases, a pilot phase and a main phase.:  
1) Pilot trial phase (COMPLETED) was divided in two additional parts 
a) Pyrimethamine only Arm (Arm 1): to assess safety and to ensure that the increased number 
of Sanaria® PfSPZ Challenge (50,000 or 100,000 or target dose 200,000) and 
pyrimethamine dosing regimen on days 2 and 3 (50 mg or 75 mg) will not result in 
subpatent parasitemia in any of the subjects (NIH malaria real time PCR and retrospective 
LMIV qPCR negative in 2 of 2 subjects);
b) Chloroquine only Arm (Arm 5): to assess safety and to ensure that the increased number 
of Sanaria® PfSPZ Challenge (100,000 or target dose 200,000) while on weekly 
chloroquine regimen will not result in diagnosis of clinical malaria during CVac requiring 
treatment with another antimalaria medication 
 
 2) Main trial phase (ONGOING) will assess the safety of Sanaria® PfSPZ-CVac conducted 
with pyrimethamine alone or chloroquine alone with increased Sanaria® PfSPZ Challenge doses. 
In addition, the main arm will assess the protective efficacy against homologous challenge 
(NF54) for the pyrimethamine Arm 2a and heterologous challenge (7G8) for the pyrimethamine 
Arm 2b and chloroquine Arm 3. Subjects in the main study will receive three successive CVac 
administrations (NF54 Sanaria® PfSPZ Challenge + pyrimethamine OR chloroquine) at 4 week 
intervals followed by CHMI.  Antibody and cellular immune responses and transcriptomic 
analyses will be assessed periodically after vaccination in order to identify correlates of 
protection. 
 Study Objectives 
2.1 
Primary Objective 
The primary objectives of this study are twofold:  
x Safety:  
 To monitor the safety and tolerability of Sanaria® PfSPZ Challenge (NF54) via 
DVI with pyrimethamine (PfSPZ-CVac pyrimethamine) or chloroquine (PfSPZ-
CVac chloroquine). (Arms 1, 2, 3, 5) 
 To evaluate whether pyrimethamine on days 2, 3 prevents patent parasitemia post 
Sanaria® PfSPZ Challenge (NF54) via DVI at increasing PfSPZ Challenge doses 
(maximum 200,000 PfSPZ).  (Arm 1, 2) 
 To evaluate whether weekly chloroquine prevents clinical malaria diagnosis 
requiring additional antimalaria medication post Sanaria® PfSPZ Challenge 
(NF54) via DVI at increasing PfSPZ Challenge doses (maximum of 200,000 
PfSPZ).  (Arm 3, 5) 
 


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2.2 
Secondary Objective 
The secondary objective of this study is: 
Protective Efficacy:  
 
To assess the protective efficacy of PfSPZ-CVac pyrimethamine against 
homologous (NF54) CHMI. (Arm 2a and 4a) 
 
To assess the protective efficacy of PfSPZ-CVac pyrimethamine or chloroquine 
against heterologous (7G8) CHMI. (Arm 2b, 3 and 4b) 
2.3 
Exploratory Objectives 
The exploratory objectives of this study are: 
 
To assess the humoral and cell mediated immune responses to PfSPZ and to 
known pre-erythrocytic and blood stage antigens 
 
To assess the humoral and cell mediated immune responses to novel 
pre-erythrocytic antigens 
 
To assess the kinetics of subpatent parasitemia during PfSPZ-CVac 
 
To describe changes in γδ T cells in malaria naïve individuals after CVac 
and CHMI 
 
To explore new diagnostic testing platforms (qPCR, rapid diagnostics, 
ELISA) 
 
 
Study Design 
3.1 
Overall Design 
This is a Phase 1 study to investigate the safety, immunogenicity, and protective efficacy following 
pre-erythrocytic stage only parasite exposure by DVI with aseptic, purified, cryopreserved Pf 
sporozoites (NF54 Sanaria® PfSPZ Challenge), under pyrimethamine treatment, to induce stage 
specific homologous and heterologous sterile protection.  Additionally, we look to evaluate the 
heterologous protective efficacy of PfSPZ-CVac with chloroquine with increasing doses of PfSPZ 
Challenge during PfSPZ-CVac. 
3.2 
Pilot Study 
The goal of the pilot study was to ensure increasing the number of Sanaria® PfSPZ Challenge (NF54) 
to 200,000 under pyrimethamine or chloroquine is safe and well tolerated. The pilot study  occured in 
two parts:  
1. Pyrimethamine pilot study (Arm 1a, 1b and 1d)   assessed whether increasing the number of 
Sanaria® PfSPZ Challenge (NF54) to 200,000 while under pyrimethamine dosing will 
prevent the development of asexual erythrocytic stages of the malaria (qPCR negative). 
The highest number of Sanaria® PfSPZ Challenge administered by DVI in PfSPZ CVac 
with pyrimethamine to date is 51,200 PfSPZ (in NIAID protocol #15-I-0169). The goal of 
this study, is in a stepwise manner increase the number of sporozoites injected during 
Sanaria® PfSPZ-CVac to 200,000 PfSPZ while monitoring closely for safety, tolerability, 
and presence of detectable parasitemia (Figure 6).  
x Arm 1a (n=2) is designed to confirm that injecting 50,000 of Sanaria® PfSPZ 
Challenge with pyrimethamine on 2 and 3 days post DVI without chloroquine 


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prophylaxis will prevent the development of either subpatent parasitemia 
(negative retrospective LMIV qPCR result; a positive result is defined as two 
consecutive samples > 40 parasites/mL or a single positive retrospective 
LMIV qPCR ≥ 100 parasites/mL AND negative NIH malaria real time qPCR) 
in Ӌ1/2 subjects. In our previous study (NIAID protocol #15-I-0169), 
participants who received pyrimethamine and chloroquine did not develop 
subpatent parasitemia during PfSPZ-CVac whereas all those who received 
chloroquine alone were parasitemic post CVac #1.  
Subjects will be defined as having definitively negative subpatent parasitemia if through  Day 
14 post injection all qPCRs have remained negative (NIH malaria real time qPCR AND 
retrospective LMIV qPCR). For safety monitoring, concurrent NIH malaria real time qPCR 
will be performed and participants will be treated with Malarone® for 3 consecutive days if 
they have one positive qPCR result regardless of symptoms. All pilot subjects (Arm 1x) who 
remain negative through day 14 post PfSPZ Challenge will be treated at the end of the study. 
Retrospective LMIV qPCR will be completed as outlined in Section 8.13.  
The NIH malaria real time and LMIV retrospective qPCR results will determine which pilot 
arm will be assessed next as outlined in Figure 6.    
x Arm 1b (n=2) 100,000 Sanaria® PfSPZ Challenge with 50 mg of pyrimethamine  
x Arm 1c (n=2) 50,000 Sanaria® PfSPZ Challenge with 75 mg of pyrimethamine  
x Arm 1d (n=2) 200,000 Sanaria® PfSPZ Challenge with 50 mg of pyrimethamine  
x Arm 1e (n=2) 100,000 Sanaria® PfSPZ Challenge with 75 mg of pyrimethamine  
x Arm 1f (n=2) 200,000 Sanaria® PfSPZ Challenge with 75 mg of pyrimethamine 
  
The ideal pilot pathway is doubling the number of Sanaria® PfSPZ Challenge stepwise while 
maintaining pyrimethamine dose at 50 mg without detection of subpatent parasitemia in 2/2 
subjects per arm. Hence, if subjects in Arm 1a do not develop subpatent parasitemia (2/2 subjects 
with negative qPCRs until day 14 post DVI), Arm 1b will follow. If Arm 1 b meets criteria, it 
will be followed by Arm 1d. Before continuing with Arm 1d to the main arm, we will confirm the 
results with an additional 2 subjects to ensure that this regimen does prevent development of 
subpatent parasitemia. (Figure 6 – solid line pathway).  
 
If 1 or both (≥1/2) subjects in Arms 1a are qPCR positive (less than 2/2 have negative qPCR), we 
will first increased the dose of pyrimethamine to 75 mg (Arm 1c) and maintain the dose of 
Sanaria® PfSPZ Challenge at 50,000 sporozoites (Arm 1c). If Arm 1c meets criteria, we will 
continue with doubling the number of sporozoites while maintaining the dose of pyrimethamine 
at 75 mg (Figure 6 – dotted line pathway). If subpatent parasitemia is detected in either Arm 1c 
or 1e, the study will halt for reassessment.  
 
If the pilot reaches Arm 1f, and subpatent parasitemia is not detected in 2/2 subjects, then 2 more 
subjects will be enrolled in Arm 1f for confirmation. If the second group enrolled meets criteria, 
the regimen in Arm 1f will be used for the main study. If 1/2 subjects develops subpatent 
parasitemia (1/2 negative qPCR), then we will step down the dose of Sanaria® PfSPZ Challenge 
and Arm 1e regimen will be used for the main study. If both subjects in the second group of Arm 
1f develop subpatent parasitemia (0/2 negative LMIV qPCR), we will step down to Arm 1e but 
repeat by enrolling 2 more subjects for confirmation before continuing to the main study. If any 


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of the subjects in Arm 1e develop subpatent parasitemia (less than 2/2 negative qPCR), the study 
will halt for reassessment (Figure 6).   
 
Figure 6: Pyrimethamine Pilot Study Up-Down Schema 
50,000 PfSPZ
50mg PYR
Day 2,3
Arm 1a; n=2
100,000 PfSPZ
50mg PYR
Day 2,3 
Arm 1b; n=2
50,000 PfSPZ
75 mg PYR 
Day 2,3
Arm 1c; n=2
200,000 PfSPZ
50mg PYR
Day 2,3
Arm 1d; n=2
100,000 PfSPZ
75 mg PYR
Day 2,3 
Arm 1e; n=2
REPEAT*
200,000PfSPZ
50mg PYR
Day 2,3
Arm 1d, n=2
2/2 -ve
2/2 -ve
HALT, Reassess
2/2 -ve
MAIN
100,000 
PfSPZ
75 mg PYR
Arm 1e
MAIN
200,000 
PfSPZ
50mg PYR
Arm 1d
2/2 -ve
2/2 -ve
<2/2 -ve
HALT, Reassess
<2/2 -ve
200,000 PfSPZ
75 mg PYR
Day 2,3
Arm 1f; n=2
2/2 -ve
<2/2 -ve
2/2 -ve
REPEAT*
200,000 PfSPZ
75 mg PYR
Day 2,3
Arm 1f, n=2
REPEAT*
100,000  PfSPZ
75 mg PYR
Day 2,3 
Arm 1e; n=2
<2/2 -ve
MAIN
200,000 
PfSPZ
75 mg PYR
Arm 1f
2/2 -ve
2/2 -ve
<2/2 -ve
1/2 -ve
<2/2 -ve
<2/2 -ve
<2/2 -ve
0/2 -ve
 
Solid line (       ) = 50 mg of pyrimethamine; Dotted line (           ) = 75 mg pyrimethamine.  
–ve = NEGATIVE 
* Two additional volunteers will be enrolled into the pilot study to confirm that the selected regimen does not result in 
development of subpatent parasitemia before the main study starts 
Note: LMIV qPCR will be done daily from days 6-14 post DVI and the results will be used to determine the pilot path 
to be followed. 
 
 
 
     )
       ) =


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Figure 7: Ideal Pyrimethamine Pilot Study Pathway Schema 
 
* Two additional volunteers will be enrolled into the pilot study to confirm that the selected regimen does not result in development 
of subpatent parasitemia before the main study starts 
 
2) Chloroquine pilot study (Arms 5a and 5b)  assessed safety and ensured that the increased 
number of PfSPZ of Sanaria® PfSPZ Challenge (100,000 or target dose 200,000) while on weekly 
chloroquine regimen will not result in diagnosis of clinical malaria requiring treatment with another 
antimalaria medication. The goal of this study, is in a stepwise manner increase the number of 
sporozoites injected during Sanaria® PfSPZ-CVac to 200,000 PfSPZ while monitoring closely for 
safety, tolerability, and development of clinical malaria requiring treatment with another 
antimalaria medication (Figure 8).  
x Arm 5a (n=2) is designed to confirm that injecting 100,000 PfSPZ of Sanaria® 
PfSPZ Challenge with weekly chloroquine starting with loading dose 2 days 
prior to DVI and second and last dose 5 days post DVI will prevent the 
development of clinical malaria diagnosis requiring treatment with another 
antimalarial medication. Clinical malaria diagnosis is per definition outlined in 
LMIV SOP 25: Diagnosing Malaria; in which subject will be evaluated for 


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clinical malaria diagnosis requiring treatment if clinically ill and has a 
T>38.0°C without other symptoms or has two or more signs and symptoms 
(fever T>38.0°C, headache; chills; sweats; malaise; myalgia; arthralgia; 
nausea; vomiting; abdominal pain; diarrhea; low back pain; or non-
musculoskeletal chest pain). If the subject meets these criteria, in discussion 
with the PI, a work up with a symptomatic blood smear and/or real-time NIH 
qPCR and blood smear set will be initiated. If the work up is positive and in 
agreement with the PI, the subject will be treated with Malarone® for 3 
consecutive days and withdrawn from the study. 
 
Subjects will be monitored daily with blood smears, routine qPCRs will not be 
done as it is expected that with PfSPZ-CVac with chloroquine, subpatent 
parasitemia is detectable. In our previous study (NIAID protocol #15-I-0169) 
in which subjects received 51,200 Sanaria® PfSPZ Challenge, subjects who 
received chloroquine only developed subpatent parasitemia (qPCR positive). 
None of the subjects developed patent parasitemia (positive blood smear). It is 
however expected that with higher numbers to 200, 000 PfSPZ of Sanaria® 
PfSPZ Challenge, subjects may develop patent parasitemia, thus the presence 
of parasitemia alone will not determine treatment with another antimalaria 
medication (Malarone®). 
Subjects will be defined as not been diagnosed with clinical malaria if they are 
negative through day 12 post injection. All pilot subjects (Arm 5x) who remain 
negative through day 12 post PfSPZ Challenge will be treated with Malarone® 
for 3 consecutive days at the end of the study. Retrospective LMIV qPCR will 
be completed as outlined in Section 8.13.  
 
If both subjects in Arm 5a do not develop clinical malaria requiring treatment with another 
antimalaria medication, then Arm 5b will be assessed. If both subjects in Arm 5b do not 
develop clinical malaria, two additional subjects will be enrolled into this Arm for 
confirmation. If 1 or both (≥1/2) subjects in Arm 5a develop clinical malaria then, we will not 
continue to Arm 5b and the chloroquine pilot will halt for reassessment. If 1 or both (≥1/2) 
subjects in Arm 5b develop clinical malaria requiring treatment with another antimalaria 
medication, then the regimen in Arm 5a will first be repeated in additional 2 subjects before 
being used in the main study (Figure 8 and 9).  
x Arm 5b (n=2) 200,000 Sanaria® PfSPZ Challenge with weekly chloroquine (2 doses 
total) 
Since the goal of the study is to use the same dose of Sanaria® PfSPZ Challenge in both Arms 2 
and 3, the chloroquine pilot (Arm 5a) will only be started after the pyrimethamine pilot has 
completed 100,000 Sanaria® PfSPZ Challenge Arm (Arm 1b or 1e). Arm 5b will only be enrolled 
after the pyrimethamine pilot has completed the first round of 200,000 Sanaria® PfSPZ Challenge 
(Arm 1d or Arm 1f) (Figure 8). If the chloroquine pilot study is unsuccessful to progress through 
100,000 Sanaria® PfSPZ Challenge (Arm 5a), the study will halt for reassessment.  
 
 


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Figure 8:Chloroquine Pilot Flow Chart 
100,000  PfSPZ
Arm 5a; n=2
200,000PfSPZ
Arm 5b; n=2
REPEAT*
200,000PfSPZ
Arm 5b, n=2
2/2 -ve
MAIN
200,000 
PfSPZ
Arm 5b
2/2 -ve
2/2 -ve
Enrolled ONLY after PYR pilot  has 
completed 100,000  PfSPZ, 
Arm 1b or 1e
Enrolled ONLY after PYR pilot  has 
completed 200,000PfSPZ
Arm 1d or 1f
HALT, Reassess
<2/2 -ve
REPEAT*
100,000  PfSPZ
Arm 5a; n=2
<2/2 -ve
MAIN
100,000 
PfSPZ
Arm 5a
HALT, Reassess
2/2 -ve
<2/2 -ve
  
 
 
 
 
Note: Solid line – Ideal pathway; Dash line – alternative pathway 
 
 


PfSPZ-CVac PYR #2                Version 10.0    Nov. 12, 2019      Page 58 of 169 
 
 
Figure 9: Chloroquine Pilot Ideal Pathway 
 
.  
 Pilot Study Outcomes 
The pilot study was completed in October 2017 and the goal dose of 200, 000 PfSPZ Challenge 
SPZ was achieved in both the pyrimethamine group and the chloroquine group. A total of 13 
volunteers completed the study, 1 volunteer is still being followed Grade 1 laboratory 
abnormality. The two regimens were found to be safe and well tolerated. Briefly, the safety 
results are summarized below: 
 
In the pyrimethamine pilots (n=8), all doses from 50,000 to 200,000 PfSPZ 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 100,000 PfSPZ Challenge SPZ that was not related to study participation. A second 
Grade 3 AE of costochondritis, unrelated to study participation, occurred in a subject who 
received 200,000 PfSPZ Challenge SPZ. All AEs resolved without sequalae. Safety labs were 
also followed closely per protocol, there was one Grade 1 white count decreased and one Grade 1 
ALT increased observed in one asymptomatic subject who received 200,000 PfSPZ Challenge 
SPZ. The WBC decreased has resolved, ALT increase is stable and being followed by the study 
team (Table 15). 
  
Legend
Screening Period
Pre-erythrocytic stage
Subpatent/patent period
PfSPZ
PfSPZ Challenge DVI
0
3
6
9
Study Weeks
CHLOROQUINE IDEAL PILOT SCHEMA
200,000 PfSPZ
Arm 5b (N=2)
EOS
BS
Malarone Tx
THIS REGIMEN 
TO MAIN STUDY
100,000 PfSPZ
Arm 5a (N=2)
EOS
BS
Malarone Tx
200,000 PfSPZ
EOS
Malarone Tx
BS
12
Arm 5b (N=2) Additional
2/2 -ve for clinical 
malaria requiring 
additional 
treatment
CQ
CQ
CQ
2/2 -ve for clinical 
malaria requiring 
additional 
treatment
2/2 -ve for clinical 
malaria requiring 
additional 
treatment


PfSPZ-CVac PYR #2                Version 10.0    Nov. 12, 2019      Page 59 of 169 
 
 
Table 15: Summary of AEs by severity and frequency observed 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 pilots (n=6), overall, there have been more reported AEs and related AEs 
compared to the pyrimethamine pilots. 
However, the majority have remained Grade 1, expected AEs (Table 16). At the dose of 100,000 
PfSPZ of PfSPZ Challenge, neither participant had a positive blood smear (0/2). At the dose of 
200,000 PfSPZ of PfSPZ Challenge, the majority of individuals (3/4; 75%) had a single positive 
blood smear (symptomatic blood smear which is 15 passes versus 5 passes for routine 
asymptomatic read) with associated expected symptoms related to induced malaria such, 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 without 
sequalae and symptoms were at Grade 3 for less than 24 hours during the period of expected peak 
parasitemia. All CQ Arm subjects were LMIV research qPCR positive for at least 2 consecutive 
days (day 7 and 8 post PfSPZ Challenge) as expected, with those with 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 16: 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 SMC per protocol and ad hoc and after the careful 
review, it was determined that administration with 200,000 PfSPZ Challenge SPZ with either 
pyrimethamine or chloroquine was safe and tolerable. The SMC recommended continuation to 
the main study at this dose with added monitoring of Arm 3 (chloroquine only Arm) on the days 
of peak parasitemia. Additional clinic visits and pre-emptive administration of NSAIDs to Arm 3 
in the main study have thus been added to this protocol in order to increase tolerability of this 
regimen. 
 
All participants were treated with Malarone® per protocol and completed their study participation 
per protocol.   
PfSPZ Challenge with PYR Grade 1
Grade 2
Grade 3
Grade 4 Total AEs
Arm 1a ; 50000 (n=2)
4 (2) 100% 0 (0) 0%
0 (0) 0%
0 (0) 0% 4 (2) 100% 
Arm 1b;  100000 (n=2)
9 (2) 100% 0 (0) 0%
1 (1) 50% 0 (0) 0% 10 (2) 100% 
Arm 1d; 200000 (n= 4)
8 (3) 75% 
1 (1) 25% 1 (1) 25% 0 (0) 0% 10 (3) 100% 
PfSPZ Challenge with CQ
Grade 1
Grade 2
Grade 3
Grade 4 Total AEs
Arm 5a ; 100000 (n=2)
16 (2) 100% 1 (1) 50% 0 (0) 0%
0 (0) 0% 17 (2) 100%
Arm 5b ; 200,000 (n= 4)
19 (4) 100% 5 (2) 50% 3 (2) 50% 0 (0) 0% 27 (4) 100%


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3.3 
Main Study 
Since the pilot study has completed and the optimal regimens have been determined as outlined in 
Section 3.2, the main study can start. As outlined above in Section 3.2.1 the following regimens 
will be executed during the main phase: 
x Arm 2a (n=17) and Arm 2b (n=10) will receive the selected regimen of Sanaria® PfSPZ 
Challenge (NF54 at 200,000) via DVI and pyrimethamine 50mg on 2, 3 days post PfSPZ 
Challenge without chloroquine prophylaxis. These participants will receive three 
successive PfSPZ-CVac immunizations administered approximately 4 weeks apart. 
Participants in Arm 2 will be monitored in real time daily by NIH malaria real time 
qPCR on days 6-14 post DVI after the first CVac.  NIH malaria real time qPCR will be 
done in real time from days 7-9 post DVI (most likely time period to detect parasitemia) 
during second and third CVacs.  NIH malaria real time qPCR will be done at other times 
outside this window if clinically indicated. If there is a single positive NIH malaria real 
time qPCR detected during the first PfSPZ-CVac, NIH malaria real time qPCR will be 
done in real time from days 6-10 (and longer if CVac #1 qPCR positive greater than day 
10) post DVI during second and third CVac immunizations for all subjects in this Arm.  
 
We did not observe subpatent parasitemia (negative by blood smear and qPCR) in the 
PfSPZ-CVac with pyrimethamine + chloroquine arms in protocol #15-I-0169, indicating 
the concurrent administration of chloroquine may not be needed. Thus the 
pyrimethamine arms in this study (Arm 1 and 2) will not receive chloroquine, but as an 
added safety measure, as outlined above, we will use NIH real time (“real time” meaning 
performed the day that the sample is drawn to provide immediate results) malaria qPCR 
for subpatent parasitemia monitoring during the pilot study and during PfSPZ-CVac of 
the main study in Arm 2 only as outlined above (days 6-14 post DVI during CVac #1 and 
days 7-9 post DVI during CVac #2 and #3). If subpatent parasitemia is not detected 
during the first vaccination (NIH real time malaria qPCR and LMIV retrospective 
malaria qPCR are negative in ALL subjects at ALL timepoints), based on previous 
studies, it is expected that subpatent parasitemia will likely be prevented in subsequent 
vaccinations. If ≥ 1 subject has a positive NIH real time malaria qPCR during PfSPZ-
CVac #1, then NIH real time malaria qPCR will be conducted from days 6-10 post DVI 
during CVac #2 and #3 for all subjects in this Arm (see Appendix A). 
 
Arm 3 (n=10) will receive the same number of Sanaria® PfSPZ Challenge determined for 
Arm 2 (NF54 at 200,000) via DVI while under weekly chloroquine prophylaxis without 
pyrimethamine. These participants will receive three successive vaccinations 
administered approximately 4 weeks apart. These subjects will be monitored by daily 
blood smears from days 6-14 during CVac #1 and from days 6-10 during CVac #2 and 
#3. NIH real time malaria PCR will not be completed routinely in this arm during CVac 
unless clinically indicated given it is expected that Arm 3 will be qPCR positive on 
multiple days following CVac #1, #2, and even #3.  
 
As already noted, weekly chloroquine prophylaxis in the CVac model protected all 
subjects from development of patent parasitemia following CHMI (Roestenberg, McCall 
et al. 2009). More recently, chloroquine prophylaxis was used with Sanaria’s injectable 
infectious PfSPZ (Sanaria® PfSPZ Challenge) and again, all subjects were protected (9/9) 


PfSPZ-CVac PYR #2                Version 10.0    Nov. 12, 2019      Page 61 of 169 
 
 
using a regimen of three monthly injections of 51,200 PfSPZ (Mordmuller, Surat et al. 
2017).  In a LMIV protocol (NIAID Protocol #15-I-0169), these results were replicated in 
which 4/5 participants were protected against homologous CHMI.  
 
In the proposed study, chloroquine prophylaxis will be similarly employed as the 
previous study (at standard dosing, unless clinically indicated, due to adverse events, to 
adjust to weight based dosing) in Arm 3 subjects only. This chloroquine arm will serve as 
CVac phase positive controls as well as a comparison arm to assess differences in 
immunological responses induced by exposure to SPZ and liver stages (PfSPZ-CVac 
pyrimethamine) only versus SPZ, liver and blood stage exposures (PfSPZ-CVac 
chloroquine). Additionally, with the plan to undergo 7G8 challenge, Arm 2b and Arm 3 
serve to explore if increasing the PfSPZ Challenge exposure can result in heterologous 
protection. 
 
As noted above, the pilot study was completed in November 2017 and the goal dose of 
PfSPZ Challenge of 200,000 NF54 SPZ was achieved in both the pyrimethamine group 
(pyrimethamine dosed at 50mg on days 2 and 3 post DVI) and the chloroquine group; 
thus, PfSPZ Challenge 200,000 NF54 SPZ dose will be administered in both Arms 2 and 
3 during CVac 1, 2 and 3.  Arm 3 will continue to have daily blood smears during 
prepatent period, but will also have closer monitoring (and more frequent blood smears if 
clinically indicated) with twice a day visits during peak parasitemia post CVac #1 (see 
Appendix A). In addition, all subjects in Arm 3 will be given around the clock 
nonsteroidal anti-inflammatory drugs (NSAIDs) pre-emptively during expected peak 
parasitemia (days 7 and 8 post PfSPZ Challenge DVI) to improve tolerability.  
 
x Arm 4a (n=8) will serve as CHMI infectivity controls for Arm 2a and will only receive 
one dose of 3,200 Sanaria® PfSPZ Challenge (NF54) via DVI at CHMI.  
 
x Arm 4b (n=8) will serve as infectivity controls for Arm 2b and 3 and will only receive 
one dose of 3,200 (or other determined dose based on results from studies discussed in 
Section 1.4.4) Sanaria® PfSPZ Challenge (7G8) via DVI at CHMI.  
Subjects in Arms 1a, 1b, 1d (and if need Arms 1c, 1e and 1f) will be considered enrolled upon 
receipt of Sanaria® PfSPZ Challenge. Subjects in Arm 2, 3, 4a, and 4b, will be considered enrolled 
into the study upon blood draw of first baseline research samples (approximately 2 days prior to 
administration of first dose of Sanaria® PfSPZ Challenge). Subjects in Arms 5a and 5b will be 
considered enrolled into the study upon receipt of loading dose of chloroquine (2 days prior to 
administration of first dose of Sanaria® PfSPZ Challenge). Enrolled subjects who withdraw prior 
to administration of first dose of Sanaria® PfSPZ Challenge, will be replaced. A Main study 
schema is represented in Figure 10. 
Antibody and cellular immune responses and transcriptomic profiles will be assessed periodically 
after vaccination and in unvaccinated infection controls (Arm 4a & 4b) as per Appendix A. 
 
 


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Figure 10: Main Study Schema  
 
  
 Preliminary results of the first cohort of the main phase 
The main study began enrollment in January 2018. Due to logistical reasons, staffing and slow 
recruitment, only half of the planned participants were enrolled in the first cohort. The participants 
were split into all three Arms, the following number of subjects in each arm were enrolled and 
received at least one vaccination: 
x Arm 2a (PfSPZ CVac with 200,000 PfSPZ Challenge (NF54) under PYR 
chemoprophylaxis; then homologous NF54 challenge; n=8) 
x Arm 2b (PfSPZ CVac with 200,000 PfSPZ Challenge (NF54) under PYR 
chemoprophylaxis; then heterologous 7G8 challenge; n=5) 
x Arm 3 (PfSPZ CVac with 200,000 PfSPZ Challenge (NF54) under weekly CQ 
chemoprophylaxis, then heterologous 7G8 challenge; n=5) 
In the pyrimethamine Arms, (Arms 2a and 2b, n=13), vaccinations were safe and well tolerated, 
93% of the reported AEs were Grade 1. The most commonly reported AEs were fatigue, 
palpitations, arthralgia, and myalgia. There were 4 Grade 2 AEs (myalgia, gastroenteritis, 
creatinine increased and hemoglobin decreased). There were 2 Grade 3 AEs, a gastroenteritis that 


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was deemed not related to the study and a neutrophil decrease that was considered related to the 
study.  
NIH Malaria qPCRs were completed 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 only monitored with blood smears on days 6 and 10 post PfSPZ Challenge injection after 2nd 
and 3rd vaccinations.  All NIH malaria qPCRs (performed real time; used for safety follow up) and 
the LMIV qPCRs (performed retrospectively throughout pre-patency period; used to confirm no 
blood stage exposure) were negative at every timepoint (Figure 11). These results in a larger group 
of participants confirmed that the pyrimethamine regimen prevents the development of blood stage 
parasites even with higher number of sporozoites used for vaccination (200,000 PfSPZ Challenge 
NF54). Therefore subsequent participants enrolled in either Arm 2a or 2b will be monitored by 
daily by real time qPCR on days 6-10 post DVI (rather than days 6-14) during 1st vaccination 
(Appendix A) . 
In the chloroquine Arm, (Arm 3, n=5), vaccinations were safe and well tolerated. The majority of 
reported AEs (96%) were Grade 1, expected AEs. There was a decrease in 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 pre-emptive NSAIDs starting day 7 post vaccination. We also observed a 
decrease in frequency of AEs with each successive vaccination as has been observed in prior 
studies. There were no Grade 2 AEs reported. There were two Grade 3 SAEs. The first SAE was 
that of acute change in mental status 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. The second SAE was that of acute spontaneous pneumothorax 
that occurred 59 days after third vaccination and was determined not to be related to study 
procedures.  
Thick blood smears were completed daily from days 6-14 post PfSPZ Challenge after 1st  
vaccination and from days 6-10 post PfSPZ Challenge after 2nd and 3rd vaccination. 
One blood smear was positive in one participant on day 8 post the first vaccination. Otherwise, all 
blood smears remained negative throughout. A sensitive RNA based LMIV qPCR was performed 
retrospectively throughout the pre-patency period to confirm no blood stage exposure. As 
expected, due to the action of chloroquine, all subjects had detectable subpatent parasitemia after 
the first vaccination (Figure 11). Fewer subjects had detectable subpatent parasitemia with 
subsequent vaccinations, likely reflecting the developing immunity. The decreasing frequency and 
density of parasitemia in second and third immunizations was expected as has been demonstrated 
in similar studies at LMIV and elsewhere. Therefore subsequent participants enrolled in Arm 3 will 
be monitored daily by blood smear days 6-10 post DVI (rather than days 6-14) during 1st 
vaccination (Appendix A). 
 
 


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Figure 11: LMIV qPCR results during vaccination phase 
 
 
 
CHMI was performed ~12 weeks post third vaccination with 3.2x103 sporozoites of either PfSPZ 
Challenge NF54 (homologous, Arm 2a, n=8) or PfSPZ Challenge 7G8 (heterologous, Arm 2b n=4; 
Arm 3 n=3). In addition, half of the originally planned unvaccinated controls were enrolled to 
undergo either PfSPZ Challenge NF54 (Arm 4a, n=4) or PfSPZ Challenge 7G8 (Arm 4b, n=4). 
CHMI was safe and well tolerated. The majority of reported AEs (92%) were Grade 1 expected 
AEs that were due to malaria symptoms. The most common reported AEs were fatigue, headache 
and myalgia. There were 8 Grade 2 AEs, commonly fatigue and decreased hemoglobin. There 
were no reported Grade 3 AEs or SAEs reported during CHMI. All AEs were transient and 
resolved without sequaelae.   
 
Participants were monitored for malaria diagnosis with NIH malaria PCR daily starting 6 days post 
CHMI. All unvaccinated controls became infected with malaria within 12 days of CHMI. In the 
vaccinated groups, only 1/8 participant in Arm 2a (homologous CHMI) became infected. All other 
vaccinated participants remained uninfected throughout 28 days post CHMI per protocol (Table 
17). Our preliminary results show, for the first time,  that homologous and heterologous immunity 
can be achieved in the absence of blood stage exposure in a chemoprophylaxis vaccination model. 
 
 
Figure 11: X/Y indicate number of 
participants with positive 
qPCR/Total participants in the 
chloroquine Arm. One participant 
in the chloroquine Arm was 
withdrawn prior to vaccination #3. 
None of the participants in the 
pyrimethamine Arms had a positive 
qPCR during vaccination.   


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 Rationale for staggered enrollment  
Due to ongoing slow progress in recruitment, the study split the remainder of the main phase into 
two cohorts. The second cohort  enrolled into heterologous Arms only (Arms 2b and 3 n=10). The 
third cohort was planned to be started at a later date, would enroll into the homologous Arm (Arm 
2a; n=9). The order of the 2nd and 3rd cohorts was determined after reviewing the results of the first 
cohort and literature review as outlined below.  
 
This study is a follow up study from a prior chemoprophylaxis vaccination with pyrimethamine 
that used 50, 000 sporozoites of PfSPZ Challenge NF54 for vaccination, NIAID protocol #15-I-
0169. In that study, 1/9 participants was protected against homologous CHMI (Section 1.4.3.2.1). 
One premise of this current study was that increasing number of sporozoites four fold (to 200,000 
per vaccination), thus increasing the antigen dose, would increase the level of protective efficacy 
against homologous CHMI as had been observed with vaccinations with irradiated sporozoites. 
Our preliminary results in which 7/8 participants in Arm 2a remained uninfected after homologous 
CHMI show that indeed this higher dose of sporozoites may lead to significant increase in the level 
of protective efficacy observed.  
 
 
Table 17: Results of CHMI for the first Cohort  
 
 
Arm 
Number of 
subjects 
challenged 
Number 
of 
subjects 
infected 
Days post 
CHMI to 
diagnosis 
Median 
days post 
CHMI to 
diagnosis 
Positive 
Blood 
Smear 
Percent 
UNinfected 
2a; 
pyrimethamine 
with homologous 
CHMI (NF54)  
8 
1 
12 
12 
0 
87.5% 
2b; 
pyrimethamine 
with heterologous 
CHMI (7G8) 
4 
0 
N/A
N/A 
0 
100% 
3; chloroquine 
with heterologous 
CHMI (7G8) 
3 
0 
N/A
N/A 
0 
100% 
4a; NF54 Control 
4 
4 
9,11, 11, 11 
11 
1 
0% 
4b; 7G8 Control 
4 
4 
9, 9, 12, 12 
10.5 
0 
0% 


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The study is also designed to explore development of immunity against heterologous CHMI. 
Immunity against heterologous CHMI is a more stringent test of efficacy. In an earlier study 
malaria naïve participants were immunized by 5 doses of 2.7 × 105 PfSPZ Vaccine. CHMI 
performed three weeks after last vaccination showed 12/13 participants (92.3% [95% CI: 48.0, 
99.8]) were protected against homologous CHMI while 4/5 participants (80.0% [95% CI: 10.4, 
99.5]) were protected against heterologous CHMI. The difference in protective efficacy was more 
pronounced when CHMI was performed twenty four weeks post last immunization during which 
7/10 participants (70.0% [95% CI: 17.3, 93.3]) were protected against homologous CHMI where as 
only 1/10 participants (10.0% [95% CI: –35.8, 45.6]) were protected against heterologous CHMI 
(Epstein, Paolino et al. 2017). This 5-dose regimen was also used in an LMIV study in an endemic 
area in which the hazard ratio for vaccine efficacy was 0. 712 (0528将0918) by proportional 
analysis (p=0.006) (Sissoko, Healy et al. 2017). In another study, malaria naïve individuals were 
immunized by bites of Plasmodium falciparum NF54 infected mosquitoes. CHMI by infected 
mosquitoes performed 14 weeks post last immunization also showed  lower efficacy against 
heterologous CHMI (2/10 and 1/9 remained uninfected against two different strains, NF135.C10 
and NF166.C8 respectively) compared to homologous CHMI (5/5 remained uninfected) (Walk, 
Reuling et al. 2017).  
 
In this current study, some participants in the pyrimethamine Arm also underwent heterologous 
challenge with PfSPZ Challenge 7G8, Arm 2b (n=4). All participants in this group remained 
uninfected post CHMI. Although the numbers are small, this is an unprecedented result for a 
chemoprophylaxis vaccination study with or without blood stage exposure. These preliminary 
results indicate that this regimen has the potential to provide protection against heterologous CHMI 
and subsequent vaccine efficacy in the field.  
 
In view of these preliminary results and existing limitations, for the second cohort of the main 
study, the plan was to first enroll into heterologous Arms, Arm 2b and 3. At that juncture, evidence 
of development of protective efficacy of PfSPZ-CVac under pyrimethamine against homologous 
(NF54) CHMI had been demonstrated. However, there was more evidence that heterologous 
CHMI better predicts vaccine efficacy in field trials where heterogeneous strains circulate. 
Therefore Arms that would undergo heterologous (7G8) CHMI (Arms 2b and 3) were prioritized.  
The number of participants planned to be enrolled in each Arm in the 2nd and 3rd cohort is outlined 
below (Table 18). 
  
 In the 2nd cohort:  
x Arm 2b (PfSPZ CVac with 200,000 PfSPZ Challenge (NF54) under PYR 
chemoprophylaxis; then heterologous 7G8 challenge; n=5) 
x Arm 3 (PfSPZ CVac with 200,000 PfSPZ Challenge (NF54) under weekly CQ 
chemoprophylaxis, then heterologous 7G8 challenge; n=5) 
x Arm 4b (Control participants for 7G8 CHMI n=4) 
In the 3rd cohort: 
x Arm 2a (PfSPZ CVac with 200,000 PfSPZ Challenge (NF54) under PYR 
chemoprophylaxis; then homologous NF54 challenge; n=9) 
x Arm 4a (Control participants for NF54 CHMI n=4) 


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Table 18: Number of participants in each Arm during first, second and third cohort 
ARMS 
Participants in 1st Cohort 
enrolled and received at 
least 1 vaccination 
(COMPLETED CHMI) 
Participants 
in 2nd 
Cohort  
Participants in 
3rd Cohort 
Total # of 
participants 
2a, PYR 
+NF54 
8 (8) 
 
0 
9 
17 
2b, PYR +7G8 
5 (4)* 
 
5 
 
0 
10 
3, CQ +7G8 
5 (3)# 
 
5 
0 
10 
4a, NF54 
Control 
4 (4) 
 
0 
4 
8 
4b, 7G8 
Control 
4 (4) 
 
4 
 
0 
8 
*One participant withdrew consent after 1 vaccination 
# Two participants were withdrawn due to SAEs prior to CHMI 
 Rationale for stopping the study prior to full enrollment 
Cohort 2 was enrolled in September 2018 and completed the study in March 2019. Heterologous 
(7G8) CHMI was conducted approximately 12 weeks post the 3rd vaccination and showed that high 
protective efficacy can be induced by both regimens. In the vaccinated groups, 3/5 (60%) in Arm 
2b and 3/3 (100%) participants in Arm 3 remained uninfected post CHMI whereas in the control 
group, Arm 4b, 0/4 (0%) participants were protected. Combined results of cohorts 1 and 2 are 
shown in Table 19. With this high significant level of protection against heterologous CHMI, and 
since heterologous CHMI is a more stringent test of assessing development of immunity (see 
section 3.3.2), we can already fully answer the secondary objectives of the study. Therefore, the 
study will not complete enrollment in Arms 2a and 4a to undergo homologous CHMI (cohort 3, 
Table 18). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Table 19: Combined Results of CHMI for the first and Second Cohort  
 
 
 
 
 
 
 
 
 
 
Arm 
Number of 
subjects 
challenged 
Number 
of 
subjects 
infected 
Days 
post 
CHMI to 
diagnosis 
Median 
days post 
CHMI to 
diagnosis 
Positive 
Blood 
Smear 
Estimated 
Vaccine 
Efficacy 
95% CI p- value 
2a; 
pyrimethamine 
with 
homologous 
CHMI (NF54) 
8 
1 
12 
12 
0 
0.875 
0.425, 1 0.003 
2b; 
pyrimethamine 
with 
heterologous 
CHMI (7G8) 
9 
2 
10, 10 
10 
0 
0.778 
0.398, 1 <0.001 
3; chloroquine 
with 
heterologous 
CHMI (7G8) 
6 
0 
N/A 
N/A 
N/A 
1 
0.541, 1 <0.001 
4a; NF54 
Control 
4 
4 
9,11, 11, 
11 
11 
1 
N/A 
N/A 
N/A 
4b; 7G8 
Control 
8 
8 
9, 9,9, 9, 
10, 12, 
12, 16 
9.5 
0 
N/A 
N/A 
N/A 


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 Description of Investigational Products and Plan 
4.1 
Chloroquine Phosphate 
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). Chloroquine phosphate 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 [1g (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 
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, pyrimethamine (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, pyrimethamine and pyrimethamine combinations 
have fallen out of favor for clinical use. Since the 1950’s, pyrimethamine has been FDA approved 
for acute treatment and chemoprophylaxis of malaria due to susceptible strains of plasmodia. 
Should circumstances arise, pyrimethamine may be used alone for acute malaria at 50 mg orally 
for two days.  Pyrimethamine 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).  
 
The approved dosage of pyrimethamine 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.  With doses of 
pyrimethamine 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.   
Similar to our previous study, the plan is to utilize the dose of pyrimethamine at 50 mg orally for 
two days, as approved for adult dosage of acute malaria. If subpatent parasitemia is seen during the 
pilot phase, at most we will increase the pyrimethamine dose to 75 mg daily orally for two days, 
similar to pyrimethamine doses utilized in toxoplasmosis treatment, but for a significantly shorter 
period of time.   


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The current approved package insert for pyrimethamine and additional details on safety of 
pyrimethamine is provided in the Investigator’s Brochure. 
4.3 
Sanaria® PfSPZ Challenge  
Sanaria Inc has manufactured two strains of Sanaria® PfSPZ Challenge: NF54 and 7G8. The 
Sanaria® 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, pyrimethamine, atovaquone, 
artesunate, but not mefloquine. Sanaria® PfSPZ Challenge (7G8) is known to be susceptible to 
mefloquine, atovaquone, artemether and artesunate but not to chloroquine or pyrimethamine.    
More detailed Sanaria® PfSPZ Challenge product information can be found in Section 7.1 and the 
Investigator’s Brochure. 
 
The clinical studies for Sanaria® PfSPZ Challenge are detailed in Sections 1.4.2, 1.4.3, and 1.4.4. 
4.4 
Description of Intervention 
The intervention in this study is induction of stage specific immunity (pre-erythrocytic immunity) 
to malaria following DVI with aseptic, purified, vialed, cryopreserved, fully infectious NF54 
PfSPZ (referred to as Sanaria® PfSPZ Challenge) when exposure is limited to the SPZ and liver-
stages of the parasite life cycle (as summarized in Table 19). 
 
Pilot Study 
x Pyrimethamine pilot [Arm 1a, 1b and 1d] will receive 1 dose of NF54 Sanaria® PfSPZ 
Challenge AND 2 doses of pyrimethamine (50 mg and if needed 75 mg) administered on 
2 and 3 days post injection timed to eliminate parasites in the liver-stage of development, 
after hepatocyte invasion and development, but prior to full maturation and release into 
the bloodstream. This regimen is intended to prevent parasitemia detectable by qPCR 
(subpatent parasitemia) and by blood smear (patent parasitemia). Subjects will be 
followed daily with NIH real time malaria qPCR from day 6 through day 14 post PfSPZ 
Challenge. All subjects will be treated with standard, approved treatment dosing of 
Malarone® for three consecutive at the time of a single qPCR detection of parasites or at 
the conclusion of follow-up post PfSPZ-CVac. See Section 3.2 for more details. 
 
x Chloroquine pilot (Arms 5a and 5b): will receive 1 dose of NF54 Sanaria® PfSPZ 
Challenge AND chloroquine weekly dosing beginning 2 days prior to Sanaria® PfSPZ 
Challenge (1g (600 mg base) loading dose then 2nd and last dose of 500 mg (300 mg 
base) on day 5 after the third NF54 Sanaria® PfSPZ Challenge to prevent development of 
patent parasitemia detectable by thick blood smear and clinical malaria. Subjects will be 
followed daily with blood smears from day 6 through day 12 post PfSPZ Challenge. All 
subjects will be treated with standard, approved treatment dosing of Malarone® for three 
consecutive at the time of clinical malaria diagnosis or at the conclusion of follow-up 
post PfSPZ-CVac. See Section 3.2 for more details. 


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Main study 
x Arm 2a and 2b will receive three vaccinations, 28 days apart, with the 200,000 NF54 
Sanaria® PfSPZ Challenge AND pyrimethamine, 50mg per dose, given on days 2 and 3 
post injection. Pyrimethamine is timed to eliminate parasites in the liver-stage of 
development, after hepatocyte invasion and development, but prior to full maturation 
and release into the bloodstream. This group will not receive chloroquine. This regimen 
is intended to prevent parasitemia detectable by qPCR (subpatent parasitemia) and by 
blood smear (patent parasitemia) (see Section 3.3 for more details). This group will not 
receive chloroquine. 
x Arm 3 will receive three vaccinations, 28 days apart, using 200,000 NF54 Sanaria® 
PfSPZ Challenge with continuous suppressive prophylaxis with the blood-stage 
antimalarial drug chloroquine [weekly dosing beginning 2 days prior to first Sanaria® 
PfSPZ Challenge (1g (600 mg base) loading dose then 500 mg (300 mg base) 
maintenance dosing weekly) thereafter] and continuing throughout CVac, last dose on 
day 5 after the third NF54 Sanaria® PfSPZ Challenge to prevent development of patent 
parasitemia detectable by thick blood smear and clinical malaria. This regimen will not 
prevent parasitemia detectable by qPCR, but will eliminate the blood stage parasites 
before they can replicate in erythrocytes. This group will not receive pyrimethamine. 
x Arms 4a and 4b (Infectivity Controls for CHMI) will be the challenge control group. 
This group will join the study for administration of Sanaria® PfSPZ Challenge, NF54 
and 7G8 respectively, at CHMI. This group will not receive chloroquine or 
pyrimethamine. Upon diagnosis with malaria infection per protocol (one positive NIH 
real time malaria qPCR or single positive blood smear) subjects will be treated with 
standard, approved treatment dosing of Malarone®. 
 
Sanaria® PfSPZ Challenge (3,200 PfSPZ) produced by Sanaria®, Inc will be given to all enrolled 
and eligible subjects in the absence of any antimalarial drugs during CHMI. Subjects in Arms 2a 
and 4a will be challenged with Sanaria® PfSPZ Challenge (NF54) whereas Arms 2b, 3 and 4b will 
be challenged with Sanaria® PfSPZ Challenge (7G8) (Figure 10). Subjects who develop patent 
parasitemia (one positive real time NIH qPCR OR 2 unambiguous parasites seen in a single thick 
blood smear) will be treated with a standard, FDA approved treatment dosing of Malarone® (1g 
atovaquone/400 mg proguanil hydrochloride) daily for three days. Subjects who demonstrate 
sterile immunity, meaning they do not develop a positive real time NIH qPCR or blood smear, will 
also be treated with Malarone® daily for three days at the end of the study, starting on day 27 post 
CHMI. 
 
The chemoprophylactic agents used in the current study are pyrimethamine and chloroquine. Both 
pyrimethamine and chloroquine are commercially available Food and Drug Administration (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 will be administered 2 days prior to NF54 Sanaria® PfSPZ Challenge DVI and 
continued weekly until 5 days after the third PfSPZ Challenge. In prior studies, administering the 


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last dose of chloroquine in this manner has been sufficient to eliminate parasitemia in all study 
subjects during CVac, as described in Section 1.4.3.1 (Mordmuller, Surat et al. 2017) and observed 
in our previous study, NIAID protocol #15-I-0169 as described in Section 1.4.3.2.1.  
 
Pyrimethamine dosing in this study will be daily dosing of 50 mg for two consecutive days dosed 
on days 2 and 3 after NF54 Sanaria® PfSPZ Challenge DVI. This uses the FDA recommended 
dosing regimen for treatment of malaria due to susceptible parasites. In Arms 2a and 2b, where 
PfSPZ Challenge will be administered three times, there will be a total of 6 doses of 
pyrimethamine received during the study.  Treatment with pyrimethamine on 2 and 3 days post 
NF54 Sanaria® PfSPZ Challenge DVI is chosen to successfully maximize antigenic exposure of 
liver-stage parasites to the host while preventing maturation and release into the bloodstream, thus 
achieving the aims of the study.  
 
 
 


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Table 20: Chemoprophylaxis regimen and assignment 
Intervention 
Arm Assignment 
Dosing 
Antimalarials 
Pyrimethamine  
       
 
Arm 11: n=8-12 
Arm 2a: n=17 
Arm 2b: n=10 
 
Two  single strength pills (50  mg pyrimethamine total) 
given2, 3 days following each administration of Sanaria® 
PfSPZ Challenge by DVI during PfSPZ-CVac 
immunization. In total, 2 daily doses given for Arms 1 
and 6 daily doses given for Arm 2. 
Chloroquine  
          
 
 
 
Arm 3: n=10 
Arm 5: n=2-6 
 
Loading dose of approximately 1 g chloroquine 
phosphate (600 mg chloroquine base) 2 days prior to first 
administration of NF54 Sanaria® PfSPZ Challenge by 
DVI. Then 500 mg (300 mg base) weekly until 5 days 
after 1st injection (Arm 5, 2 doses total) OR until 5 days 
after 3rd injection (Arm 3, 10 doses total).  
P. falciparum sporozoites (PfSPZ), non-attenuated 
Sanaria® PfSPZ 
Challenge (NF54) 
administered by DVI 
for CVac 
       
 
 
     
Arm 11: n= 8-12 
Arm 2a: n=17 
Arm 2b: n=10 
Arm 3: n=10 
Arm 5: n=2-6 
 
 
Aseptic, purified, vialed, cryopreserved, non-attenuated 
PfSPZ (NF54 Sanaria® PfSPZ Challenge) administered 
by DVI of either 50,000, 100,000 or 200,000 PfSPZ in 
pilot arms (Arm 5 will not receive 50,000 dose). 200,000 
PfSPZ dose will be administered to Arm 2a, 2b and Arm 
3.  
Sanaria® PfSPZ 
Challenge (NF54) 
administered by DVI 
for CHMI 
Arm 2a: n=17 
Arm 4a: n=8 
 
Aseptic, purified, vialed, cryopreserved, non-attenuated 
PfSPZ (NF54 Sanaria® PfSPZ Challenge) administered 
by DVI of 3,200 PfSPZ  
Sanaria® PfSPZ 
Challenge (7G8) 
administered by DVI 
for CHMI 
 
 
Arm 2b: n=10 
Arm 3: n=10 
Arm 4b: n=8 
 
Aseptic, purified, vialed, cryopreserved, non-attenuated 
PfSPZ (7G8 Sanaria® PfSPZ Challenge) administered by 
DVI of  3,200 PfSPZ (this dose may be adjusted 
depending on the results of other studies; if adjusted an 
amendment will be submitted) 
1 Arm 1 represents pilot phase with administration of 50 mg pyrimethamine and 50,000 Sanaria® PfSPZ Challenge 
during CVac.  Depending on whether primary Pf infection objective is achieved or not, arms 1b to 1f may be followed 
as outlined in Section 3.1.  The target regimen is Arm 1d. All pilot subjects only receive 1 CVac and will not undergo 
homologous challenge via CHMI per Appendix A. 


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4.5 
Presumptive Antimalarial Treatment 
Subjects who develop patent parasitemia (defined as one positive real time NIH qPCR or a single 
positive blood smear) following CHMI (all Arms) or PfSPZ-CVac (Arms 1 and 2) will be 
immediately treated with a standard, FDA-approved treatment dosing of Malarone® (1 g 
atovaquone/ 400 mg proguanil hydrochloride) daily for three consecutive days. Treated subjects 
will continue to be monitored with daily visits and blood smears until three consecutive daily blood 
smears are negative and any residual symptoms of malaria are mild or resolved. 
 
Subjects who remain real time NIH qPCR and blood smear negative will be treated empirically 
with a standard dose of Malarone® at 27 days after CHMI with Sanaria® PfSPZ Challenge, at 14 
days after Sanaria® PfSPZ-CVac (Arm 1) and at 12 days after Sanaria® PfSPZ-CVac (Arm 5).  
Subjects will return to the clinical site for two additional consecutive days to receive the second 
and third doses of Malarone® under direct observation by study staff. 
 Study Endpoints 
 Primary Endpoints 
Safety 
x Incidence and severity of local and systemic adverse events (AEs) and serious 
adverse events (SAEs) occurring after PfSPZ-CVac. (Arms 1, 2, 3,5) 
x P. falciparum blood stage infection defined as detection of P. falciparum 
parasites by qPCR (real time NIH qPCR and sensitive retrospective LMIV 
qPCR) following Sanaria® PfSPZ Challenge. (Arm 1, 2) 
x Incidence of clinical malaria diagnosis occurring after PfSPZ-CVac – 
chloroquine requiring treatment with additional antimalaria (Arms 3 and 5) 
 
 Secondary Endpoints 
           Protective Efficacy  
x P. falciparum blood stage infection defined as detection of at least 2 P. 
falciparum parasites by microscopic examination of 0.5 μL of blood or one 
positive real time NIH qPCR after homologous PfSPZ CHMI (NF54) via DVI. 
(Arm 2a and 4a) 
x P. falciparum blood stage infection defined as detection of at least 2 P. 
falciparum parasites by microscopic examination of 0.5 μL of blood or one 
positive real time NIH qPCR after heterologous PfSPZ CHMI (7G8) via DVI. 
(Arm 2b, 3 and 4b) 
 
 Exploratory Endpoints 
 
x Humoral immune responses after PfSPZ-CVac regimens by assessing antibodies to 


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PfSPZ, Pf asexual erythrocytic stages (AES), and specific Pf sporozoite, liver and 
blood-stage antigens such as CSP, EXP-1, LSA-1, MSP-3, MSP-1, AMA-1 in 
subjects with no patent and/or subpatent parasitemia and those with patent and/or 
subpatent parasitemia, among drug groups. 
x Cellular immune responses after PfSPZ-CVac regimen to PfSPZ, PfAES, and specific 
Pf sporozoite, liver and blood-stage antigens, such as CSP, EXP-1, LSA-1, MSP-3, 
MSP-1, AMA-1, in subjects with no patent and/or subpatent parasitemia and those 
with patent and/or subpatent parasitemia, among drug groups. 
x Subpatent parasitemia and degree of subpatent parasitemia by sensitive retrospective 
LMIV qPCR following Sanaria® PfSPZ-CVac pyrimethamine and Sanaria® PfSPZ-
CVac chloroquine. 
x Comparison of γδ T cells before and after Sanaria® PfSPZ-CVac and CHMI using 
ex vivo whole blood staining 
x Comparison of time to positivity of various malaria diagnostic platforms (qPCR, 
rapid diagnostics, ELISA). 
5.2 
Sample Size and Estimated Duration of Study 
A total of up to 67 subjects are expected to be enrolled in this trial, if the pyrimethamine and 
chloroquine ideal pilot pathway is followed in which only four pilot arms will be conducted 
(Figures 6, 7).  Subjects in the pilot arms and control arm will be monitored actively for about 1 
month whereas subjects in the main arms will be actively monitored for approximately 7 months. 
Up to 200 subjects will be screened to accommodate possible screen failures. 
Study Population
6.1 
Description of Population and Site 
 
The study will be conducted at the NIH Clinical Center (outpatient facilities) in Bethesda, 
Maryland.  NIH Clinical Center has successfully supported healthy volunteer subject clinical trials, 
including malaria vaccine and controlled human malaria infection trials.  Healthy malaria-naïve 
subjects will be recruited from the surrounding community. 
6.2 
Recruitment 
 
Healthy adult male and non-pregnant female subjects will be recruited from a variety of sources 
including those previously screened or enrolled in other vaccine trials at the NIH clinical center or 
by the use of an Institutional Review Board (IRB)-approved screening protocol and study- specific 
print or media advertising.  After an initial phone screen (using an IRB approved Phone Screen), a 
screening visit will be scheduled. 
 
During the screening process, which may require more than one visit, the subject will read the 
consent form, be encouraged to ask questions, and then complete a written comprehension 
evaluation questionnaire (Malaria Comprehension Exam).  The questionnaire is used to identify the 
areas of the study and consent that the subject may not fully understand.  The person administering 
consent reviews the answers with the subject.  If the subject gets a question wrong, the person 
administering the consent reviews the portion of the consent form that relates to that particular 


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question with the subject. The subject may either sign the consent form during the screening visit, 
or return after further consideration. 
6.3 
 Participation of NIH Staff 
NIH staff and members of their immediate families may participate in this protocol. We will follow 
the Guidelines for the Inclusion of NIH staff in NIH Research Studies and will give each staff 
member a copy of the “NIH information sheet on Staff Research Participation.” 
  
For NIH staff: 
x Neither participation nor refusal to participate will have an effect, either beneficial or 
adverse, on the participant’s employment or work situation. 
x The NIH information sheet regarding NIH staff research participation will be distributed 
to all potential subjects who are NIH staff. 
x The staff subject’s privacy and confidentiality will be preserved in accordance with NIH 
Clinical Center and NIAID policies, which define the scope and limitations of the 
protections.  
x For NIH staff subjects, consent will be obtained by an individual independent of the 
staff’s team. Those in a supervisory position to any staff and co-workers of the staff will 
not obtain consent.  
x The importance of maintaining confidentiality when obtaining potentially sensitive and 
private information from co-workers or subordinates will be reviewed with the study staff 
at least annually and more often if warranted. 
6.4 
Inclusion Criteria 
 
All of the following criteria must be fulfilled for a subject to participate in this trial: 
 
1. Age ≥ 18 and ≤ 50 years.   
2. In good general health and without clinically significant medical history  
3. Malaria comprehension exam completed, passed (a score of Ӌ80% or per 
investigator’s discretion) and reviewed prior to enrollment 
4. Reliable access to the clinical trial center and availability to participate for duration 
of study 
5.  Females of childbearing potential must be willing to use reliable contraception (as 
defined below) from 21 days prior to study day -2 to 28 days following last Sanaria® 
PfSPZ Challenge exposure 
 
x Subject to the judgment and discretion of the PI, female participants who meet 
ANY ONE of the criteria listed immediately below, may not be required to take any 
additional measures to avoid pregnancy. Such participants will be counseled on 
risks at the time of consent and at appropriate points (e.g. when pregnancy testing 
occurs) during the study: 
 


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o Females who have had their uterus, and/or BOTH ovaries removed 
o Females who have had BOTH fallopian tubes surgically ‘tied’ or removed 
o Females who are above the age of 45 and have spontaneously had no menses 
at any point during the past 12 or more consecutive months (i.e. have reached 
menopause) 
o Females who, in the conservative and reasonable judgment of the PI (e.g. 
due to sexual orientation or serious life choice (such as being celibate clergy 
or transgender), during the entire trial will NOT participate in any potentially 
reproductive sexual contact  
o Females who, in the conservative and reasonable judgment of the PI, are in 
a monogamous stable relationship with a male who has undergone 
vasectomy at least 4 months prior or another procedure/medical condition 
that deems the male sterile 
 
x Subject to the judgment and discretion of the PI, female participants who DO 
NOT meet ANY of the criteria listed above, will be appropriately counseled on 
reproductive risks and pregnancy avoidance, and will be required to adhere to the 
following measures and agree to 2 methods of pregnancy prevention as noted below: 
 
CATEGORY 1: 
x a highly effective hormonal method to prevent pregnancy [e.g. CONSISTENT, 
CONTINUOUS use of contraceptive pill, patch, ring, implant or injection], and/or  
x IUD or equivalent  
 
IN ADDITION TO 
 
 
 
CATEGORY 2: 
 
x a barrier method to be used at the time of potentially reproductive sexual activity (e.g. 
[male/female condom, ‘cap,’ or diaphragm] + spermicide).   
 
6.5 
Exclusion Criteria 
 
A subject will be excluded from participating in this trial if any one of the following criteria is 
fulfilled: 
 
1. Currently is breast-feeding (if female). 
2. Pregnancy as determined by a positive urine or serum human choriogonadotropin (β-
hCG) test at any point during the study (if female). 
3. Recent travel to a malaria endemic area within 5 years of enrollment (Endemic areas 
are defined per the CDC website. Factors such as  but not limited to use of antimalaria 
prophylaxis during travel, length of stay, activities during the travel, history of illnesses 
within 30 days of travel will be considered to determine the likelihood that the subject 
was exposed to malaria) 
4. Planned travel to a malaria endemic area (as defined by the Center for Disease Control) 
during the study period 


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5. Reported history of confirmed malaria diagnosis on peripheral blood smear or by 
clinical history in the past 10 years. 
6. Hemoglobin, WBC, platelets, ALT, and creatinine outside of local lab normal range 
(subjects may be included at the investigator’s discretion for ‘not clinically 
significant’ values outside of normal range) 
7. Abnormal urinalysis as defined by positive urine glucose, protein, and red blood 
cells. Subject can be included if investigator determine the abnormality is “not 
clinically significant”. 
8. BMI < 17 or BMI > 35 
9. Anticipated use during the study period, or use within the following periods prior 
to enrollment: 
a. Investigational malaria vaccine within the last five years 
b. Malaria chemoprophylaxis within 6 months  
c. Chronic systemic immunosuppressive medications (>14 days) within 6 months 
(e.g.cytotoxic medications, oral/parental corticosteroids >0.5 mg/kg/day 
prednisone or equivalent). Corticosteroid nasal spray for allergic rhinitis and 
topical corticosteroids for mild, uncomplicated dermatitis are allowed. 
d. Blood products or immunoglobulins within 6 months 
e. Systemic antibiotics with antimalarial effects within 30 days (such as clindamycin, 
doxycycline) 
f. Investigational or non-registered product or vaccine within 30 days 
g. Receipt of a live vaccine within 28 days or a killed vaccine within the 14 days prior 
to Sanaria® PfSPZ Challenge  
h. Medications known to interact with pyrimethamine and/or chloroquine (for the 
main and pilot study participants ONLY); atovaquone, proguanil (ALL 
participants) 
10. Reported history of: 
a. Sickle cell disease, sickle cell trait, or other hemoglobinopathies 
b. Splenectomy or functional asplenia 
c. Systemic anaphylaxis 
d. Any allergic reactions to study drugs (pyrimethamine, chloroquine) or NSAIDs, 
atovaquone, proguanil 
e. Documented history of chronic or active neurologic disease (including seizures, 
uncontrolled migraine headaches) 
f. Psoriasis or porphyria 
g. Ocular diseases including retinopathy or visual field defects 
11. Clinically significant medical condition, physical examination findings, other clinically 
significant abnormal laboratory results, or past medical history that may have clinically 
significant implications for current health status and participation in the study in the 
opinion of the Investigator.  A clinically significant condition or process includes but is 
not limited to: 
a. A process that would affect the immune response, or requires medication that 
affects the immune response 
b. Any contraindication to repeated phlebotomy 
c. A condition or process in which signs or symptoms could be confused with 
reactions to malaria challenge and/or infection, including dermatologic 
abnormalities at the site of sporozoite inoculation 


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d. A chronic or subclinical condition which could be exacerbated by 
administration of any of the PfSPZ-CVac components or malaria infection 
12. History of, or known active cardiac disease including: (1) prior myocardial infarction 
(heart attack); (2) angina pectoris; (3) congestive heart failure; (4) valvular heart 
disease; (5) cardiomyopathy; (6) pericarditis; (7) stroke or transient ischemic attack; 
(8) exertional chest pain or shortness of breath; or ( 9) other heart conditions under the 
care of a doctor 
13. Clinically significant ECG findings, as determined by the expert study cardiologist 
14. Moderate or high risk for coronary heart disease (CHD) based on NHANES I 
cardiovascular risk assessment (Appendix B)  
15. Acute illness at the time of enrollment 
16. Infection with HIV, Hepatitis B, Hepatitis C 
17. Psychiatric condition that precludes compliance with the protocol including but 
not limited to: 
a. Psychosis within the past 3 years 
b. Ongoing risk for suicide, or history of suicide attempt or gesture within the past 3 
years 
18. Suspected or known current alcohol or drug abuse as defined by the American 
Psychiatric Association in the DSM V at the discretion of the PI 
19. Clinical trial staff and/or Sanaria® Inc. employees with direct involvement in the 
conduct of the trial are excluded from participation. 
20. Participating in other clinical trials involving investigational interventions or off label 
medication use during the study period (excluding participation in the optional long 
term follow up visits). Participation in other trials such as observational or imaging 
studies will be discussed with the investigators.  
21. Any other finding that, in the judgment of the Investigator, would interfere with, or 
serve as a contraindication to, protocol adherence, assessment of safety or 
reactogenicity, or a subject’s ability to give informed consent, or increase the risk of 
having an adverse outcome from participating in the study 
 Study Agents 
7.1 
Sanaria® PfSPZ Challenge  
 Sanaria® PfSPZ Challenge (NF54) 
 
Sanaria® PfSPZ Challenge consists of aseptic, cryopreserved P. falciparum 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 


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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 of the well- characterized Pf isolate NF54. Infected adult mosquitoes are 
maintained under aseptic conditions. Pf SPZ migrate to the salivary glands. The salivary glands 
from the Pf SPZ infected mosquitoes are removed by hand dissection under aseptic conditions. 
Salivary glands are then triturated to release the P. falciparum sporozoites. The sporozoites are 
purified, counted, and, at a specified concentration, cryopreserved. Cryopreservation commences 
with the addition of cryoprotective additives to the purified sporozoites to produce the Sanaria® 
PfSPZ Challenge product. Sanaria® 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.  
 Sanaria® PfSPZ Challenge (7G8)  
PfSPZ Challenge (7G8) is manufactured and characterized by essentially the same methods as 
NF54 PfSPZ Challenge except that is comprised of PfSPZ from the 7G8 clone of P. falciparum. 
The P. falciparum gametocyte-infected blood is produced from cultures of the Pf isolate 7G8 (Pf 
7G8) derived from a Working Cell Bank of the well-characterized Pf 7G8.The 7G8 clone of Pf (Pf 
7G8) was derived from a Brazilian isolate, IMTM22, obtained from a 12 year old male near 
Manaus, Brazil on 12 March 1980. The isolate was cryopreserved and adapted to continuous 
culture. After 19 weeks of continuous culture, a sample was cloned by limiting dilution. The clone 
7G8 was selected for ability to produce microgametes, exflagellate, and infect A. freeborni 
resulting in oocysts and sporozoites. It was tested for drug sensitivity by the Division of 
Experimental Therapeutics, Walter Reed Army Institute of Research (WRAIR) and was found to 
be resistant to chloroquine, and sensitive to mefloquine.  
 
Pf 7G8 has a different genomic sequence than does Pf NF54 and a distinct microsatellite map. 
Thus, if Pf 7G8 is used to assess protective immunity by CHMI in subjects immunized with 
Sanaria® PfSPZ Challenge (NF54), it can assess the capacity of Sanaria® PfSPZ Challenge (NF54) 
to protect against heterologous parasites. All the procedures are described in more detail in the 
investigator’s brochure. 
 Phosphate buffered saline and human serum albumin diluent 
 
The diluent for Sanaria® 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 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. 


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 Storage and Handling of Sanaria® PfSPZ Challenge  
 
Shipment of Sanaria® 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 Sanaria® PfSPZ 
Challenge from its storage site to the clinical trial site will follow standard operating procedures 
provided by the Sanaria®. During transport and at the study site, the LNVP shipper will be 
monitored. Receipt of the Sanaria® PfSPZ Challenge will be documented on a tracking log by 
study staff. 
7.2 
Malarone®, Pyrimethamine and Chloroquine  
Chloroquine, pyrimethamine, and Malarone® are a commercially available antimalarial drugs. 
Package inserts for all three drugs are provided.  
Chloroquine phosphate is supplied and manufactured in tablets of 250 mg or 500 mg (equivalent to 
150 or 300 mg chloroquine phosphate base). Based on availability of chloroquine phosphate in the 
U.S., chloroquine phosphate from Europe may be obtained under approval from the FDA, which is 
supplied and manufactured in tablets of 250 mg (equivalent to 155 mg chloroquine phosphate 
base).  
Pyrimethamine is manufactured in tablets of 25 mg.  
Malarone® is manufactured in tablets of 250mg atovaquone/ 100 mg proguanil hydrochloride. 
 Malarone®, Pyrimethamine and Chloroquine Packaging and Labeling 
Pyrimethamine and chloroquine tablets will be maintained in the manufacturer’s original packaging 
until prepared for dispensing in the trial. 
 
Malarone®, though not a drug under study but a drug that all participants will receive, will be 
maintained in the same manner as pyrimethamine and chloroquine. 
 Malarone®, Pyrimethamine and Chloroquine Accountability 
7.2.2.1 Receipt 
Pyrimethamine, chloroquine, and Malarone® tablets will be purchased from commercial 
sources and provided by the NIH Clinical Center Pharmacy. Drug accountability will be 
managed as outlined in the Pharmacy Manual. 
7.2.2.2 Preparation and Administration 
Pyrimethamine, chloroquine, and Malarone® 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 pyrimethamine, chloroquine, and Malarone® will be prepared by 
study staff as described in the Pharmacy Manual.


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7.2.2.3 Storage and Handling 
Pyrimethamine, chloroquine, and Malarone® tablets will be maintained in the manufacturer’s 
original packaging and stored at the clinic under recommended storage conditions until prepared 
for dispensing. 
7.2.2.4 Return of Study Product 
Final accountability of drug supplies will be performed at the conclusion of the study. Final 
disposition of any remaining pyrimethamine, chloroquine, and Malarone® will be determined 
and documented. 
 Study Procedures 
After signing the informed consent, study procedures are conducted prior to and following 
enrollment on all subjects (unless otherwise noted) at the time points indicated in Appendix A. 
Associated laboratory assays and blood volumes are also specified in Appendix A. 
8.1 
Screening 
 
The purpose of the screening visit is to determine subject eligibility for study participation. 
Subjects who are diagnosed with a medical condition during the screening process (e.g., test 
positive for hepatitis B, hepatitis C, or HIV) will be notified and referred for medical care, 
reportable positive results will be reported to the Maryland Department of Health.  
  
The following screening procedures and evaluations for this study must be completed within 56 
days prior to study enrollment.
 
x Explain the study and Informed Consent to the subject. Ensure the subject has signed the 
Informed Consent prior to any study procedures and receives a signed copy of the 
Informed Consent.  
 
x Ensure the subject has successfully completed the Malaria Comprehension Exam (scoring ≥ 
80% correct and those questions answered in error reviewed) 
 
x Ensure that HIV pre-test counseling has been performed and ensure that the subject has 
agreed to HIV testing (required by Maryland state law). 
 
x Elicit a complete medical history, including menstrual and contraceptive history and/or 
history of surgical sterility for females. 
 
x Pregnancy prevention counseling will be performed. 
 
x Administer a complete physical examination, including vital signs (height, weight, blood 
pressure, temperature, respirations, and pulse). 
 
x Complete NHANES I Classification (see Appendix B) 
 
x Obtain blood samples for complete blood count (CBC) with differential and platelet count, 
ALT, Creatinine, Hepatitis B surface antigen, Hepatitis C antibody, and HIV antibody. 
 
x Obtain urine or blood for pregnancy testing  
 


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x Obtain urine for urinalysis testing for protein and blood. 
 
x Obtain electrocardiogram (ECG), review, initial and date. ECG to be transmitted to study 
cardiologist for final read. 
 
Note: Laboratory studies and ECG completed under a different NIH protocol can be used for 
screening purposes as long as they are within 56 days prior to study enrollment. 
8.2 
Malaria Comprehension Exam 
 
To ensure the subject fully comprehends key concepts related to the study and to highlight areas 
that may need additional discussion or clarification, a Malaria Comprehension Exam will be 
administered to the subject before signing informed consent. 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. 
Discussions of understanding will be documented in the subject’s source documentation. 
8.3 
Assignment to Groups 
 
Arm 1a, 1b and 1d (and Pilot Arms 1c, 1e and 1f if necessary) as well as Arm 5a and 5b will 
be filled first and on an available basis. Arm 2a, 2b and 3 will be enrolled following 
completion of the Pilot Arms and will be randomized for enrollment into Arm 2a, 2b or 3.   
 
Subjects for Arm 4a and 4b will be randomized for enrollment into Arms 4a or 4b.  
8.4 
Study Schedule 
 
The study schedule is outlined below. Details of specific laboratory samples and approximate 
amounts of blood drawn are summarized in Appendix A.  
Note: 
1. To ensure safety and compliance of enrolled subjects, all study drug administration 
(chloroquine, pyrimethamine and Malarone®) will be completed under directly observed 
therapy. The subject will swallow the study drugs with liquid and study staff will inspect the 
mouth to verify that the entire dose of medication was swallowed. 
2. Although some doses of NSAIDs in Arm 3 may be given in clinic, directly observed therapy 
is not required. 
3.  A complete physical exam will be performed during the screening visit. During ALL other 
visits, physical exam will only be performed as clinically indicated. 
Chemoprophylaxis Vaccination for the pyrimethamine (Arm 1) and chloroquine (Arm 5) 
PILOT studies 
 
Study Day -2 [Arm 5 ONLY] (Enrollment/ Chloroquine Load) 
1. Verify that Informed Consent and up-to-date version of the Informed Consent was 


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obtained. 
2. Ensure that all inclusion criteria are met and exclusion criteria are not met and Eligibility 
Checklist completed and signed. 
3. Ensure that CBC with differential, ALT, and creatinine measurements from 
screening tests are within protocol-defined limits (see Exclusion Criteria).  
4. Ensure that Hepatitis B, Hepatitis C, and HIV testing are negative from screening visit. 
5. During the indicated physical examination, study staff will educate the subject regarding 
signs and symptoms of potential AEs and indications for contacting the site. 
6. For females, obtain a urine or serum sample for E-hCG (pregnancy) testing.  Ensure that 
the test is negative before administering chloroquine load dose; a positive test will exclude 
the subject from the trial. 
7. Record vital signs (blood pressure, temperature, heart rate). 
8. Provide chloroquine loading dose (approximately 1 gram) by directly observed therapy 
(Arm 5 only). 
a. Observe subject for at least 30 minutes following chloroquine loading 
dose to evaluate for immediate adverse reactions. (Only required after the 
first dose) 
9. Record AEs and concomitant medications, if applicable 
 
Study Day 1 [Arm 1 and 5](Enrollment for Arm 1/ Sanaria® PfSPZ Challenge #1) 
1. 
Verify that Informed Consent and up-to-date version of the Informed Consent was 
obtained. 
2. 
Ensure that all inclusion criteria are met and exclusion criteria are not met and Eligibility 
Checklist completed and signed. 
3. 
Ensure that CBC with differential, ALT, and creatinine measurements from 
screening tests are within protocol-defined limits (see Exclusion Criteria).  
4. 
Ensure that Hepatitis B, Hepatitis C, and HIV testing are negative from screening visit. 
5. 
During the indicated physical examination, study staff will educate the subject regarding 
signs and symptoms of potential AEs and indications for contacting the site. 
6. 
Obtain approximately 10 mL of blood for baseline labs including: retrospective LMIV 
qPCR, safety labs, and research labs. 
x Arm 1 only: Obtain approximately additional 10ml of blood for research labs. 
x Arm 5 only: Obtain approximately 3ml of blood and 1 ml of urine for research 
labs. 
7. 
For females, obtain a urine or serum sample for E-hCG (pregnancy) testing.  
Ensure that the test is negative before Sanaria® PfSPZ Challenge; a positive test 
will exclude the subject from the trial. 
8. 
Record AEs and concomitant medications, if applicable. 
9. 
Verify that no contraindications for Sanaria® PfSPZ Challenge or deferral of Sanaria® 
PfSPZ Challenge have been met. 
10. Confirm emergency contact information and complete medical emergency release form 
11. Perform basic history and focused physical examination, emphasizing examination of 
any acute complaints. 
12. Educate the subject regarding signs and symptoms of potential adverse events (AEs), 
clinical malaria, and indications for use of antipyretics if fever, headache, or malaise 
occurs. 
13. For females, ensure continued agreement and compliance with pregnancy prevention 


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before Sanaria® PfSPZ Challenge. 
14. Record vital signs (blood pressure, temperature, and heart rate). 
15. Review Inclusion/Exclusion criteria and confirm final eligibility. 
16. Administer Sanaria® PfSPZ Challenge via DVI. 
a. Observe subject for at least 30 minutes after to evaluate for immediate 
adverse reactions  
17. Dispense diary, thermometer and ruler to subject and demonstrate use and assess 
understanding of use. 
18. Review instructions for contacting the site to report adverse experiences and/or inquire 
about study procedures.  
19. Study number will be the same as screening number.  
20. Provide subject with Do’s and Don’t’s handout. 
 
Study Day 2 [Arm 1 and 5] (Phone Follow-Up; 1 Day after Sanaria® PfSPZ Challenge #1)  
1. Perform basic history and AE review (including DVI site), emphasizing review of any acute 
complaints.  
2. Complete diary review. 
3. Record AEs and concomitant medications, if applicable. 
4. Provide pregnancy prevention counseling for female subjects. 
 
Study Day 3 [Arm 1 ONLY] (pyrimethamine dose; 2 Days after Sanaria® PfSPZ Challenge #1) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review. 
4. Record AEs and concomitant medications, if applicable. 
5. Provide pregnancy prevention counseling for female subjects. 
6. Provide pyrimethamine dose by directly observed therapy (Arm 1 only) 
Note:  2 tablets (50mg) for Arms 1a, 1b and 1d;  
3 tablets (75mg) for Arms 1c, 1e and 1f 
7. Observe subject for at least 30 minutes following pyrimethamine to evaluate for immediate 
adverse reactions (Only required after the first dose) 
 
Study Day 4 [Arm 1 and 5] (pyrimethamine dose (Arm 1 only); 3 Days after Sanaria® PfSPZ 
Challenge #1) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review. 
4. Record AEs and concomitant medications, if applicable. 
5. Provide pregnancy prevention counseling for female subjects. 
6. Obtain approximately 7 mL of blood for safety labs and research labs. 
7. Provide pyrimethamine dose by directly observed therapy (Arm 1 only)  
Note:  2 tablets (50mg) for Arms 1a, 1b and 1d;  
3 tablets for (75mg) Arms 1c, 1e and 1f 
 
Study Day 6 [Arm 5 ONLY] (Chloroquine dose; 5 Days after Sanaria® PfSPZ Challenge #1) 


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1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review. 
4. Record AEs and concomitant medications, if applicable. 
5. Provide pregnancy prevention counseling for female subjects. 
6. Provide chloroquine dose (approximately 500 mg) by directly observed therapy (Arm 5 
only). Note: if a subject has problems related to tolerability of chloroquine, a lower 
weight-based dose (as defined in the PI) would be clinically appropriate for maintenance 
dosing, this adjustment may be made on a case by case basis.  
 
 
***NOTE for all visits between Study Day: 7 - 14: If a subject develops malaria defined in Arm 
1 as 1 positive NIH malaria qPCR and in Arm 5 as Clinical malaria as outlined by LMIV SOP 25. 
1. Administer Malarone® dose (4 tablets) by directly observed therapy. 
2. Observe subject for at least 30 minutes following Malarone® dose to evaluate for immediate 
adverse reactions. (Only required after the first dose) 
3. Complete Malarone® dose treatment for 3 consecutive days 
4. Arm 1: Do not repeat NIH malaria qPCR until end of study, study day 29 
Study Day 7 [Arm 1 and 5] (Clinic Follow-Up; 6 Days after Sanaria® PfSPZ Challenge #1) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review. 
4. Complete malaria symptom review. 
5. Record AEs and concomitant medications, if applicable. 
6. Provide pregnancy prevention counseling for female subjects. 
7. Arm 1 ONLY: Obtain approximately 4 mL of blood for real time NIH and retrospective 
LMIV qPCR. Blood smear completed if indicated.  
Arm 5 ONLY: Obtain approximately 3 mL of blood for retrospective LMIV qPCR and 
Blood smear. 
8. Confirm subject and emergency contact information to follow up malaria diagnostic result. 
 
Study Day 8 [Arm 1 and 5] (Clinic Follow-Up; 7 Days after Sanaria® PfSPZ Challenge #1) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review. 
4. Complete malaria symptom review. 
5. Record AEs and concomitant medications, if applicable. 
6. Provide pregnancy prevention counseling for female subjects. 
7. Arm 1 ONLY: Obtain approximately 11 mL of blood for safety labs, real time NIH and 
retrospective LMIV qPCR, and research labs. Blood smear completed if indicated.   
Arm 5 ONLY: Obtain approximately 10 mL of blood for safety labs, retrospective LMIV 
qPCR, blood smear and research labs. 
8. Confirm subject contact information to follow up malaria diagnostic result. 


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Note:  
Arm 1 will have daily prepatent follow up until Study Day 14, Malarone treatment (unless 
otherwise indicated) will start Study Day 15 
Arm 5 will have daily prepatent follow up until Study Day 12, Malarone treatment (unless 
otherwise indicated) will start Study Day 13 
 
Study Day 9 – 14 [Arm 1 ONLY]; 9 – 12 [Arm 5 ONLY] (Clinic Follow-Up; 8 – 13; 8-11 Days 
after Sanaria® PfSPZ Challenge #1) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review. Complete and Collect diary on Study Day 11. 
4. Complete malaria symptom review. 
5. Record AEs and concomitant medications, if applicable. 
6. Provide pregnancy prevention counseling for female subjects. 
7.  Arm 1 ONLY Obtain approximately 3 mL of blood for real time NIH and retrospective 
LMIV qPCR. Blood smear completed if indicated. 
Arm 5 ONLY: Obtain approximately 3 mL of blood for retrospective LMIV qPCR and 
Blood smear. 
8. Confirm subject contact information.  
 
Study Day 15 (Arm 1); Day 13 (Arm 5) (Treatment visit; 14; 12 Days after Sanaria® PfSPZ 
Challenge #1) 
1.  Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2.  Record vital signs (blood pressure, temperature, and heart rate). 
3.  Complete malaria symptom review. 
4.  Record AEs and concomitant medications, if applicable. 
5.  Provide pregnancy prevention counseling for female subjects. 
6. Arm 1 ONLY: Obtain approximately 14 mL of blood for real time NIH and retrospective 
LMIV qPCR, and research labs. Blood smear completed if indicated.               
Arm 5 ONLY: Obtain approximately 3 ml of blood for retrospective LMIV qPCR and Blood 
smear. 
7. Administer Malarone® dose (4 tablets) by directly observed therapy. 
a. Observe subject for at least 30 minutes following Malarone® dose to 
evaluate for immediate adverse reactions. (Only required after the first 
dose) 
8. Confirm subject contact information to follow up malaria diagnostic result. 
 
Study Day 16 (Arm 1); Day 14 (Arm 5)   (Treatment visit; 15; 13 Days after Sanaria® PfSPZ 
Challenge #1) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Provide pregnancy prevention counseling for female subjects 


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5. Arm 1 ONLY: Obtain approximately 3 mL of blood for real time NIH and retrospective 
LMIV qPCR. Blood smear completed if indicated.               
Arm 5 ONLY: Obtain approximately 3 mL of blood for retrospective LMIV qPCR, blood 
smear and research labs. 
6. Administer Malarone® dose (4 tablets) by directly observed therapy 
7. Confirm subject contact information  
Study Day 17 (Arm 1); Day 15 (Arm 5)    (Treatment visit; 16; 14 Days after Sanaria® PfSPZ 
Challenge #1) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Provide pregnancy prevention counseling for female subjects. 
5. Arm 1 ONLY: Obtain approximately 10 mL of blood for safety, real time NIH and 
retrospective LMIV qPCR. Blood smear completed if indicated.               
Arm 5 ONLY: Obtain approximately 10 mL of blood for safety, retrospective LMIV qPCR 
and Blood smear. 
6. Administer Malarone® dose (4 tablets) by directly observed therapy 
7. Confirm subject contact information 
 
Study Day 29 [Arm 1 and 5] (Final visit; 28 Days after Sanaria® PfSPZ Challenge #1) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Provide pregnancy prevention counseling for female subjects. 
5. Obtain approximately 10 mL of blood for real time NIH qPCR and retrospective LMIV 
qPCR, and safety labs. 
6. Confirm subject contact information 
Chemoprophylaxis Vaccination for the MAIN study, Arms 2 and 3  
Note: Subjects will be vaccinated by NF54 Sanaria® PfSPZ Challenge DVI  
 
Study Day -7 (Day of Enrollment, Baseline labs #1; 7 days prior to Sanaria® PfSPZ Challenge) 
1. Verify that Informed Consent and up-to-date version of the Informed Consent was obtained. 
2. Ensure that all inclusion criteria are met and exclusion criteria are not met and Eligibility 
Checklist completed and signed. 
3. Ensure that CBC with differential, ALT, and creatinine measurements from 
screening tests are within protocol-defined limits (see Exclusion Criteria).  
4. Ensure that Hepatitis B, Hepatitis C, and HIV testing are negative from screening visit. 
5. Perform basic history and focused physical examination, emphasizing examination of 
any acute complaints. 
6. During the physical examination, study staff will educate the subject regarding signs and 
symptoms of potential AEs, clinical malaria, and indications for use of antipyretics if fever, 


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headache, or malaise occurs. 
7. Record vital signs (blood pressure, temperature, heart rate). 
8. For females, ensure continued agreement and compliance with pregnancy prevention 
9. Obtain approximately 57 mL of blood for research labs.  
10. Record AEs and concomitant medications, if applicable 
11. Enrollment study number will be the same as screening number.  
 
Note: If screening tests were done outside the 56 days window of enrollment, they will be 
repeated during this visit 
 
Study Day -2 (Baseline labs #2; chloroquine load; 2 days prior to Sanaria® PfSPZ Challenge) 
1. Perform basic history and focused physical examination, emphasizing examination of 
any acute complaints.  
2. Study staff will educate the subject regarding signs and symptoms of potential AEs and 
indications for contacting the site. 
3. Obtain approximately 51 mL of blood for baseline labs including: retrospective LMIV 
qPCR, safety labs, and research labs. 
a. Obtain additional 1 mL of blood for drug assays (Arm 2 only) 
4. For females, obtain a urine or serum sample for E-hCG (pregnancy) testing.  Ensure that 
the test is negative before administering chloroquine; a positive test will exclude the 
subject from the trial. Pregnancy prevention counseling will be performed. 
5. Record vital signs (blood pressure, temperature, heart rate). 
6. Provide chloroquine loading dose (approximately 1 gram) by directly observed therapy 
(Arm 3 only). 
a. Observe subject for at least 30 minutes following chloroquine loading 
dose to evaluate for immediate adverse reactions. (Only required after 
the first dose) 
7. 
Record AEs and concomitant medications, if applicable 
 
Study Day 1 (Sanaria® PfSPZ Challenge #1) 
1. Verify that no contraindications for Sanaria® PfSPZ Challenge or deferral of Sanaria® PfSPZ 
Challenge have been met. 
2. Confirm emergency contact information and complete medical emergency release form 
3. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
4. During the physical examination, study staff will educate the subject regarding signs and 
symptoms of potential adverse events (AEs), clinical malaria, and indications for use of 
antipyretics if fever, headache, or malaise occurs. 
5. For females, obtain a urine or serum sample for E-hCG (pregnancy) testing.  Ensure 
that the test is negative before Sanaria® PfSPZ Challenge; a positive test will exclude 
the subject from the trial. 
6. For females, ensure continued agreement and compliance with pregnancy prevention before 
Sanaria® PfSPZ Challenge. 
7. Obtain approximately 3ml of blood and 3 ml of urine for drug assays (Arm 3 only) 
8. Record vital signs (blood pressure, temperature, and heart rate). 
9. Review Inclusion/Exclusion criteria and confirm final eligibility. 
10. Administer Sanaria® PfSPZ Challenge via DVI. 


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a. Observe subject for at least 30 minutes after to evaluate for immediate 
adverse reactions. 
11. Record AEs and concomitant medications, if applicable.  
12. Review instructions for contacting the site to report adverse experiences and/or inquire 
about study procedures. 
13. Dispense diary, thermometer and ruler to subject and demonstrate use and assess 
understanding of use.  
Study Day 2 (Phone Follow-Up; 1 Day after Sanaria® PfSPZ Challenge #1)  
1. Perform basic history and AE review (including DVI site), emphasizing review of any acute 
complaints. 
2. Complete diary review. 
3. Record AEs and concomitant medications, if applicable.  
4. Provide pregnancy prevention counseling for female subjects. 
Study Day 3 (pyrimethamine dose; 2 Days after Sanaria® PfSPZ Challenge #1) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review. 
4. Record AEs and concomitant medications, if applicable. 
5. Provide pregnancy prevention counseling for female subjects. 
6. Provide pyrimethamine dose by directly observed therapy  
Note for Arm 2 only:   
2 tablets if 50mg dose  
7. Observe subject for at least 30 minutes following pyrimethamine to evaluate for immediate 
adverse reactions (Only required after the first dose) 
8. Record AEs and concomitant medications, if applicable. 
 
Study Day 4 (pyrimethamine dose; 3 Days after Sanaria® PfSPZ Challenge #1) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review. 
4. Record AEs and concomitant medications, if applicable. 
5. Provide pregnancy prevention counseling for female subjects. 
6. Obtain approximately 45 mL of blood for safety labs and research labs. 
7. Provide pyrimethamine dose by directly observed therapy  
Note for Arm 2 only:   
2 tablets if 50mg dose  
Study Day 6 (chloroquine dose; 5 Days after Sanaria® PfSPZ Challenge #1) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review. 
4. Record AEs and concomitant medications, if applicable. 
5. Provide pregnancy prevention counseling for female subjects. 
6. Provide chloroquine dose (approximately 500 mg) by directly observed therapy (Arm 3 


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only). Note: if a subject has problems related to tolerability of chloroquine, a lower 
weight-based dose (as defined in the PI) would be clinically appropriate for maintenance 
dosing, this adjustment may be made on a case by case basis.  
 
Study Day 7 (Clinic Follow-Up; 6 Days after Sanaria® PfSPZ Challenge #1) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review. 
4. Complete malaria symptom review. 
5. Record AEs and concomitant medications, if applicable. 
6. Provide pregnancy prevention counseling for female subjects. 
7. Obtain approximately 3 mL of blood for malaria infection assays (see below).  
x Arm 2: real time NIH and retrospective LMIV qPCR. Blood smear if indicated 
x Arm 3: retrospective LMIV qPCR and blood smear. Real time NIH PCR if 
indicated. 
x Obtain additional approximately 1 mL of blood for drug assays. (Arm 2 only) 
8. Confirm subject and emergency contact information to follow up malaria diagnostic result. 
 
Study Day 8 (Clinic Follow-Up; 7 Days after Sanaria® PfSPZ Challenge #1) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review. 
4. Complete malaria symptom review. 
5. Record AEs and concomitant medications, if applicable. 
6. Provide pregnancy prevention counseling for female subjects. 
7. Obtain approximately 59 mL of blood for safety labs, malaria infection assays (see below) 
and research labs. 
x Arm 2: real time NIH and retrospective LMIV qPCR. Blood smear if indicated. 
x Arm 3: retrospective LMIV qPCR and blood smear. Real time NIH PCR if 
indicated. 
8. Confirm subject contact information to follow up malaria diagnostic result. 
9. Arm 3 ONLY:  
a. Administer at least Ibuprofen 400 mg (or another NSAID if indicated) from the 
outpatient prescription 
b. Provide the remainder of outpatient prescription of NSAIDs for the subject to take 
home 
c. Instruct subject to bring NSAIDs prescription to all study Day 8 and 9 clinic visits 
 
 
Study Day 8 PM; [Arm 3 ONLY] (PM Clinic Follow-Up; 7 Days after Sanaria® PfSPZ 
Challenge #1) 
1. Perform basic history and focused physical examination, emphasizing examination of 
any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete malaria symptom review. 


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4. Record AEs and concomitant medications, if applicable. 
5. Provide pregnancy prevention counseling for female subjects. 
6. Confirm subject contact information to follow up malaria diagnostic result. 
7. Provide at least Ibuprofen 400mg dose (or another NSAID is indicated) 
 
 
Study Day 9 (Clinic Follow-Up; 8 Days after Sanaria® PfSPZ Challenge #1) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review. 
4. Complete malaria symptom review. 
5. Record AEs and concomitant medications, if applicable. 
6. Provide pregnancy prevention counseling for female subjects. 
7. Obtain approximately 3 mL of blood for malaria infection assays (see below). 
x Arm 2: real time NIH and retrospective LMIV qPCR. Blood smear if indicated. 
x Arm 3: retrospective LMIV qPCR and blood smear. Real time NIH PCR if 
indicated.  
8. Confirm subject contact information to follow up malaria diagnostic result. 
9. Arm 3 ONLY: Administer at least Ibuprofen 400 mg dose (or another NSAID if indicated) 
from the outpatient prescription 
 
Study Day 9 PM; [Arm 3 ONLY] (PM Clinic Follow-Up; 8 Days after Sanaria® PfSPZ 
Challenge #1) 
1. Perform basic history and focused physical examination, emphasizing examination of 
any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete malaria symptom review. 
4. Record AEs and concomitant medications, if applicable. 
5. Provide pregnancy prevention counseling for female subjects. 
6. Confirm subject contact information to follow up malaria diagnostic result. 
7. Provide at least Ibuprofen 400mg dose (or another NSAID is indicated) 
 
 
Study Day 10 (Clinic Follow-Up; 9 Days after Sanaria® PfSPZ Challenge #1) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review. 
4. Complete malaria symptom review. 
5. Record AEs and concomitant medications, if applicable. 
6. Provide pregnancy prevention counseling for female subjects. 
7. Obtain approximately 3 mL of blood for malaria infection assays (see below). 
x Arm 2: real time NIH and retrospective LMIV qPCR. Blood smear if indicated. 
x Arm 3: retrospective LMIV qPCR and blood smear. Real time NIH PCR if 
indicated.  


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8. Confirm subject contact information to follow up malaria diagnostic result. 
 
 
 
Study Day 11 (Clinic Follow-Up; 10 Days after Sanaria® PfSPZ Challenge #1) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review, enter information for today’s visit in diary, and collect diary 
(window for diary collection +2 days) 
4. Complete malaria symptom review. 
5. Record AEs and concomitant medications, if applicable.  
6. Obtain approximately 3 mL of blood for malaria infection assays (see below). 
x Arm 2: real time NIH and retrospective LMIV qPCR. Blood smear if indicated. 
x Arm 3: retrospective LMIV qPCR and blood smear. Real time NIH PCR if 
indicated. 
 
x Obtain additional approximately 1 mL of blood for drug assays. (Arm 2 only) 
7. Confirm subject contact information to follow up malaria diagnostic result. 
 
Study Day 13 (chloroquine dose; 12 Days after Sanaria® PfSPZ Challenge #1) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete malaria symptom review. 
4. Record AEs and concomitant medications, if applicable. 
5. Provide pregnancy prevention counseling for female subjects.  
6. Provide chloroquine dose (approximately 500 mg by directly observed therapy (Arm 3 
only). Note: if a subject has issues related to tolerability of chloroquine and a lower weight-
based dose (as defined in the PI) would be clinically appropriate for maintenance dosing, 
this adjustment may be made on a case by case basis. 
7. Confirm subject contact information to follow up malaria diagnostic result. 
 
Study Day 15 (14 Days after Sanaria® PfSPZ Challenge #1) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete malaria symptom review. 
4. Record AEs and concomitant medications, if applicable. 
5. Provide pregnancy prevention counseling for female subjects 
6. Obtain approximately 53 mL of blood for research labs. 
7. Confirm subject contact information to follow up malaria diagnostic result. 
Study Day 20 (chloroquine dose; 19 Days after Sanaria® PfSPZ Challenge #1) 
1. Perform basic history and focused physical examination, emphasizing examination of any 


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acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Provide pregnancy prevention counseling for female subjects. 
5. Provide chloroquine dose (approximately 500mg) by directly observed therapy (Arm 3 
only). Note: if a subject has issues related to tolerability of chloroquine and a lower weight-
based dose (as defined in the PI) would be clinically appropriate for maintenance dosing, 
this adjustment may be made on a case by case basis. 
 
Study Day 27 (chloroquine dose; 26 Days after Sanaria® PfSPZ Challenge #1) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Provide pregnancy prevention counseling for female subjects. 
5. Obtain approximately 24 mL of blood for safety labs, retrospective LMIV qPCR, and 
research labs. 
a. Obtain additional approximately 1 mL of blood for drug assays (Arm 2 only). 
6. Provide chloroquine dose (approximately 500 mg) by directly observed therapy (Arm 3 
only). Note: if a subject has issues related to tolerability of chloroquine and a lower weight-
based dose (as defined in the PI) would be clinically appropriate for maintenance dosing, 
this adjustment may be made on a case by case basis. 
Study Day 29(Sanaria® PfSPZ Challenge #2) 
1. Verify that no contraindications for Sanaria® PfSPZ Challenge or deferral of Sanaria® PfSPZ 
Challenge have been met. 
2. Confirm emergency contact information.  
3. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
4. During the physical examination, study staff will educate the subject regarding signs and 
symptoms of potential adverse events (AEs), clinical malaria, and indications for use of 
antipyretics if fever, headache, or malaise occurs. 
5. For females, obtain a urine or serum sample for E-hCG (pregnancy) testing.  Ensure 
that the test is negative before Sanaria® PfSPZ Challenge; a positive test will exclude 
the subject from the trial. 
6. For females, ensure continued agreement and compliance with pregnancy prevention before 
Sanaria® PfSPZ Challenge. 
7. Record vital signs (blood pressure, temperature, and heart rate). 
8. Review Inclusion/Exclusion criteria and confirm eligibility. 
9. Administer Sanaria® PfSPZ Challenge via DVI. 
a. Observe subject for at least 30 minutes after administration to evaluate for 
immediate adverse reactions. 
10. Record AEs and concomitant medications, if applicable. 
11. Dispense diary, thermometer, and ruler to subject and demonstrate use and assess 
understanding of use. 
12. Review instructions for contacting the site to report adverse experiences and/or inquire 
about study procedures. 


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Study Day 31 (pyrimethamine dose; 2 Days after Sanaria® PfSPZ Challenge #2) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review. 
4. Record AEs and concomitant medications, if applicable. 
5. Provide pregnancy prevention counseling for female subjects. 
6. Provide pyrimethamine dose by directly observed therapy  
Note for Arm 2 only:   
2 tablets if 50mg dose  
 
 
Study Day 32 (pyrimethamine dose; 3 Days after Sanaria® PfSPZ Challenge #2) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review. 
4. Record AEs and concomitant medications, if applicable. 
5. Provide pregnancy prevention counseling for female subjects. 
6. Obtain approximately 55 mL of blood for safety labs and research labs. 
7. Provide pyrimethamine dose by directly observed therapy  
Note for Arm 2 only:   
2 tablets if 50mg dose  
 
Study Day 34 (chloroquine dose, 5 Days after Sanaria® PfSPZ Challenge #2) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review. 
4. Record AEs and concomitant medications, if applicable. 
5. Provide pregnancy prevention counseling for female subjects. 
6. Provide chloroquine dose (approximately 500 mg) by directly observed therapy (Arm 3 
only). Note: if a subject has issues related to tolerability of chloroquine and a lower 
weight-based dose (as defined in the PI) would be clinically appropriate for maintenance 
dosing, this adjustment may be made on a case by case basis. 
 
Study Day 35 (Clinic Follow-Up; 6 Days after Sanaria® PfSPZ Challenge #2) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review. 
4. Complete malaria symptom review. 
5. Record AEs and concomitant medications, if applicable. 
6. Obtain approximately 3 mL of blood for malaria infection assays (see below) and research 
labs: 


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x Arm 2: retrospective LMIV qPCR and blood smear. Real time NIH PCR if 
indicated. 
x Arm 3: retrospective LMIV qPCR and blood smear. Real time NIH PCR if 
indicated. 
x Obtain additional approximately 1 mL of blood for drug assays (Arm 2 only). 
7. Confirm subject contact information to follow up malaria diagnostic result..   
 
Study Day 36 (Clinic Follow-Up; 7 Days after Sanaria® PfSPZ Challenge #2) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review. 
4. Complete malaria symptom review 
5. Record AEs and concomitant medications, if applicable. 
6. Provide pregnancy prevention counseling for female subjects 
7. Obtain approximately 59 mL of blood for safety labs, malaria infection assays (see below), 
and research labs. 
x Arm 2: real time NIH and retrospective LMIV qPCR.Blood smear if indicated. 
x Arm 3: retrospective LMIV qPCR and blood smear. Real time NIH PCR if 
indicated. 
8. Confirm subject contact information to follow up malaria diagnostic result. 
 
Study Day 36 PM [Arm 3 ONLY] (PM Phone Follow-Up; 7 Days after Sanaria® PfSPZ 
Challenge #2)  
1. Perform basic history and AE review (including DVI site), emphasizing review of any acute 
complaints.  
2. Complete malaria symptom review. 
3. Record AEs and concomitant medications, if applicable. 
4. Provide pregnancy prevention counseling for female subjects. 
 
 
Study Day 37 (Clinic Follow-Up; 8 Days after Sanaria® PfSPZ Challenge #2) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate) 
3. Complete diary review. 
4. Complete malaria symptom review 
5. Record AEs and concomitant medications, if applicable. 
6. Provide pregnancy prevention counseling for female subjects 
7. Obtain approximately 3 mL of blood for malaria infection assays (see below) and research 
labs: 
x Arm 2: real time NIH and retrospective LMIV qPCR. Blood smear if indicated. 
x Arm 3: retrospective LMIV qPCR and blood smear. Real time NIH PCR if 
indicated. 
8. Confirm subject contact information to follow up malaria diagnostic result. 


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Study Day 37 PM [Arm 3 ONLY] (PM Phone Follow-Up; 8 Days after Sanaria® PfSPZ 
Challenge #2)  
1. Perform basic history and AE review (including DVI site), emphasizing review of any acute 
complaints.  
2. Complete malaria symptom review. 
3. Record AEs and concomitant medications, if applicable. 
4. Provide pregnancy prevention counseling for female subjects. 
 
 
Study Day 38 (Clinic Follow-Up; 9 Days after Sanaria® PfSPZ Challenge #2) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, heart rate, and respiratory rate). 
3. Complete diary review. 
4. Complete malaria symptom review. 
5. Record AEs and concomitant medications, if applicable. 
6. Provide pregnancy prevention counseling for female subjects. 
7. Obtain approximately 3 mL of blood for malaria infection assays (see below) and research 
labs: 
x Arm 2: real time NIH and retrospective LMIV qPCR. Blood smear if indicated. 
x Arm 3: retrospective LMIV qPCR and blood smear. Real time NIH PCR if 
indicated. 
8. Confirm subject contact information to follow up malaria diagnostic result. 
 
Study Day 39 (Clinic Follow-Up 10 Days after Sanaria® PfSPZ Challenge #2) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review, enter information for today’s visit in diary, and collect diary 
(window for diary collection “+2” days) . 
4. Complete malaria symptom review. 
5. Record AEs and concomitant medications, if applicable. 
6. Provide pregnancy prevention counseling for female subjects. 
7. Obtain approximately 3 mL of blood for malaria infection assays(see below) and research 
labs: 
x Arm 2: retrospective LMIV qPCR and blood smear. Real time NIH PCR if 
indicated. 
x Arm 3: retrospective LMIV qPCR and blood smear. Real time NIH PCR if 
indicated. 
x Obtain additional approximately 1 mL of blood for drug assays (Arm 2 only). 
8. Confirm subject contact information to follow up malaria diagnostic result. 
 
Study Day 41 (chloroquine dose; 12 Days after Sanaria® PfSPZ Challenge #2) 


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1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete malaria symptom review. 
4. Record AEs and concomitant medications, if applicable. 
5. Provide pregnancy prevention counseling for female subjects. 
6. Provide chloroquine dose (approximately 500 mg) by directly observed therapy (Arm 3 
only). Note: if a subject has issues related to tolerability of chloroquine and a lower weight-
based dose (as defined in the PI) would be clinically appropriate for maintenance dosing, 
this adjustment may be made on a case by case basis. 
 
Study Day 43 (Clinic Follow-Up; 14 Days after Sanaria® PfSPZ Challenge #2) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3.  Complete malaria symptom review. 
4.  Record AEs and concomitant medications, if applicable. 
5. Provide pregnancy prevention counseling for female subjects. 
6. Obtain approximately 52 mL of blood for research labs. 
 
Study Day 48 (chloroquine dose; 19 Days after Sanaria® PfSPZ Challenge #2) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Provide pregnancy prevention counseling for female subjects. 
5. Provide chloroquine dose (approximately 500 mg) by directly observed therapy (Arm 3 
only). Note: if a subject has issues related to tolerability of chloroquine and a lower weight-
based dose (as defined in the PI) would be clinically appropriate for maintenance dosing, 
this adjustment may be made on a case by case basis. 
 
Study Day 55 (chloroquine dose; 26 Days after Sanaria® PfSPZ Challenge #2)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Provide pregnancy prevention counseling for female subjects. 
4. Obtain approximately 24 mL of blood for safety labs, retrospective LMIV qPCR, and 
research labs. 
a. Obtain additional approximately 1 ml of blood for drug assays. (Arm 2 only) 
5. Provide chloroquine dose (approximately 500 mg) by directly observed therapy (Arm 3 
only). Note: if a subject has issues related to tolerability of chloroquine and a lower weight-
based dose (as defined in the PI) would be clinically appropriate for maintenance dosing, 
this adjustment may be made on a case by case basis. 
 
Study Day 57 (Sanaria® PfSPZ Challenge #3) 
1. Verify that no contraindications for Sanaria® PfSPZ Challenge or deferral of Sanaria® 
PfSPZ Challenge have been met. 


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2. Confirm emergency contact information.  
3. Perform basic history and focused physical examination, emphasizing examination of 
any acute complaints. 
4. During the physical examination, study staff will educate the subject regarding signs and 
symptoms of potential AEs, clinical malaria, and indications for use of antipyretics if 
fever, headache, or malaise occurs. 
5. For females, obtain a urine or serum sample for E-hCG (pregnancy) testing.  
Ensure that the test is negative before Sanaria® PfSPZ Challenge; a positive test 
will exclude the subject from the trial. 
6. For females, ensure continued agreement and compliance with pregnancy prevention 
before Sanaria® PfSPZ Challenge. 
7. Record vital signs (blood pressure, temperature, 
8.  and heart rate). 
9. Review Inclusion/Exclusion criteria and confirm final eligibility. 
10. Administer Sanaria® PfSPZ Challenge via DVI. 
a. Observe subject for at least 30 minutes after administration to evaluate for 
immediate adverse reactions. 
11. Record AEs and concomitant medications, if applicable. 
12. Dispense diary, thermometer and ruler to subject and demonstrate use and assess 
understanding of use. 
13. Review instructions for contacting the site to report adverse experiences and/or inquire 
about study procedures. 
 
Study Day 59 (pyrimethamine dose; 2 Days after Sanaria® PfSPZ Challenge #3) 
1. Perform basic history and focused physical examination, emphasizing examination of 
any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review. 
4. Record AEs and concomitant medications, if applicable. 
5. Provide pregnancy prevention counseling for female subjects. 
6. Provide pyrimethamine dose by directly observed therapy  
Note for Arm 2 only:   
2 tablets if 50mg dose  
 
Study Day 60 (pyrimethamine dose; 3 Days after Sanaria® PfSPZ Challenge #3) 
1.  Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review 
4. Record AEs and concomitant medications, if applicable. 
5. Provide pregnancy prevention counseling for female subjects 
6. Obtain approximately 55 mL of blood for safety labs and research labs.  
7. Provide pyrimethamine dose by directly observed therapy  
Note for Arm 2 only:   
2 tablets if 50mg dose  
 
Study 62 (chloroquine dose; 5 Days after Sanaria® PfSPZ Challenge #3) 


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1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review. 
4. Record AEs and concomitant medications, if applicable. 
5. Provide pregnancy prevention counseling for female subjects. 
6. Provide chloroquine dose (500 mg) by directly observed therapy (Arm 3 only). Note: if a 
subject has issues related to tolerability of chloroquine and a lower weight-based dose (as 
defined in the PI) would be clinically appropriate for maintenance dosing, this adjustment 
may be made on a case by case basis. 
 
Study Day 63 (Clinic Follow-Up; 6 Days after Sanaria® PfSPZ Challenge #3) 
1. Perform basic history and focused physical examination, emphasizing examination of 
any acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review. 
4. Complete malaria symptom review. 
5. Record AEs and concomitant medications, if applicable. 
6. Provide pregnancy prevention counseling for female subjects. 
7. Obtain approximately 3 mL of blood for malaria infection assays (see below) and 
research labs. 
x Arm 2: retrospective LMIV qPCR and blood smear. Real time NIH PCR if 
indicated. 
x Arm 3: retrospective LMIV qPCR and blood smear. Real time NIH PCR if 
indicated. 
Obtain additional approximately 1 mL of blood for drug assays (Arm 2 only). 
8. Confirm subject contact information to follow up malaria diagnostic result. 
 
Study Day 64 (Clinic Follow-Up; 7 Days after Sanaria® PfSPZ Challenge #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate).  
3. Complete diary review. 
4. Complete malaria symptom review. 
5. Record AEs and concomitant medications, if applicable. 
6. Provide pregnancy prevention counseling for female subjects. 
7. Obtain approximately 59 mL of blood for malaria infection assays (see below), safety and  
research labs. 
x Arm 2: real time NIH and retrospective LMIV qPCR.  .Blood smear if indicated. 
x Arm 3: retrospective LMIV qPCR and blood smear. Real time NIH PCR if 
indicated. 
8. Confirm subject contact information to follow up malaria diagnostic result. 
 
Study Day 64 PM [Arm 3 ONLY] (PM Phone Follow-Up; 7 Days after Sanaria® PfSPZ 
Challenge #3)  
1. Perform basic history and AE review (including DVI site), emphasizing review of any acute 


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complaints.  
2. Complete malaria symptom review. 
3. Record AEs and concomitant medications, if applicable. 
4. Provide pregnancy prevention counseling for female subjects 
 
Study Day 65 (Clinic Follow-Up; 8 Days after Sanaria® PfSPZ Challenge #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review. 
4. Complete malaria symptom review. 
5. Record AEs and concomitant medications, if applicable. 
6. Provide pregnancy prevention counseling for female subjects. 
7. Obtain approximately 3 mL of blood for malaria infection assays (see below) and research 
labs. 
x Arm 2: real time NIH and retrospective LMIV qPCR. .Blood smear if indicated. 
x Arm 3: retrospective LMIV qPCR and blood smear. Real time NIH PCR if 
indicated. 
8. Confirm subject contact information to follow up malaria diagnostic result. 
 
Study Day 65 PM [Arm 3 ONLY] (PM Phone Follow-Up; 8 Days after Sanaria® PfSPZ 
Challenge #3)  
1. Perform basic history and AE review (including DVI site), emphasizing review of any acute 
complaints.  
2. Complete malaria symptom review. 
3. Record AEs and concomitant medications, if applicable. 
4. Provide pregnancy prevention counseling for female subjects 
 
Study Day 66 (Clinic Follow-Up; 9 Days after Sanaria® PfSPZ Challenge #3 via DVI) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review. 
4. Complete malaria symptom review. 
5. Record AEs and concomitant medications, if applicable. 
6. Provide pregnancy prevention counseling for female subjects. 
7. Obtain approximately 3 mL of blood for malaria infection assays (see below) and research 
labs 
x Arm 2: real time NIH and retrospective LMIV qPCR. .Blood smear if indicated. 
x Arm 3: retrospective LMIV qPCR and blood smear. Real time NIH PCR if 
indicated. 
8. Confirm subject contact information to follow up malaria diagnostic result. 
 
Study Day 67 (Clinic Follow-Up; 10 Days after Sanaria® PfSPZ Challenge #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 


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3. Complete diary review, enter information for today’s visit in diary, and collect diary (window 
for diary collection “+2” days). 
4. Complete malaria symptom review. 
5. Record AEs and concomitant medications, if applicable. 
6. Provide pregnancy prevention counseling for female subjects. 
7.  Obtain approximately 3 mL of blood for malaria infection assays (see below) and research 
labs  
 
x Arm 2: retrospective LMIV qPCR and blood smear. Real time NIH PCR if 
indicated. 
x Arm 3: retrospective LMIV qPCR and blood smear. Real time NIH PCR if 
indicated. 
x Obtain additional approximately 1 mL of blood for drug assays (Arm 2 only). 
8. Confirm subject contact information to follow up malaria diagnostic result. 
 
Study Day 71 (Clinic Follow-Up; 14 Days after Sanaria® PfSPZ Challenge #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete malaria symptom review. 
4. Record AEs and concomitant medications, if applicable. 
5. Provide pregnancy prevention counseling for female subjects. 
6. Obtain approximately 52 mL of blood for research labs. 
 
Study Day 84 (Clinic Follow-Up; 27 Days after Sanaria® PfSPZ Challenge #3) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Provide pregnancy prevention counseling for female subjects. 
4. Record AEs and concomitant medications, if applicable. 
5. Obtain approximately 15 mL of blood for research labs. 
Study Day 112 (Phone Follow Up; 55 Days after Sanaria® PfSPZ Challenge #3) 
1. Perform basic history and AE review, emphasizing review of any acute or new chronic 
complaints.  
2. Record AEs and concomitant medications, if applicable. Provide pregnancy prevention 
counseling for female subjects. 
3. Provide pregnancy prevention counseling for female subjects. 
 
Study Day 141 (Pre Challenge Visit; 84 Days after Sanaria® PfSPZ Challenge #3)  
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Provide pregnancy prevention counseling for female subjects. 


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5. Obtain approximately 72 mL of blood for safety labs, malaria infection assays- retrospective 
LMIV qPCR and research labs.  
a. Obtain additional approximately 1 ml of blood for drug assays. (Arm 2 only) 
Note: This blood draw can be split into two separate draws within the visit window and prior to 
Study Day 147 (Day of CHMI). 


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Controlled Human Malaria Infection (CHMI) Arms 2a, 2b, 3, 4a and 4b 
 
Study Day -7--Arm 4a and 4b ONLY (Day of Enrollment, Baseline #1 labs; 7 days prior to 
Sanaria® PfSPZ Challenge) 
 
1. Verify that Informed Consent and up-to-date version of the Informed Consent was obtained. 
2. Ensure that all inclusion criteria are met and exclusion criteria are not met and Eligibility 
Checklist completed and signed. 
3. Ensure that CBC with differential, ALT, and creatinine measurements from 
screening tests are within protocol-defined limits (see Exclusion Criteria).  
4. Ensure that Hepatitis B, Hepatitis C, and HIV testing are negative from screening visit. 
5. Perform basic history and focused physical examination, emphasizing examination of 
any acute complaints. 
6. During the physical examination, study staff will educate the subject regarding signs and 
symptoms of potential AEs, clinical malaria, and indications for use of antipyretics if fever, 
headache, or malaise occurs. 
7. Record vital signs (blood pressure, temperature, heart rate). 
8. For females, ensure continued agreement and compliance with pregnancy prevention 
9. Obtain approximately 47 mL of blood for research labs.  
10. Record AEs and concomitant medications, if applicable 
11. Enrollment study number will be the same as screening number.  
 
Study Day -2 Arm 4a and 4b ONLY (Baseline labs #2; 2 days prior to Sanaria® PfSPZ 
Challenge) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints.  
2. Study staff will educate the subject regarding signs and symptoms of potential AEs and 
indications for contacting the site. 
3. Obtain approximately 51 mL of blood for baseline labs including: safety labs, 
retrospective LMIV qPCR, and research labs. 
4. For females, ensure continued agreement and compliance with pregnancy prevention 
5. Record vital signs (blood pressure, temperature, heart rate). 
6. Record AEs and concomitant medications, if applicable. 
 
Study Day 147 Arms 2a, 2b &3, Study Day 1 Arm 4a and 4b  (CHMI with Sanaria® PfSPZ 
Challenge)  
 
NOTE: Arm 2a and 4a will receive homologous CHMI with NF54 Sanaria® PfSPZ Challenge 
 Arm 2b, 3 and 4b will receive heterologous CHMI with 7G8 Sanaria® PfSPZ Challenge 
 
1. Verify that no contraindications for Sanaria® PfSPZ Challenge or deferral of Sanaria® PfSPZ 
Challenge have been met. 
2. Confirm emergency contact information and complete Medical Emergency Release Form 
(Arm 4a and 4b only). 


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3. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
4. During the physical examination, study staff will educate the subject regarding signs and 
symptoms of potential AEs and indications for use of antipyretics if fever, headache, or 
malaise occurs. 
5. For females, obtain a urine or serum sample for E-hCG (pregnancy) testing.  Ensure 
that the test is negative before vaccinating; a positive test will exclude the subject 
from the trial. 
6. For females, ensure continued agreement and compliance with pregnancy prevention before 
vaccinating. 
7. Record vital signs (blood pressure, temperature, and heart rate). 
8. Review Inclusion/Exclusion criteria and confirm final eligibility.  
9. Administer Sanaria® PfSPZ Challenge via DVI. 
a. Observe subject for at least 30 minutes after administration to evaluate for 
immediate adverse reactions. 
7. Record AEs and concomitant medications, if applicable. 
8. Dispense diary, thermometer and ruler to subject and demonstrate use and assess 
understanding of use. 
9. Review instructions for contacting the site to report adverse experiences and/or inquire 
about study procedures. 
10. Provide subject with Do’s and Don’t’s handout. 
 
Study Day 148 Arms 2a, 2b  &3, Study Day 2 Arm 4a and 4b  (Phone visit; 1 Day after Sanaria® 
PfSPZ Challenge) 
1. Perform basic history and AE review (including DVI site), emphasizing review of any acute 
complaints.  
2. Complete diary review. 
3. Record AEs and concomitant medications, if applicable. 
4. Pregnancy prevention counseling for females. 
 
Study Day 150 Arms 2a, 2b  &3, Study Day 4 Arm 4a and 4b (3 Days after Sanaria® PfSPZ 
Challenge) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review. 
4. Record AEs and concomitant medications, if applicable. 
5. Provide pregnancy prevention counseling for female subjects. 
6. Obtain approximately 55 mL of blood for safety labs and research labs. 
 
Study Day 152 Arms 2a, 2b  &3, Study Day 6 Arm 4a and 4b  (Phone visit; 5 Days after 
Sanaria® PfSPZ Challenge) 
1. Perform basic history and AE review (including DVI site), emphasizing review of any acute 
complaints.  
2. Complete diary review. 
3. Record AEs and concomitant medications, if applicable. 
4. Pregnancy prevention counseling for females. 


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Study Day 153 Arms 2a, 2b  &3, Study Day 7 Arm 4a and 4b  (Clinic Follow-Up; 6 Days after 
Sanaria® PfSPZ Challenge) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review. 
4. Complete malaria symptom review. 
5. Record AEs and concomitant medications, if applicable. 
6. Provide pregnancy prevention counseling for female subjects. 
7. Obtain approximately 3 mL of blood for malaria infection assays (see below)  
a. Real time NIH and retrospective LMIV qPCR. Blood smear if indicated. 
8. Confirm subject’s emergency contact information to follow up real time NIH qPCR and 
blood smear results. 
 
 
Study Day 154 Arms 2a, 2b  &3, Study Day 8 Arm 4a and 4b (Clinic Follow-Up; 7 Days after 
Sanaria® PfSPZ Challenge) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review. 
4. Complete malaria symptom review. 
5. Record AEs and concomitant medications, if applicable. 
6. Provide pregnancy prevention counseling for female subjects. 
7. Obtain approximately 58 mL of blood for safety labs, malaria infection assays (see below) 
and research labs 
a. Real time NIH and retrospective LMIV qPCR. Blood smear if indicated. 
. 
8. Confirm subject’s emergency contact information to follow up real time NIH qPCR and 
blood smear results. 
9.  If a subject is diagnosed with malaria infection,  
a. Administer Malarone® dose (4 tablets) by directly observed therapy. 
b. Observe subject for at least 30 minutes following first Malarone® dose to 
evaluate for immediate adverse reactions. 
c. Complete Malarone® dose treatment for 3 consecutive days. 
d. Following 3rd (final) Malarone® dose, subject’s next visit will be Study Day 
176 for Arms 2 &3 and Study Day 30 for Arm 4 to complete final visit and 
lab draws. 
 
Study Day 155-160 Arms 2a, 2b  &3, Study Day 9-14 Arm 4a and 4b  (Clinic Follow-Up; 8-13 
Days after Sanaria® PfSPZ Challenge) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete diary review (For Study Day 157 – Arms 2a, 2b,&3; Study Day 11-Arms 4a &4b 
enter information for the day’s visit in the diary and collect diary (diary collection has a 


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window of +2 days). 
4. Complete malaria symptom review. 
5. Record AEs and concomitant medications, if applicable.  
6. Provide pregnancy prevention counseling for female subjects. 
7. Obtain approximately 3 mL of blood for malaria infection assays (see below)   
a. Real time NIH and retrospective LMIV qPCR. Blood smear if indicated. 
8. Confirm subject’s emergency contact information to follow up real time NIH qPCR and 
blood smear results. 
9. If a subject is diagnosed with malaria infection,  
a. Administer Malarone® dose (4 tablets) by directly observed therapy. 
b. Observe subject for at least 30 minutes following first Malarone® dose to 
evaluate for immediate adverse reactions. 
c. Complete Malarone® dose treatment for 3 consecutive days. 
d. Following final Malarone® dose, subject’s next visit will be Study Day 176 
for Arms 2 &3 and Study Day 30 for Arm 4 to complete final visit and lab 
draws. 
 
 
Study Day 161 Arms 2a, 2b  &3, Study Day 15 Arm 4a and 4b  (Clinic Follow-Up; 14 Days 
after Sanaria® PfSPZ Challenge) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete malaria symptom review. 
4. Record AEs and concomitant medications, if applicable. 
5. Provide pregnancy prevention counseling for female subjects. 
6. Obtain approximately 62 mL of blood for safety labs, malaria infection assays and research 
labs. 
a.  Real time NIH and retrospective LMIV qPCR. Blood smear if indicated. 
7. Confirm subject’s emergency contact information to follow up real time NIH qPCR and 
blood smear results. 
8. If a subject is diagnosed with malaria infection,  
a) Administer Malarone® dose (4 tablets) by directly observed therapy. 
b) Observe subject for at least 30 minutes following first Malarone® dose to evaluate 
for immediate adverse reactions. 
c) Complete Malarone® dose treatment for 3 consecutive days. 
d) Following final Malarone® dose, subject’s next visit will be Study Day 176 for 
Arms 2 &3 and Study Day 30 for Arm 4 to complete final visit and lab draws. 
 
Study Day 162-168, 170, 172 Arms 2a, 2b  &3, Study Day 16-22, 24, 26 Arm 4a and 4b   
(Clinic Follow-Up; 15 – 21, 23, 25,Days after Sanaria® PfSPZ Challenge) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete malaria symptom review. 
4. Record AEs and concomitant medications, if applicable. 
5. Provide pregnancy prevention counseling for female subjects. 


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6. Obtain approximately 3 mL of blood malaria infection assays (see below). 
a. Real time NIH and retrospective LMIV qPCR. Blood smear if indicated. 
7. Confirm subject’s emergency contact information to follow up real time NIH qPCR and 
blood smear results. 
8. If a subject is diagnosed with malaria infection,  
a. Administer Malarone® dose (4 tablets) by directly observed therapy. 
b. Observe subject for at least 30 minutes following first Malarone® dose to 
evaluate for immediate adverse reactions. 
c. Complete Malarone® dose treatment for 3 consecutive days. 
d. Following final Malarone® dose, subject’s next visit will be Study Day 176 
for Arms 2 &3 and Study Day 30 for Arm 4 to complete final visit and lab 
draws. 
 
Study Day 174-175 Arms 2a, 2b  &3, Study Day 28-29 Arm 4a and 4b  (Treatment Visit; 27- 28 
Days after Sanaria® PfSPZ Challenge) 
1. Perform basic history and focused physical examination, emphasizing examination of any 
acute complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Complete malaria symptom review. 
4. Record AEs and concomitant medications, if applicable. 
5. Obtain approximately 3 mL of blood for malaria infection assays (see below). 
a. Real time NIH and retrospective LMIV qPCR. Blood smear if indicated. 
 
6. ALL subjects that have been real time NIH qPCR and blood smear NEGATIVE receive 
Malarone® dose (4 tablets) by directly observed therapy on Study Day 174 and Day 175 for 
Arm 2 &3 and Study Day 28 and Day 29 for Arm 4. 
7. Observe subject for at least 30 minutes following first Malarone® dose to evaluate for 
immediate adverse reactions only upon receipt of the first dose of Malarone®. 
 
Study Day 176 Arms 2a, 2b  &3, Study Day 30 Arm 4a and 4b  (Final Visit, Treatment; 29 Days 
after Sanaria® PfSPZ Challenge) – ALL subjects (Arms 2, 3 & 4) return for lab draw on this 
visit. 
1. Perform basic history and focused physical examination, emphasizing examination of any acute 
complaints. 
2. Record vital signs (blood pressure, temperature, and heart rate). 
3. Record AEs and concomitant medications, if applicable. 
4. Subjects that have been real time NIH qPCR and blood smear NEGATIVE receive 3rd dose of 
Malarone® dose (4 tablets) by directly observed therapy. In addition to above subjects, rest of 
Arm 2, 3, & 4 subjects return for LAB draw on this day. 
5. Obtain approximately 73 mL of blood for safety labs, malaria infection assays (see below) and 
research labs 
a. Real time NIH and retrospective LMIV qPCR. Blood smear if indicated. 
b. Obtain additional approximately 1 ml of blood for drug assays. (Arm 2 only) 
Note: This blood draw can be split into two separate draws occurring after receipt of 
first dose of Malarone®. 


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8.5 
Large Volume Blood Draw 
Blood samples will be drawn at multiple time points throughout the study as outlined in 
Appendix A.  Throughout the study, there are three large volume draws.  The first occurs before 
Sanaria® PfSPZ Challenge #1, the second before CHMI, and the last at the end of the study. In 
addition, there are multiple medium volume blood draws, 2 baseline samples and throughout 
vaccination and CHMI period that will be used for deep immunological studies at the Center for 
Human Immunology (CHI). These time points are identified as crucial to obtain the maximum 
amount of sample as the current trial presents an opportunity to determine the frequency, function 
and phenotype of the cellular and humoral immune system required for conferring protection 
against Plasmodium infection. We anticipate that the levels of antigen specific cells will be less 
than 1% of the total cells collected. Hence, the large blood draws at baseline, before CHMI, and 
after CHMI are required to thoroughly dissect the (protective) immune response. The samples 
will also serve as a Biobank that can be used for studies on future technological platforms as they 
become available.  
 
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 clinical center). Subjects will be offered to split the large blood draw in 
two days if desired. If blood volume collection is limited, specimen collection will be prioritized 
for safety and malaria assays (blood smears, real time NIH qPCR), followed by secondary 
endpoint assessments (retrospective LMIV qPCR, immunology labs), and lastly long term 
storage.  
8.6 
Symptom Memory Enhancement Card (Diary) 
 
Subjects will be asked to keep daily symptom memory enhancement diary (IRB approved paper 
or electronic diary) for recording oral temperature once during the day, as well as solicited 
questions regarding and signs/symptoms the subject has experienced that may be related to 
Sanaria® PfSPZ Challenge, malaria infection subsequent to Sanaria® PfSPZ Challenge, 
chloroquine, and pyrimethamine. In the pilot study and the main study, subjects will complete a 
daily symptom memory enhancement diary through 10 days post Sanaria® PfSPZ Challenge. 
The type of information (solicited symptoms: direct question of known possible side effects of 
the product; unsolicited: open-ended questioning such as “do you have any other symptoms”), 
duration of collected information, and format information is captured are summarized below in 
Section 12.3. 
 
The size of any injection-site reaction will be measured using a standardized plastic measurement 
tool and recorded in the memory enhancement diary.  Clinical site staff will review these diaries 
with the subjects and capture reported symptoms in CRIMSON.  The memory enhancement 
diaries will be collected by the study staff or electronically. 
 


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8.7 
Photographs of Rash or Injection Site Reactions 
If a subject develops a rash or injection site reaction, photographs may be taken by the 
subject or NIH division of medical arts.  These photographs will not include the subject’s 
face or any identifying scars, marks, or tattoos. 
8.8 
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: 
 
x Oral temperature >38.0˚C at the time of vaccination will warrant deferral of injection until 
fever resolves (within protocol-defined vaccination window). 
 
x 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. 
8.9 
Treatments that Could Potentially Interfere with Vaccine-induced Immunity 
Treatment with any of the following medications during the study may exclude a subject from 
receiving further doses of Sanaria® PfSPZ Challenge.  However, the subject will be encouraged 
to remain in the study for the duration of the study for safety evaluations. 
 
x Licensed vaccine in the 2-week period (4 weeks for live vaccines) prior to and following 
each vaccination. 
x Receipt of immunoglobulins and/or any blood products up to 3 months prior to the first 
Sanaria® PfSPZ Challenge through 30 days after administration of the last Sanaria® 
PfSPZ Challenge. 
 
x Chronic oral or intravenous administration (≥14 days) of immunosuppressive doses of 
steroids, i.e., prednisone >10 mg per day, immunosuppressants or other immune-modifying 
drugs from each day of Sanaria® PfSPZ Challenge to 2 weeks following each Sanaria® 
PfSPZ Challenge. 
 
x Any investigational drug or investigational vaccine during the study period. 
 
x Required surgical removal of the spleen or the development of a hematologic or other disease 
that would interfere with normal immunity. 
 
x Receipt of antibiotics with antimalarial activities during the study period. Specifically, 
antibiotic use will be assessed on a case by case basis with the study Sponsor and if needed, 
Safety Monitoring Committee, given the variable half lives of antibiotics with antimalarial 
activities. 


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8.10 Clinical Laboratory Testing 
 
Using standard techniques, the clinical laboratory will perform the following tests: 
1. Complete blood count (CBC) plus white blood cell differential and platelet count. 
The following CBC parameters will be assessed for safety throughout the trial: WBC, 
absolute neutrophil count (ANC), hemoglobin, and platelet count. 
2. Serum creatinine (Cr). 
3. Alanine aminotransferase (ALT) 
4. HbsAg test (ELISA, PCR if indicated). 
5. HCV test (ELISA, PCR if indicated). 
6. HIV test (ELISA, Western Blot if indicated). 
7. Urine dipstick/urinalysis (at screening only). 
8. Urine and/or serum pregnancy testing (β-hCG) at screening and prior to every Sanaria® 
PfSPZ Challenge via DVI or as clinically indicated. 
9. Malaria real time NIH qPCR at the NIH Clinical Center (NIH Malaria Genus Species (4-
plex) PCR). 
 
Note: Laboratory studies completed under a different NIH protocol can be used for screening 
purposes as long as they are within 56 days prior to study enrollment. 
8.11 Electrocardiograms 
Electrocardiograms (12-lead ECGs) will be performed during screening and as clinically indicated 
throughout the study in the cardiology department and read by study cardiologist or representative. 
Subjects with clinically significant abnormalities will be excluded from the study. 
 
Note: ECG completed under a different NIH protocol can be used for screening purposes as long 
as they are within 56 days prior to study enrollment. 
8.12 Malaria Blood Smears 
The gold standard for malaria diagnosis and evaluation of vaccine efficacy endpoints is the 
detection of malaria parasites on Giemsa-stained thick blood films. Blood smears are prepared in 
duplicate according to standard malaria challenge procedures and evaluated by trained study 
microscopists, and the results reported to the study Principal Investigator within 24 hours 
(usually within 6 hours), and is prioritized to be read immediately if a subject is symptomatic. At 
least 0.5μl are scanned for the presence of malaria parasites. This method allows for detection of 
a parasite density of approximately 3 parasites/μl and early diagnosis, often before subjects 
become symptomatic for malaria.  
 
For symptomatic subjects, (including all subjects during CHMI and Arm 2a/2b subjects during 
CVac #2 and #3; for Arm 3 only when clinically ill),at least 1.5μl are evaluated.  


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Blood smears are considered positive if at least two unambiguous parasites per slide are 
identified and confirmed by a second microscopist. Blood smears will be performed as outlined 
in Appendix A and as noted below. 
1. Arm 1 (pyrimethamine pilot): blood smears will only routinely be done for symptomatic 
subjects. 
2. Arm 5 (chloroquine pilot): routine daily blood smears from days 6-12 post injection. 
3. Arms 2a & 2b: 
a. CVac #1: blood smears will only routinely be done for symptomatic subjects. 
b. CVac #2 & #3: routine daily blood smears on day 6 and day 10  post injection 
regardless of symptoms. 
4. Arm 3: 
a. CVac #1: routine daily blood smears from days 6-10 post injection. 
b. CVac #2 &3: routine daily blood smears from days 6-10 post injection. 
 
5. CHMI (Arms 2a, 2b, 3, 4a, 4b): blood smears will only be done for symptomatic 
subjects OR if real time NIH qPCR cannot be resulted on the same day. 
Note: 
1. Regardless on timing, at any time point post PfSPZ Challenge 
injection, if clinically indicated, a blood smear may be completed and 
subjects evaluated and managed per protocol and LMIV malaria 
diagnosis SOP. 
2. A positive blood smear will be followed daily until 3 consecutive 
negative blood smears have been recorded. 
8.13 qPCR (Retrospective LMIV and Real Time NIH) 
 
While detection of parasites on thick blood smears remains the most common primary endpoint in 
human challenge trials, both PCR- and nucleic acid sequence-based amplification (NASBA)- 
based methods have been increasingly used to support blood smear data in malaria vaccine trials 
(Walther, Dunachie et al. 2005, Webster, Dunachie et al. 2005, Walther, Thompson et al. 2006, 
Roestenberg, McCall et al. 2009). These research molecular assays have significantly increased 
sensitivity for detection of P. falciparum blood-stage infection approaching 20 parasites/mL, 
often resulting in detection 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 a retrospective LMIV 
qPCR that detects 18s of Pf with a lowest detection threshold of approximately 40 parasites/mL 
that will be used during PfSPZ-CVac immunization period and during the CHMI period for 
comparison to the real time NIH qPCR performed by the NIH molecular diagnostics department 
and to other malaria challenge sites performing qPCR.   
 
In the past few years, qPCR detection of malaria infection has become more acceptable parasite 
detection tool. The molecular diagnostics department, part of the clinical laboratory, at the NIH 
has developed a NIH Malaria Genus Species (4-plex) PCR with a sensitivity of 500 parasites/mL 
of whole blood (0.5 parasites/μl), detecting parasitemia approximately 2 days before clinical 


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symptoms develop. This assay has been externally validated and is the malaria molecular assay at 
the NIH clinical center currently.  
 
In this study, retrospective LMIV qPCR will be used for evaluation of endpoints, specifically 
to characterize the presence and kinetics of subpatent parasitemia following Sanaria® PfSPZ 
Challenge exposure and pyrimethamine dosing during CVac.  Retrospective LMIV qPCR will 
be completed in batches during days 6-14 in the pilot and first CVac in the main study; then 6-
10 in the second and third CVac in the main arms post Sanaria® PfSPZ Challenge.  
Retrospective LMIV qPCR will also be completed daily starting on day 6 post DVI following 
PfSPZ CHMI. If a blood smear is clinically indicated during the CVac time period of the 
study, a retrospective LMIV qPCR will also be collected at this time. 
 
NIH Malaria Genus Species 4-plex qPCR will be used for malaria diagnosis and treatment 
decisions during the pilot, first CVac for Arm 2, and following Sanaria® PfSPZ CHMI (all 
Arms) and for any visits for which a subject presents symptomatic and requires rapid qPCR 
results. One positive NIH real time malaria qPCR result or a single peripheral thick blood 
smear during pilot study, at any time for Arm 2, and at any time post-CHMI will be used for 
diagnosis and clinical decisions to treat the subject. Subjects in Arm 3 are expected to have 
evidence of blood stage exposure given they are only on chloroquine, thus the presence of 
asexual parasites alone in these subjects will not warrant automatic treatment during PfSPZ-
CVac. The NIH qPCR will be performed as outlined in Appendix A.  
x Arm 1 (pyrimethamine pilot): daily real time NIH qPCR from day 6-14 post 
injection 
x Arm 5 (chloroquine pilot): NIH real time qPCR will only be done if clinically 
indicated.  
x Arm 2: 
o CVac #1: daily real time NIH qPCR from day 6-10 post injection 
o CVac #2 &3: daily real time NIH qPCR from days 7-9 post injection 
x Arm 3: NIH real time qPCR will only be done if clinically indicated.  
x CHMI (Arms 2, 3, 4a, 4b): daily real time NIH qPCR from day 6 post injection 
until diagnosis, then at the end of study. 
 
Any blood left over from qPCR sampling time points may be sent to collaborators with 
established malaria qPCR or point of care diagnostics for further quantification of parasite 
loads, validation of assay results, and inter-assay comparison.   
8.14 Drug Assays (Chloroquine, Desethylchloroquine, and Pyrimethamine) 
Chloroquine levels will be sent for Arm 3 and 5 only on study Day 1 (approximately 2 days after 
loading dose) and will be analyzed in real time by NMS Labs (Willow Grove, PA) or by another 
laboratory with an equivalent capability. The results of the chloroquine levels will be available by 
day 7 post administration of Sanaria® PfSPZ Challenge (Study Day 8). If the chloroquine level is 
<10 ng/mL in the serum and undetectable in the urine, the subject will be treated with Malarone® 
and withdrawn from the study regardless of their clinical presentation. If the subject has a lower 
than expected chloroquine level, but one that is detectable above 10 ng/mL by the blood assay, 
that subject will continue to be followed per protocol with daily histories, diary symptom review, 
blood smears and qPCR assays, as we would expect chloroquine to still be effective in clearing 


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parasitemia. If clinically indicated, the subject will have additional thick blood smears and/or real 
time NIH PCRs completed. The decision to withdraw the subject and treat the subject with 
Malarone® will be based primarily on the clinical presentation and parasitology results, but the 
specific chloroquine level will be strongly taken into consideration. If there appears to be an issue 
with the chloroquine testing (multiple subjects having undetectable levels) repeat samples will be 
sent immediately for testing from all enrolled subjects at risk of parasitemia (depending on 
timing). In the protocol #15-I-0169 in which subjects were dosed with chloroquine similarly, all 
subjects (n=20) had detectable chloroquine levels above 10 ng/mL. 
 
Demonstrating that a subject has adequate chloroquine levels after the first administration will rule 
out poor absorption, unusual metabolism, or surreptitious non-compliance (e.g., purposeful 
regurgitation).  For this reason, there are no plans to re-test chloroquine levels after subsequent 
doses of chloroquine.  However, individual subjects may be retested at any point based on clinical 
need. 
For the majority of sampling time points, whole blood levels will be obtained by venous draw and 
stored on filter paper until analysis is performed following the last study visit or sooner if clinically 
indicated. Pyrimethamine (Arm 2 only) and chloroquine (Arm 3 only) levels will be tested 
retrospectively by high performance liquid chromatography/tandem mass spectrometry throughout 
the study as outlined in Appendix A.  
8.15 Immunology 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 Sanaria® PfSPZ 
Challenge will be performed at the Laboratory of Malaria Immunology and Vaccinology, 
NIAID, NIH, Sanaria Inc, NIH CHI, and Antigen Discovery, Inc. according to standard 
laboratory procedures. 
 
These assays may include but are not limited to: 
1. ELISA for antibodies to P. falciparum liver stage and blood stage antigens 
(PfCSP, PfMSP-1, PfAMA-1, PfLSA-1, PfMSP-5, PfEBA-175, EXP-1) 
2. (IFN-γ) ELISPOT assay and multi-parameter flow cytometry with intracellular 
cytokine staining on peripheral blood mononuclear cells in P. falciparum liver- stage 
antigens (CSP, LSA-1) pre-/post- Sanaria® PfSPZ Challenge  administration 
3. B and T cells studies to analyze immunologic responses  
4. Immunofluorescence assay for antibodies to Pf sporozoites, asexual, and sexual 
erythrocytic stage parasites 
5. Inhibition of sporozoite invasion assay for antibodies exhibiting functional activity 
against sporozoites 
6. Protein microarrays 
 
In addition Sanaria Inc will assess antibodies to whole PfSPZ by immunofluorescence assay 
(IFA) and inhibition of sporozoite invasion assay (ISI) and to asexual erythocytic stage parasites 
by IFA as described (Epstein, Tewari et al. 2011, Seder, Chang et al. 2013). 
 
Laboratory assays to assess immune responses to novel pre-erythrocytic antigens will be performed 
in the PEVA Consortium laboratories at the Laboratory of Malaria Immunology and Vaccinology, 


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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 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 indicating 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 CVac with 
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 provides protective immunity 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. IFN-γ ELISPOT/Intracellular cytokine staining (ICS) assay on peripheral blood 
mononuclear cells in P. falciparum pre-erythrocytic antigens (PFL1995c, 
PFE0305w, LISP1(PF14_0179), SAP1 (PF11_0480), MAL7P1.164, PF14_0113) 
pre-/post-Sanaria ®PfSPZ Challenge administration 
 
The Center for Human Immunology will collaborate with LMIV to assess the innate and humoral 
immune responses after vaccination and CHMI using but not limited to flow cytometry, 
Somalogic, Luminex and RNA-Seq.   
 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 to PfSPZ, chloroquine, or pyrimethamine. Genetic 
testing may be performed in accordance with the genetic testing information that is included in the 
study informed consent. 
Storage: Access to stored research samples will be limited using either a locked room or a locked 
freezer. Temporary storage of samples may occur at the LMIV laboratory in Building 10 at the 
NIH Clinical Center, prior to shipment to LMIV.  Samples will be stored at the LMIV in 
Rockville, MD or at LMIV’s designated repository, Thermo Scientific, Rockville, MD.  Samples 
and data will be stored using codes assigned by the investigators or their designees. Data will be 
kept in password-protected computers. Only investigators or their designees will have access to 
the samples and data.  
Tracking: Samples will be tracked using a sample-tracking software program, e.g., Freezerworks.  
Disposition at the Completion of the Protocol: In the future, other investigators (both at the 
NIH and outside) may wish to study these samples and/or data. In that case, IRB approval must be 


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sought prior to any sharing of samples and/or data. Any clinical information shared about the 
sample with or without patient identifiers would similarly require prior IRB approval.   
At the completion of the protocol (termination), samples and data will either be destroyed or, after 
IRB 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 IRB.  
Consent to allow long term storage of study samples is a part of the inclusion criteria for this 
study.  However, subjects may decide at any point not to have their samples stored.  In this case, 
the principal investigator will destroy all known remaining samples and report what was done to 
both the subject and to the IRB.  This decision will not affect the subject’s participation in this 
protocol or any other protocols at NIH. 
 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, or 
transferred to another existing protocol, “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 National Institutes of Health (NIH) may send samples to the other investigators.  In that case, 
IRB approval must be sought prior to any sharing of samples.  Any clinical information shared 
about the sample with or without patient identifiers would similarly require prior IRB approval.  
The research use of stored, unlinked, or unidentified samples (for example, as a standard for 
immunological analyses) may be exempt from the need for prospective IRB review and approval.  
Exemption requests will be submitted in writing to the NIH Office of Human Subjects Research 
Protections (OHSRP), which is authorized to determine whether a research activity is exempt. 
 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. 


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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.  
 Assessment of Safety 
12.1 Documenting, Recording, and Reporting Adverse Events 
At each contact with the subject, information regarding adverse events will be elicited by 
appropriate questioning and examinations and will be: 
x immediately documented in the subject’s medical record/source document, 
x recorded on the electronic database, and 
x reported as outlined below (e.g., IND Sponsor, IRB, FDA). 
 
A study clinician will be available during the study period and will be available to the study 
subjects at all times. Should a subject call 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, 
phone call summaries, survey tools and data collection tools. Source documents will be reviewed 
in a timely manner by the research team. All reportable adverse events that are identified will be 
recorded in CRIMSON. 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 be recorded in CRIMSON. 
12.2 Definitions 
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) 


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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 
x results in death; 
x is life-threatening (places the subject at immediate risk of death from the event as it 
occurred); 
x results in inpatient hospitalization or prolongation of existing hospitalization; 
x results in a persistent or significant disability/incapacity 
x results in a congenital anomaly/birth defect; or 
x 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  
Any change, divergence, or departure from the IRB approved study procedures in a research 
protocol. Protocol deviations are designated as serious or non-serious and further characterized as 
1. Those that occur because a member of the research team deviates from the protocol. 
2. Those that are identified before they occur, but cannot be prevented. 
3. Those that are discovered after they occur. 
 
Serious Protocol Deviation 
A deviation that meets the definition of a Serious Adverse Event or compromises the safety, 
welfare or rights of subjects or others. 
 
Non-compliance  


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The failure to comply with applicable NIH HRPP policies, IRB requirements, or regulatory 
requirements for the protection of human subjects. Non-compliance is further characterized as: 
1. Serious: Non-compliance that 
a. Increases risks, or causes harm, to participants 
b. Decrease potential benefits to participants 
c. Compromises the integrity of the NIH-HRPP 
d. Invalidates the study data 
2. Continuing: Non-compliance that is recurring 
3. Minor: Non-compliance that, is neither serious nor continuing. 
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. 
Serious Unanticipated Problem (UP) 
A UP that meets the definition of a Serious Adverse Event or compromises the safety, welfare or 
rights of subjects or others. 
 
 
Unanticipated problem that is not an Adverse Event (UPnonAE): 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. 
12.3 Investigator Assessment of Adverse Events 
If a diagnosis is clinically evident (or subsequently determined), the diagnosis rather than the 
individual signs and symptoms or lab abnormalities (except for malaria, when in addition each 
laboratory value will be separately recorded) will be recorded as the AE. Assessment of safety will 


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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 Sanaria® PfSPZ Challenge. Additionally, subjects will return to the 
clinic for clinical assessments, almost daily from Day 1- 14 after Sanaria® PfSPZ Challenge via 
DVI and then weekly for two more weeks before the subsequent administration of Sanaria® PfSPZ 
Challenge (Appendix A). The pilot arm will only receive one injection of Sanaria® PfSPZ 
Challenge.  
The type of information (solicited symptoms: direct question of known possible side effects of the 
product; unsolicited: open-ended questioning such as “do you have any other symptoms”), duration 
of collected information, and format information is captured are summarized below: 
Sanaria® PfSPZ Challenge (local reactogenicity related to the injection – solicited questions 
includes pain/tenderness, redness, swelling/induration, bruising, and pruritus at the injection site): 
x Collected for 6 days post Sanaria® PfSPZ Challenge 
x Collected from the subject via daily symptom memory enhancement cards (diary) 
and solicited questions at each study visit (telephone and clinic follow up) 
 
Sanaria® PfSPZ Challenge (systemic reactogenicity related to the injection – solicited questions 
includes rash, urticaria, pruritus, edema, headache, fever, chills, malaise, myalgia, arthralgia, 
palpitations, shortness of breath, dizziness/vertigo and non-musculoskeletal chest pain): 
x Collected for 6 days post Sanaria® PfSPZ Challenge 
x Collected from the subject via daily symptom memory enhancement cards (diary) 
and solicited questions at each study visit (telephone and clinic follow up) 
 
Sanaria® PfSPZ Challenge (systemic symptoms related to parasitemia/malaria infection  – solicited 
questions includes headache, fever, chills, rigors, sweats, malaise, dizziness, palpitations, shortness 
of breath, myalgia, arthralgia, nausea, vomiting, abdominal pain, diarrhea, back pain, non-
musculoskeletal chest pain): 
x Collected from 6 days post Sanaria® PfSPZ Challenge to 14 days post Sanaria® 
PfSPZ Challenge during the PfSPZ-CVac phase (via solicited questions at study 
visits) 
x Collected from 6 days post Sanaria® PfSPZ Challenge to 27 days post Sanaria® 
PfSPZ Challenge during the CHMI phase (via solicited questions at study visits) 
x Collected from the subject via daily symptom memory enhancement cards (diary) 
(Collected through Day 10 post Sanaria® PfSPZ Challenge for the pilot and main 
Arms). 
 
Chloroquine (systemic symptoms related to chloroquine – solicited questions includes 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) 
x Collected for 6 days post chloroquine administration 
x Collected from the subject via daily symptom memory enhancement cards (diary) 


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when dispensed post Sanaria® PfSPZ Challenge and solicited questions at each 
study visit (telephone and clinic follow up) until 6 days post last chloroquine 
administration 
Pyrimethamine (systemic symptoms related to pyrimethamine– solicited questions includes nausea, 
vomiting, diarrhea, abdominal pain, anorexia, rash)  
x Collected for 6 days post pyrimethamine administration 
x Collected from the subject via daily symptom memory enhancement cards (diary) 
when dispensed post Sanaria® PfSPZ Challenge and solicited questions at each 
study visit (telephone and clinic follow up) until 6 days post last pyrimethamine 
administration 
Unsolicited adverse events 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. 
 
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 study chart, and discussed 
with the PI. 
 
All local and systemic reactions will be captured on the appropriate source documents and 
CRIMSON Data System.  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 
through day 27 after Sanaria® PfSPZ Challenge; new chronic medications will be collected for the 
remainder of the study. 
  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 post-challenge reactogenicity events 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 Sanaria® PfSPZ 
Challenge and/or diagnosis of malaria or antimalarial chemoprophylaxis. 
 
Solicited adverse events will be captured by subject daily symptom memory enhancement cards 
(diary)  and/or by direct questioning during the study. Solicited adverse events to be recorded as 
endpoints for this study are provided in Table 21. Solicited adverse events that are possibly 
related to chloroquine and/or pyrimethamine will be collected for 6 days following each drug 
administration.  Solicited adverse events that are possibly related to SPZ injection will be 
captured daily for 6 days (local and systemic symptoms).  Symptoms related to malaria 
secondary to Sanaria® PfSPZ Challenge will be solicited from 6 to 14 days (systemic 


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symptoms) following Sanaria® PfSPZ Challenge during CVac and from 6 to 27 days following 
Sanaria® PfSPZ Challenge during CHMI. 
 
Additionally, the following rare side effects have been reported with both chloroquine and 
pyrimethamine, these will not be solicited adverse events, but relationship will be determined 
on a subject by subject basis. 
 
Chloroquine: severe changes in mood, psychosis, electrocardiographic change, and hypotension 
 
Pyrimethamine: 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 
 
 
 


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 Table 21: Solicited Adverse Events 
 
Solicited Adverse Events 
Sanaria® PfSPZ Challenge (local) 
Injection pain/tenderness 
Injection swelling/edema 
Injection pruritus 
Injection erythema/redness 
Injection induration  
Bruising 
Sanaria® 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 
 
Sanaria® 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) 
 
 Severity 
Severity of AEs will be assessed by the investigator in Appendix C. AEs not included in the 
Appendix C will be graded for severity using the followings definitions as seen in Table 22. 
 
 


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Table 22: Definitions for Severity of AE Grading 
Severity 
Definition 
Grade 1 (Mild) 
No interference with activity, may use 1 dose 
of an over the counter medication 
Grade 2 (Moderate) 
Repeated use of non-narcotic pain reliever > 
24 hours or some interference with activity 
Grade 3 (Severe) 
Activities of daily living limited to <50% of 
baseline, medical evaluation/therapy required 
Grade 4 (Life-Threatening) 
Extreme limitation in activity, significant 
assistance required; immediate medical 
intervention or therapy required to prevent 
death 
Grade 5 
Death 
 
 Causality 
Causality (likelihood that the event is related to the study agent) will be assessed considering the 
factors listed under the following categories: 
Definitely Related 
x reasonable temporal relationship 
x follows a known response pattern 
x clear evidence to suggest a causal relationship 
x there is no alternative etiology 
 
Probably Related 
x reasonable temporal relationship 
x follows a suspected response pattern (based on similar agents) 
x no evidence of a more likely alternative etiology 
 
Possibly Related 
x reasonable temporal relationship 
x little evidence for a more likely alternative etiology 
 
Unlikely Related 
x does not have a reasonable temporal relationship  
 
OR 
x good evidence for a more likely alternative etiology 


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Not Related 
x does not have a temporal relationship 
 
OR 
x 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. 
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: 
x The event being temporally related with vaccination or reproduced on re-vaccination 
x A reaction of similar nature having previously been observed with this type of vaccine and/or 
formulation 
x The event having been reported in the literature for similar types of vaccines 
x 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:   
x Related - all AEs that are assessed as definitely, probably, or possibly related 
x 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). 
12.4 Investigator Reporting Responsibilities to the Sponsor 
 Adverse Events 
Line listings, frequency tables and other summary AE data will be submitted to the IND Sponsor 
when needed for periodic safety reviews, review of IND annual reports, review of IND safety 
reports, and preparation of final study reports. 
 Serious Adverse Events 
All SAEs (regardless of relationship and whether or not they are also UPs) 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 
 


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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) 
  Tel: 301-466-7943 (mobile) 
  Email: trichie@sanaria.com 
 
3. Tooba Murshedkar, MS 
SAE Fax:  240-306-0596 
Email:   tmurshedkar@sanaria.com 
 
 Unanticipated Problems 
All Unanticipated Problems that are also adverse events will be reported to the IND Sponsor on the 
NIH Problem Report Form sent by fax or e-mail attachment no later than 7 calendar days of site 
awareness of the event.   
Unanticipated Problems that are not AEs will also be reported to the IND Sponsor. 
 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: 
x Withdrawn from the study but continue in follow-up for safety. 
x PI will discuss with sponsor regarding the timing of the pregnancy from last dose of 
Sanaria® PfSPZ Challenge to determine whether treatment is warranted. If treatment 
is required, administration of Malarone® (4 tablets) daily x 3 days will be 
administered. Malarone® is pregnancy category C 
x Report to IRB as an informational item. 
x Advice research subject to notify the obstetrician of study product exposure (infectious 
SPZ, chloroquine, pyrimethamine, Malarone®). 
x Report to IND Sponsor. 
 
12.5 Reporting Procedures to the IRB 
Assessment of Safety 
AEs and other reportable events are defined in Policy 801: Reporting Research Events. 
  


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Reporting Procedures 
Unanticipated problems, non-compliance, and other reportable events will be reported to the IRB 
according to Policy 801. 
  
Reporting to the NIAID Clinical Director 
The principal investigator will report UPs, major protocol deviations, and deaths to the NIAID 
Clinical Director according to institutional timelines.
 
12.6 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 (1 month following the CHMI) 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. 
12.7 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.  
12.8 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 pyrimethamine 
components.  
 Parasitemia and Malaria Symptoms  
LMIV conducted a CVac study (NIAID protocol #15-I-0169) in which patent parasitemia 
(positive blood smear) was not detected in any participant during CVac. Subpatent parasitemia 
(positive qPCR) was not detected in any participant in Arm 2 (pyrimethamine and chloroquine) 
but was detected in Arm 3 (chloroquine only). In the proposed study, it will be the first time that a 
CVac study with increasing dose of Sanaria® PfSPZ Challenge with pyrimethamine alone is 
conducted.  
 
During the pyrimethamine pilot study (Arms 1a-1e), the participants will be monitored closely 
with NIH real time malaria qPCR and a single positive NIH real time malaria qPCR will result in 


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halting for the individual person and treatment with Malarone® per protocol. A positive NIH 
malaria qPCR and/or blood smear during PfSPZ-CVac immunization will therefore not necessarily 
provide a halt for the study as a whole, nor will it be reported as an unanticipated problem. 
However, if a positive NIH malaria real time qPCR and/or 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.  
 
The study may pause for further review during the pilot if subpatent parasitemia is detected in any 
subjects in Arms 1c and 1e as outlined in Section 3.2 and Figure 6. 
 
For Arm 2a and 2b (pyrimethamine only), a positive NIH real time malaria qPCR (during CVac 
#1) and/or blood smear will result in  treatment with Malarone® for the individual person and it 
will not be reported as an unanticipated problem. The subject will be withdrawn from the study. If 
a NIH malaria qPCR and/or 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. 
  
Again, individual subjects in Arm 2 (pyrimethamine only) will be treated with another 
antimalarial medication (Malarone®) and withdrawn if the following occurs: 
1. A positive NIH real time malaria qPCR 
2. A positive blood smear 
3. At the PI’s discretion (for example, if non-compliance with pyrimethamine ingestion is 
suspected, or if concerning signs or symptoms develop)  
 
For Arm 3 and 5 (chloroquine only), participants receiving chloroquine only have presented 
with patent parasitemia in other CVac/CPS studies during CVac phase, including positive blood 
smears at the peak of expected parasitemia. In previous study (NIAID protocol #15-I-0169), 
participants in chloroquine only arm did not develop positive blood smears although they all did 
have positive LMIV qPCR during CVac. However, in the current study, due to higher doses of 
PfSPZ Challenge, a positive blood smear may occur. Therefore, for Arm 3 and 5, a positive blood 
smear during PfSPZ-CVac immunization will not necessarily result in a halt for an individual 
participant or the study as a whole, nor will it be reported as an unanticipated problem.  
 
During the completed pilot phase of this study, in the chloroquine pilot (Arm 5b; 200,000 PfSPZ 
Challenge), we did have 3 out of the 4 subjects experience a single positive blood smear during the 
expected time period (during day 8 and 9 post PfSPZ Challenge respectively). These positive blood 
smears were associated with clinical symptoms, including preceding Grade 3 symptoms, that 
resulted in two separate study halts for further SMC review. All subjects, at the time of their 
positive blood smear, no longer had Grade 3 symptoms and were clinically well appearing, thus per 
protocol did not meet criteria for re-treatment with another antimalarial. With the SMC’s 
agreement, the study resumed in both instances. The SMC recommended closer follow up of Arm 
3 subjects during peak parasitemia period to improve tolerability of the 200,000 PfSPZ Challenge 
dose.  This amendment institutes these recommended changes. 
 
As conducted in the pilot phase, during the main phase of the study, in Arm 3 (chloroquine-only 
arm) subject will be re-evaluated within 12 hours of a positive blood smear. Individual subjects 


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will be re-treated with another antimalarial medication (Malarone®) and withdrawn if the following 
occurs: 
1. A positive blood smear outside of the range of expected peak parasitemia (on or after day 
10 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)  
 
Note: Further immunization of the entire Arm 3 will be temporarily halted if any of these criteria 
are met. 
 
Again, if a positive blood smear occurs, 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 SMC for 
evaluation and consequent further review by the SMC for determination if the subject’s patent 
parasitemia is clinically significant and warrants re-treatment. The SMC 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 (Malarone®).  
 
 
The following criteria will be used to define unacceptable parasitemia results and will result in 
an immediate hold of further administration of any Sanaria® PfSPZ Challenge to an entire 
study group pending review by the SMC to determine if the study should be terminated, 
modified, or continued: 
 
x One 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 Sanaria® PfSPZ Challenge  
OR 
 
x Four or more subjects experience a positive NIH real time malaria PCR in Arm 2 
OR 
 
x Two or more subjects experience positive blood smears outside of the expected peak 
parasitemia (on or after day 10 post PfSPZ Challenge) in Arm 3  
 
In addition to considering the optimal clinical decisions for the subject, the Sponsor and the SMC 
will determine if the event impacts the remainder of the study participants and whether any 
protocol procedures should be modified. 
 


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 Reactogenicity 
If a study product (Sanaria® PfSPZ Challenge, chloroquine, pyrimethamine) is considered 
unacceptably reactogenic (as described in the following criteria), the study will be halted.  No new 
enrollments and no further vaccinations will be administered by the Investigators until reviewed 
by the SMC and study IND Sponsor.  A report of SMC recommendations will be submitted to the 
IRB. 
 
The following criteria will be used to define unacceptable reactogenicity and will result in an 
immediate hold of further administration of any component of the investigational product to an 
entire study population pending review by the SMC to determine if the study should be 
terminated, modified, or continued: 
 
x One or more subjects experience an SAE that is determined to be possibly, probably or 
definitely related to Sanaria® PfSPZ Challenge, chloroquine, pyrimethamine, or 
 
x One or more subjects experience a hypersensitivity reaction that is probably or 
definitely related to Sanaria® PfSPZ Challenge or 
 
x Any severe clinical illness occurs that is not explained by a diagnosis that is unrelated or 
unlikely related to study product or 
 
x 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 
 
x 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 
 
x Any safety issue that the study PI or IND Sponsor determines should halt the 
study 
 
The IRB, 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 SMC or IRB, 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 SMC, 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. 
 Reporting of Study Halting 
If a halting requirement is met, a description of the adverse event(s) or safety issue must be 
reported by the Investigator by fax or email within one business day to the IND Sponsor. The 
IND Sponsor is responsible for notifying the SMC within one business day of being informed by the 
PI.  The LMIV Investigator will inform the IRB that a halting rule has been met. 


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 Resumption of a Halted Study 
The IND Sponsor, in collaboration with the PI and the SMC 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 PI will notify the IRB(s) of the decision to 
resume the study. 
12.9 Pausing Criteria for a Subject or Group 
The decision to suspend 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:  
x A subject experiences an SAE or ≥ 2 or more Grade 3 or greater AEs that is/are unexpected 
(as determined by the IND Sponsor) and is/are possibly, probably, or definitely related to 
the study agent; or 
x 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. 
 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 
PI by fax or email within 1 business day to the IND Sponsor, the IRB, and SMC. 
The PI must inform the IRB that a pausing rule has been met according to their requirements. The 
IND Sponsor will notify all sites of related Sanaria trials that the study has been paused. 
 Resumption of a Paused Study 
The IND Sponsor in collaboration with the PI and the SMC will determine if it is safe to resume 
administration of the study agent to the participant/group. The IND Sponsor will notify the PI of 
this decision. The PI will notify their IRB of the decision to resume administration of the study 
agent prior to resumption. 
12.10 Withdrawal Criteria for an Individual Participant 
A subject will not be considered to have completed the trial if any of the following reasons 
apply: 
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. 


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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 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 infections during the course of the 
study, then they meet exclusion criteria for the study. 
6. Pregnancy – If the subject becomes pregnant during the course of the study, they will be 
withdrawn for safety purposes and followed for the course of their pregnancy and for 3 
months post pregnancy outcome. 
7.   Inability to tolerate study product – inability to tolerate any component of the study 
product individually or in combination (chloroquine, pyrimethamine, Sanaria® 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 CRIMSON Data System.  Any subject 
who has received at least 1 dose of Sanaria® 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 Sanaria® 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 
analysis.  If a subject fails to complete all planned Sanaria® PfSPZ Challenges because of an AE 
or SAE, the subject will be followed until resolution or stabilization of the event.  If a subject 
withdraws, the investigator will make a reasonable effort to determine the reason. 
12.11 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. 


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12.12 Safety Oversight 
Safety Monitoring Committee (SMC) 
 
As agreed with OCRPRO, for this study a SMC chartered by the IND Sponsor, Sanaria Inc. will 
be used instead of the NIAID Intramural DSMB. The SMC will review the study twice during the 
pilot phase: 1) after the data have been collected on the first two volunteers receiving the 100,000 
PfSPZ dose 2) after the pilot study is complete and the trial is ready to move to the main study. 
The SMC will also review the study at least once during the main Sanaria® PfSPZ-CVac portion 
of the study, prior to Sanaria® PfSPZ Challenge CHMI. The Board may convene additional 
reviews as necessary, and will issue recommendations concerning continuation, modification, or 
termination of the study.  All SAEs will be reported by the PI to the IND Sponsor as outlined in 
Section 10.4.2. All reportable events (SAEs, UPs, pregnancies) as defined in Section 10.4 will be 
reported by the Sponsor to the SMC within one day of being notified of the event.  
 
The SMC will review the study data to evaluate the safety, efficacy, study progress, and conduct 
of the study. The Sponsor will notify the SMC at the time pausing criteria are met and obtain a 
recommendation concerning continuation, modification, or termination of the study. The PI will 
submit the written SMC summary reports with recommendations to the IRB(s).  
 Clinical Monitoring 
13.1 Site Monitoring Plan 
 
As per ICH-GCP 5.18 and FDA 21 CFR 312.50, clinical protocols are required to be adequately 
monitored by the study Sponsor.  The Office of Clinical Research Policy and Regulatory 
Operations (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 
monitor aspects of the trial in accordance with appropriate regulations.  The objectives of a 
monitoring visit will be: 
 
x to verify the prompt reporting of all monitored data points, and prompt reporting of 
all SAEs 
 
x to check the existence of signed informed consent documents and documentation of 
the ICF process for each monitored subject 
 
x to compare individual subject’s records (e.g. CRFs, electronic data) to the source 
documents (supporting data, laboratory specimen records, clinical notes) 
 
x 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, 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. 
 


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A specific monitoring plan will be discussed with the Principal Investigator, 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. 
 
 Statistical Considerations 
14.1 General Pilot Study 
The pilot is designed to find the largest NF54 Sanaria® PfSPZ Challenge dose that will not cause 
blood stage parasitemia in most subjects.  
14.2 General Main Study 
The main phase is designed to investigate the safety profile of the selected NF54 Sanaria® PfSPZ 
Challenge with concurrent chemoprophylaxis of pyrimethamine or chloroquine treatment in 
healthy, malaria-naïve US adults. As well, we wish to determine if this dose along with concurrent 
chemoprophylaxis of pyrimethamine can induce protective efficacy against homologous challenge 
and if this NF54 Sanaria® PfSPZ Challenge dose along with current chloroquine treatment can 
induce protective efficacy against heterologous challenge.  
14.3 Primary Objectives 
The primary objectives of this study are twofold:  
x Safety: To monitor the safety and tolerability of NF54 Sanaria® PfSPZ Challenge under 
concurrent chemoprophylaxis with pyrimethamine treatment or chloroquine treatment in 
healthy, malaria-naïve US adults 
 
x Prevention of Infection (Pilot Phase Only): To evaluate whether increasing the dose of 
Sanaria® PfSPZ Challenge (to a maximum of 200, 000 PfSPZ) under pyrimethamine 
chemoprophylaxis  in humans will  not result in the development of asexual erythrocytic 
stage parasitemia assessed by sensitive retrospective LMIV qPCR after DVI of Sanaria® 
PfSPZ Challenge in healthy, malaria-naïve adults. 
14.4 Secondary Objective 
 
The secondary objectives of this study includes assessing the protective efficacy of PfSPZ-CVac 
pyrimethamine against homologous CHMI with Sanaria® PfSPZ Challenge. 
 
Table 23 shows the decision rule for rejecting the null hypothesis that there is no significant 
difference between Arm 2a (Pyrimethamine only) and the concurrently challenged controls (Arm 
4a, n=8). Based on previous data, the team expects that the true infection rate among controls 
should be very high (≥99%).For example, if 13 participants from Arm 2a are challenged, and all 8 
of the controls become infected, we will reject the null hypothesis if and only if 10 or fewer of the 


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13in Arm 2a participants become infected. If the number of infections in the challenged Arm 2a 
participants exceed the numbers in the table below, we will not reject the null hypothesis.  
 
 


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Table 23: Decision Rule:  
The infection rate in any of the active arms (Arm 2a, 2b, 3) will be judged to be significantly 
different from the controls if the number of infections among the controls and infections among 
the challenged participants in that arm is less than or equal to the numbers in the table.  
 
Number infected out of 8 controls 
Number of 
participants 
challenged in 
Arm 2a, 2b or 3 
8/8 
7/8 
6/8 
5/8 
17 
≤11
≤6
≤5
≤3
16 
≤10
≤6 
≤5
≤2 
15 
≤9
≤6 
≤4
≤2 
14 
≤8
≤6 
≤3
≤2 
13 
≤8
≤5 
≤3
≤2 
12 
≤7
≤5 
≤1
≤1 
11 
≤6
≤4 
≤3
 
10 
≤6
≤3
≤2
≤1
9 
≤5
≤3
≤2
≤1
8 
≤4
≤3
≤1

The rule above was chosen to maximize the probability of declaring a significant difference 
if all 8 of the controls become infected, as this is considered the most likely scenario, while 
controlling the one-sided type 1 error rate at 0.025, conditioned on the number of Arm 2a, 2b 
or 3 participants challenged.   
14.5 Exploratory Objectives 
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.  
 
14.6 Sample Size and Power Calculations 
  Power calculations for primary objective (safety): Pilot Phase 
Table 24 describes the probability of break through parasitemia or infection during the pilot phase. 
For a group of two participants, there is a 75% chance of observing at least 1 infection if the true 
rate of such an event is 50% or more; the probability of observing at least 1 event among four 
participants at this level is 94% for this same true rate of 50%. Therefore, under the assumption 
that higher doses are more likely to cause infection, if we observe 4 or more subjects at higher 
doses without infection, we have good power to detect a 50% infection rate at the lower level.  
 
 
 


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Table 24: Probability of observing 0 and 2 or more break through parasitemia, among arms of 
size 2 and 4, for different true event rates 
True event rate (%) 
Pr(0/2) 
Pr(2/2) 
Pr(0/4) 
Pr(2/4) 
1 
0.98 
<0.01 
0.96 
<0.01 
5 
0.90 
<0.01 
0.81 
0.01 
10 
0.81 
0.01 
0.66 
0.05 
20 
0.64 
0.04 
0.41 
0.18 
30 
0.49 
0.09 
0.24 
0.35 
50 
0.25 
0.25 
0.06 
0.69 
  Power calculations for Main Phase 
14.6.2.1 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 Arm 2a (n =17), there 
is a 90% chance of observing at least 1 event if the true rate of such an event is 13% or more; and 
there is a greater than 90% chance of observing no events if the true rate is 0.5% or less. For Arm 
2b and 3 (n =10 each), there is a 90% chance of observing at least 1 event if the true rate of such an 
event is 20% or more; and there is a 90% chance of observing no events if the true rate is 1.0% or 
less.  
 
Probabilities of observing 0, and 2 or more events among Arms of size 17 and 10 are presented in 
Table 25 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.  
 
 


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Table 25: Probability of observing 0 events and 2 or more events, among arms of size 17 and 10, 
for different true event rates 
True event rate (%) 
Pr(0/17) 
Pr(2+/17) 
Pr(0/10) 
Pr(2+/10) 
1 
0.843 
0.012 
0.904 
0.012 
5 
0.418 
0.208 
0.599 
0.208 
10 
0.167 
0.518 
0.349 
0.518 
20 
0.023 
0.882 
0.107 
0.882 
30 
0.002 
0.981 
0.028 
0.981 
50 
<0.001 
>0.99 
0.001 
>0.99 
 
An alternative way of describing the statistical properties of the study design is in terms of the 95% 
confidence interval for the true rate of an adverse event based on the observed data Table 26 
shows the 2-sided 95% confidence intervals for the probability of an event based on a particular 
observed rate. If none of the 17 participants in Arm 2a experience a safety event, the 95% 2-sided 
upper confidence bound for the true rate of such events in the total vaccinated population is 0.2. 
For Arm 2a or 3 (n = 10), the 2-sided upper confidence bound for this rate is 0.31. 
Table 26: Two-sided 95% confidence intervals based on observing a particular rate of safety 
endpoints for arms of size 17 and 10 
Observed event rate 
95% Confidence interval (%) 
0/17 
(0.00, 0.20) 
1/17 
(0.001, 0.29) 
2/17 
(0.01, 0.36) 
0/10 
(0.00, 0.31) 
1/10 
(0.003, 0.45) 
 


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14.6.2.2 Power calculations for secondary objective (efficacy) 
We expect dropout from Arms 2 and 3 of approximately 30% before challenge. We expect no 
dropout among the control arms because they are enrolled just before challenge. Power 
calculations were considered for a range of numbers of people in Arm 2 and 3 that make it to 
challenge.  
 
We assume approximately 13 people from Arm 2a are challenged, and compared to 8 controls. The 
decision rule described in Table 23 will have good power (>80%) to detect a difference between 
the arms if the true Vaccine Efficacy (VE), defined as 1- the probability of infection in the 
treatment group divided by the probability of infection in the controls, is at least 50%. This 
provides power calculations for the comparison of Arms 2a and 4a.  
 
We assume at least 7 people from Arm 2b and 3 are challenged, and compared to 8 controls. Power 
to detect a VE of at least 70% for heterologous challenge comparing Arms 2b and 4b AND Arms 3 
and 4b, will be greater than 85%.  
14.7 Randomization 
 
Randomization for Arms 2 and 3 will be completed at the time of enrollment. For the second 
cohort, subjects will be randomized into Arms 2b and 3. Subjects will also be randomized into 
enrollment into Arm 4a or 4b in the first cohort only. In the second cohort, control subjects will 
join Arm 4b directly.. Arms 1, and 5 will not be randomized. Subjects and investigators will be 
aware of Arm assignments. The third cohort will not be enrolled. 
 Human Subject Protections and Ethical Obligations 
 
This research will be conducted in compliance with the protocol, Good Clinical Practices (GCP), 
and all applicable regulatory requirements. 
15.1 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 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. 
15.2 Informed Consent Process 
Informed consent is a process where information is presented to enable persons to voluntarily 
decide whether or not to participate as a research subject. It is an on-going conversation between 
the human research subject and the researchers which begins before consent is given and continues 
until the end of the subject's involvement in the research. Discussions about the research will 
provide essential information about the study and include: purpose, duration, experimental 


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procedures, alternatives, risks and benefits.  Subjects will be given the opportunity to ask questions 
and have them answered.  
 
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.  
15.3 Justification for Exclusion of Children 
 
This study will not enroll children, since safety of the new chloroquine and pyrimethamine 
regimen has not yet been established in adults. 
15.4 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 NIAID, the 
OHRP, Sanaria, Inc., or the sponsor’s designee. 
15.5 Risks 
Risks to the subjects are associated with venipuncture, malaria infection, Sanaria® PfSPZ 
Challenge, Chloroquine, Pyrimethamine, and large volume blood drawing.  These risks are 
outlined below: 
 Venipuncture 
Risks occasionally associated with venipuncture include pain, bruising, bleeding and infection at 
the site of venipuncture, lightheadedness, and rarely, syncope. 
 Sanaria® PfSPZ Challenge via DVI 
Possible local reactions include pain, swelling, erythema, induration, limitation of limb 
movement for several days, lymphadenopathy, or pruritus at the injection site.  Systemic 
reactions such as fever, chills, headache, fatigue, malaise, myalgia, and joint pain may also 
possibly occur, with some reactions moderate or severe. 
 
Other than mild and transient local (site of administration) reactions, the listed adverse 
reactions remain theoretical.   
 
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 


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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. 
 
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 Sanaria® 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.  
 Malaria Symptoms after Administration of PfSPZ-CVac 
In a PfSPZ-CVac study by LMIV (#15-I-0169) where subjects were injected with PfSPZ 
Challenge by DVI while under chloroquine chemoprophylaxis, all participants reported solicited or 
unsolicited symptoms that were recorded as AEs at least once during immunization phase. There 
was one SAE reported of encephalopathy NOS that was either possibly related to chloroquine or 
viral infection as described in Section 1.8.2.1. None of the participants developed patent 
parasitemia.  
 
Similar frequency AEs have been reported in other studies during CVac with chloroquine. In the 
Roestenberg study where subjects were exposed to experimental infection by the bites of Pf-
infected mosquitoes while under concurrent chloroquine prophylaxis, all subjects reported solicited 
or unsolicited symptoms that were recorded as AEs at least once during the PfSPZ-CVac 
immunization phase, although there were no statistically significant differences in AE frequency 
between the 10 vaccinees and 5 controls. There were no serious adverse events at any time and no 
evidence of blood-stage parasites (patent parasitemia) in any of the subjects at any time during the 
post-challenge follow- up period, though following that study, there have been reports of patent 
parasitemia at multiple centers in subjects during CVac/CPS while on chloroquine prophylaxis. 
 
In the study of PfSPZ-CVac in Tübingen, all subjects developed transient parasitemia after the first 
PfSPZ Challenge injection.  As in Nijmegen, there were no significant differences between the AE 


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profiles of vaccinees experiencing this subpatent parasitemia and the blinded placebo recipients. 
There was one subject with a low grade fever and concurrently the highest subpatent parasitemia 
recorded in the first stage of the study that the investigators determined may have been related. In 
the second stage of the study, there was one subject that did not develop therapeutic levels of 
chloroquine and was subsequently treated and withdrawn from the study. 
 Malaria Symptoms and Infection after PfSPZ CHMI 
In the Roestenberg study, after subsequent challenge of vaccinees with a homologous NF54 strain 
of P. falciparum following the three CVac infections, the clinical course was similar to those in 
previously reported challenge studies without CVac. 
 
In the LMIV CVac study (#15-I-0169), malaria symptoms in unprotected vaccinees was similar to 
that of unvaccinated controls with comparable severity to controls in other CVac studies. Protected 
participants developed mostly mild symptoms consistent with other CVac studies (Table 10).  
 
As with any clinical investigation, even with extensive precautions, there is always a risk of 
serious, or even life-threatening allergic reactions to administration of the investigational 
product. These risks are minimized by entry criteria that exclude subjects with a potential for 
adverse reaction to the malaria challenge, infection or study drugs. Likewise, subjects will be 
closely monitored during the administration of Sanaria® PfSPZ Challenge and/or drugs and in 
the immediate period following administration by clinical staff trained and equipped to respond 
immediately to acute systemic reactions including anaphylaxis and clinical malaria.
15.5.4.1 Cardiac Abnormalities 
 
There is no specific known cardiac risk for healthy subjects associated with CHMI or the 
antimalarial drugs at the proposed dosing used in this study. Likewise, cardiac abnormalities in 
uncomplicated clinical malaria are extremely rare (Ehrhardt, Mockenhaupt et al. 2005)and routine 
cardiac monitoring of subjects with severe malaria is not required even in subjects receiving 
treatment with antimalarials with known effects on cardiac electrical conduction (Bethell, Phuong 
et al. 1996, Bregani, Tien et al. 2004) 
 
A challenge trial subject participating in a malaria vaccine trial in the Netherlands had an 
unexplained cardiac event following receipt of an investigational vaccine, malaria challenge, 
infection and treatment with artemether/lumefantrine (Nieman, de Mast et al. 2009).  It is 
thought the temporal association of the event to malaria challenge was likely circumstantial 
(Lyke, Laurens et al. 2010). The definitive etiology of the event remains unknown and the 
subject recovered without sequelae. 
 
Another cardiac event in a malaria vaccine trial participant in the Netherlands was reported 
(van Meer, Bastiaens et al. 2014). The trial subject received a different test malaria vaccine 
than the subject reported in 2009. The subject was observed (on day 13) after challenge to have 
changes in a blood test suggesting a heart muscle problem.  Later that day the subject 
experienced chest pain and reported a heavy feeling in his left arm. After further evaluation the 
subject was diagnosed with myocarditis. It is not known whether this illness was related to 
malaria infection, the test vaccine that the subject received, the medicine used to treat malaria, 


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a viral infection unrelated to the study or something else.  Myocarditis resolved without 
treatment. 
 
Cardiac events, such as coronary vasospasm or myocarditis, are not associated with 
uncomplicated natural P. falciparum infection or experimental infection in the cumulative 
experience in over 1300 volunteers (Ehrhardt, Mockenhaupt et al. 2005) at three centers 
worldwide. However, in order to minimize the potential risk for cardiac abnormalities during the 
study, cardiac risk assessment will be conducted as part of the screening process based on the 
NHANES I study criteria as defined in Appendix B (Gaziano, Young et al. 2008) and screening 
electrocardiogram and subjects with clinically significant abnormalities on ECG or elevated risk 
(> low risk of a coronary event over the next 5 years) will be excluded from the study. 
15.5.4.2 Pregnancy 
Malaria infection can have adverse effects on both the pregnant mother and fetus.  Women who 
are pregnant, nursing or plan to become pregnant during the study are excluded from the study. 
 
Pregnancy testing is performed during screening and throughout the study including prior to each 
Sanaria® PfSPZ Challenge DVI.  If clinical history indicates recent sexual activity that may lead 
to pregnancy, a serum β-HCG test will be sent and results reviewed by a study investigator prior 
to the subject continuing in the study. Pregnancy prevention counseling and compliance is 
reinforced at every clinic visit (see Appendix A) throughout the study. 
 
  Medications used in the study 
The antimalarial medications administered in the study (pyrimethamine, chloroquine, 
Malarone®) 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 of medications with food). Specific toxicities 
for pyrimethamine, chloroquine and Malarone® are noted below and further outlined in the 
package insert for each drug. 
15.5.5.1 Chloroquine Phosphate 
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. 
 


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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, pyrimethamine 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 type of symptoms frequently and at all clinic visits. 
 
In the current study, there was one SAE (acute change in mental status) that occurred during the 
main phase of the first cohort in a participant enrolled in Arm 3; 3 weeks after second vaccination. 
The SAE was determined to be due to acute anticholinergic poisoning after participant ingested  
Datura stramonium seeds for recreational purposes.  Due to concurrent chloroquine prophylaxis 
use and unknown potentiation on effects of Datura stramonium during concurrent use, this 
episode was deemed possibly related to chloroquine. The participant recovered without sequaelae 
and was taken off study.  
 
For complete chloroquine safety information, including less commonly reported side effects, 
please refer to the Package Insert for chloroquine that is provided. 
 
15.5.5.2 Pyrimethamine 
The most commonly reported side effects of pyrimethamine 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 
pyrimethamine dose but usually disappears promptly upon reduction of dosage. Prolonged and 
higher doses of pyrimethamine, as used to treat toxoplasmosis, may produce megaloblastic 
anemia, leukopenia, thrombocytopenia, pancytopenia, atrophic glossitis, hematuria, and disorders 
of cardiac rhythms though this is 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.   
 
Pyrimethamine 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 pyrimethamine is administered 
concomitantly with a sulfonamide. The approved dosage of pyrimethamine 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 pyrimethamine safety information, including less commonly reported side effects, 
please refer to the Package Insert for pyrimethamine that is provided. 


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15.5.5.3 Malarone® (Atovaquone/proguanil) 
Though not a part of the investigational product/regimen, all subjects will receive empiric 
treatment with Malarone® prior to completion of the trial. The most commonly reported side 
effects of Malarone®  dosing include gastrointestinal disturbance, sleeping problems (insomnia), 
strange dreams, dizziness, depression, loss of appetite, fever, rash, cough and laboratory 
abnormalities such as anemia, neutropenia, transaminitis, hyponatremia or increase in amylase, 
but generally these effects do not require discontinuation of the drug.  Malarone® has 
occasionally been associated with severe hypersensitivity reactions (such as Stevens-Johnson 
syndrome, erythema multiforme, and anaphylaxis) 
 
For complete Malarone® safety information, including less commonly reported side effects, 
please refer to the Package Insert for Malarone® that is provided. 
 
15.5.5.4 Ibuprofen  
Another medication used in the study 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 3) to mitigate typical symptoms of malaria infection 
such as headache, myalgia, fever and chills.  The most commonly reported 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 creatinine 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.  
 
15.6 Risk to the Community 
Malaria is not contagious and cannot be spread by person-to-person contact. Subjects are 
routinely diagnosed and treated before the infective form of the parasite (gametocyte) appears in 
the blood. 


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15.7 Benefits 
Subjects will not receive any direct benefit from participation in this study.  It is hoped that 
information gained in this study will contribute to the development of a safe and effective malaria 
vaccine. 
15.8 Compensation 
Subjects will be compensated for time and travel to participate in this trial. Compensation is pro- 
rated based upon completion of specific visits and is not contingent upon completing the entire 
study. A compensation schedule is provided below (see Table 27). Compensation for interim visits 
to follow up clinical or laboratory abnormalities will be provided at the Investigator’s discretion, 
up to $50. If a subject withdraws from the trial, compensation will be made based on the last 
completed visit. Information about compensation, including the amount and schedule of 
payment(s) and applicable reporting to the IRS, will also be described in the informed consent 
form.                                                                                                                                          
Table 27: Compensation Table 
 
 
 
 
Pilot Study Arms PfSPZ-CVac 
Study Activity 
Compensation 
Arm 1 
Number of Visits  
Arm 1 
Compensation 
Total  
Arm 5 
Number of Visits 
Arm 5 
Compensation 
Total 
Screening Visit 
$100  
1 
$100  
1 
$100 
 
 
 
 
 
 
Sanaria® PfSPZ 
Challenge CVac 
$200  
1 
$200  
1 
$200  
Telephone 
Follow-Up 
$10  
1 
$10  
1 
$10  
Clinic Visit and 
Blood draw 
(<60ml) 
$50  
9 
$450  
8 
$400  
Clinic Visit, 
Drug Dose 
with/without 
Blood draw 
(<60ml), and  
$75  
5 
$375  
5 
$375 
Completed Diary 
(counted as visit) 
$10  
1 
$10  
1 
$10  
Total for Pilot 
Study 
N/A 
18  
$1,145  
17 
1095 


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Main Study Arms PfSPZ-CVac 
Study Activity 
Compensation 
Amount 
Arm 2a, 2b 
Number of 
Visits 
Arm 2a, 2b 
Compensation 
Total 
Arm 3 Number 
of Visits 
Arm 3 
Compensation 
Total 
Screening Visit 
$100  
1 
$100  
1 
$100  
 
Sanaria® PfSPZ Challenge CVac 
$200  
3 
$600  
3 
$600  
Telephone Follow-Up 
$10  
2 
$20  
6 
$60  
Clinic Visit  
$40  
6 
$240  
5 
$200  
Clinic Visit and Blood draw (<60 ml) 
$50  
26 
$1,300  
25 
$1,250  
Clinic Visit, Blood draw (<60 mL), and 
Drug Dose 
$75  
3 
$225  
4 
$300  
Clinic Visit, Blood draw (>60ml), and  
Drug Dose 
$100  
1 
$100  
1 
$100  
Clinic Visit and Drug Dose  
$75  
3 
$225  
6 
$450  
Completed Diary 
$10  
3 
$30  
3 
$30  
Total for CVAC 
N/A 
48 
$2,840  
54  
$3,090  
 
 
CHMI  
Study Activity 
Compensation 
Amount 
Arm 2a, 2b, 
3 Number of 
Visits 
Arm 2a, 2b, 3 
Compensation 
Total 
Arm 4a, 4b 
Number of 
Visits 
Arm 4a, 4b 
Compensation 
Total 
Screening Visit 
$100  
0 
$0  
1 
$100  
 
Sanaria® PfSPZ Challenge 
$200  
1 
$200  
1 
$200  
Telephone Follow-Up 
$10  
2 
$20  
2 
$20  
Clinic Visit and Blood Draw (<60ml) 
$50  
18 
$900  
20 
$1,000  
Clinic Visit and Blood Draw (>60 ml) 
$100  
1 
$100  
1 
$100  
Clinic Visit, Blood Draw (>60 ml) and 
Drug Dose 
$100  
1 
$100  
1 
$100  
Clinic Visit, Blood Draw (<60 ml) and 
Drug Dose  
$75  
2 
$150  
2 
$150  
Completed Diary 
$14  
1 
$14  
1 
$14  
Total CHMI  
N/A 
26 
$1,484  
29 
$1,684  
 
1 Specification of participants visits are based on Appendix A, actual visits calculated are subject to change, based on 
clinical information; however, compensation per visit will follow the guidelines in this table 
2 Clinic follow up visits are reimbursed by visits completed.  If a subject is diagnosed with malaria early this total amount 
may be less (maximum amount presented here). 
3 If enrolled in Arm 4 total compensation starts at CHMI – all subjects in Arm 4a and 4b will at least be compensated $1000 
for their participation. 
4 Subjects that require blood draw only (no clinic visit) will be compensated $25 


PfSPZ-CVac PYR #2                Version 10.0    Nov. 12, 2019      Page 148 of 169 
 
 
  Data Handling and Record Keeping 
16.1 Source Documentation 
 
Complete source documentation (laboratory test reports, hospital or medical records, 
progress notes, observations, subject diaries, etc.) is required for every study subject for the 
duration of the study.  The subject’s research record must record his/her participation in the 
clinical trial, the treatment received (with doses and frequency) or other concomitant 
medications or interventions administered, as well as any adverse reactions experienced 
during the trial. Selected data from source documentation and subject symptom memory 
enhancement cards (diaries) for subjects enrolled in the study will be entered into the 
CRIMSON Data System.  The data entry is to be completed on an ongoing basis during the 
study. Data entered into CRIMSON shall be performed by authorized individuals. 
Corrections to the data system shall be tracked electronically (password protected) with time, 
date, individual making the correction, and what was changed.  Source documentation 
should support the data collected in CRIMSON, and must be signed and dated by the person 
recording and/or reviewing the data. The Investigator is responsible for the accuracy, 
completeness and timeliness of the data reported to the Sponsor in the CRIMSON Data 
System.  All data entered into CRIMSON should be reviewed by the Investigator and signed 
as required with written or electronic signature, as appropriate. Data reported in CRIMSON 
should be consistent with source documents or the discrepancies should be explained.  
Source documentation will be made available for review or audit by the Sponsor, OCRPRO 
or their designees and any applicable Federal authorities. 
16.2 Retention of Study Records 
The investigator is responsible for retaining all essential documents listed in the ICH Good 
Clinical Practice Guideline.  Study records will be maintained by the PI for a minimum of 5 to 7 
years and in compliance with institutional, IRB, state, and federal medical records retention 
requirements, whichever is longest. All stored records will be kept confidential to the extent 
required by federal, state, and local law. 
 
Should the investigator wish to assign the study records to another party and/or move them to 
another location, the investigator will provide written notification of such intent to 
OCRPRO/NIAID with the name of the person who will accept responsibility for the transferred 
records and/or their new location. NIAID will be notified in writing and written 
OCRPRO/NIAID permission shall be obtained by the site prior to destruction or relocation of 
research records.  
16.3 Protocol Revisions 
No revisions to this protocol will be permitted without documented approval from the IRB that 
granted the original approval for the study.  Any change to the protocol will be submitted to the 
sponsor and to the participating IRB as a protocol amendment; and changes not affecting risk to 
subjects may request an expedited review.  In the event of a medical emergency, the Investigator 
shall perform any medical procedures that are deemed medically appropriate and will notify the 
IND Sponsor of all such occurrences. 


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Appendix A: Clinical Evaluation and Laboratory Procedures  
 
 
  


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Appendix B:  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: 
x 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 
x Evaluation of 5-year cardiovascular risk using Figure 11 (females) or Figure 12 
(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. 
 
Figure 12: NHANES I Classification for Women 
 


PfSPZ-CVac PYR #2                Version 10.0    Nov. 12, 2019      Page 157 of 169 
 
 
  
Figure 13: NHANES I Classification for Men 


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Appendix C:  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 
 
 
 
 
 
 
 
 
 
 
 
 


PfSPZ-CVac PYR #2                Version 10.0    Nov. 12, 2019      Page 159 of 169 
 
 
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 
hospitalizatio
n 
 
Photosensitivity/ 
Rash 
Localized rash with no 
medical intervention 
indicated 
Localized rash with 
medical intervention 
indicated  
Generalized rash with 
medical intervention 
indicated  
ER visit or 
hospitalizatio
n 
 
Abdominal Pain 
No or minimal 
interference with usual 
activities 
Greater than minimal 
interference with usual 
activities 
Inability to perform usual
activities 
ER visit or 
hospitalizatio
n 
 
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 


PfSPZ-CVac PYR #2                Version 10.0    Nov. 12, 2019      Page 160 of 169 
 
 
  
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 
hospitalizatio
n 
 
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 
hospitalizatio
n 
 
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 
hospitalizatio
n 
 
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 
hospitalizatio
n 
 
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/vert
igo  causing 
inability to 
 
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
 
Sleep disorder ( 
Insomnia, or 
Difficulty 
Sleeping, 
Drowsiness, etc) 
Difficulty sleepingSleep 
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 
hospitalizatio
n for 
hypotensive 
shock


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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 


PfSPZ-CVac PYR #2                Version 10.0    Nov. 12, 2019      Page 162 of 169 
 
 
 
     * 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 


PfSPZ-CVac PYR #2                Version 10.0    Nov. 12, 2019      Page 163 of 169 
 
 
 
Serum* 
 
Mild (Grade 1) 
 
Moderate (Grade 2) 
Severe (Grade 3) 
Potentially Life 
Threatening 
(Grade 4)** 
 
Creatinine – mg/dL 
 
1.5 – 1.7 
 
1.8 – 2.0 
2.1 – 2.5 
> 2.5 or requires 
dialysis 
Liver Function Tests – 
ALT, AST 
increase by factor 
 
1.1 – 2.5 x ULN 
 
2.6 – 5.0 x ULN 
5.1 – 10 x ULN 
> 10 x ULN 
* The laboratory values provided in the tables serve as guidelines and are dependent upon institutional normal 
parameters. Institutional normal reference ranges should be provided to demonstrate that they are appropriate. 
** The clinical signs or symptoms associated with laboratory abnormalities might result in characterization of the laboratory abnormalities as 
Potentially Life Threatening (Grade 4). For example, a low sodium value that falls within a grade 3 parameter (125-127 mE/L) should be 
recorded as a grade 4 hyponatremia event if the subject had a new seizure associated with the low sodium value. 
***ULN” is the upper limit of the normal range. 
⌃Values adjusted according to the NIH clinical laboratory normal values 
 
     * The laboratory values provided in the tables serve as guidelines and are dependent upon institutional normal 
parameters. Institutional normal reference ranges should be provided to demonstrate that they are appropriate. 
** “ULN” is the upper limit of the normal range. 
⌃Values adjusted according to the NIH clinical laboratory normal values 
 
 
Urine* 
 
Mild (Grade 1) 
 
Moderate (Grade 2) 
 
Severe (Grade 3) 
Potentially Life 
Threatening 
(Grade 4) 
 
Protein 
 
Trace 
 
1+ 
 
2+ 
Hospitalization or 
      Dialysis 
 
Glucose 
 
Trace 
 
1+ 
 
2+ 
Hospitalization for 
Hyperglycemia 
Blood (microscopic) 
– 
red blood cells per 
microliter (rbc/μL) 
 
 
17 – 30 
 
31– 60 
 
 
 
> 60 and/or gross 
blood 
 
Hospitalization or 
packed red blood 
cells (PRBC) 
transfusion 
* The laboratory values provided in the tables serve as guidelines and are dependent upon institutional normal 
parameters. Institutional normal reference ranges should be provided to demonstrate that they are appropriate. 
⌃Values adjusted according to the NIH clinical laboratory normal values 
 
Hematology * 
 
Mild (Grade 1) 
Moderate (Grade 2) 
Severe (Grade 3) 
Potentially Life 
Threatening 
(Grade 4) 
Hemoglobin 
(Female) - gm/dL 
 
10.0 -11.0 
8.5 - 9.9 
7.5 – 8.4 
< 7.5 
Hemoglobin (Male) 
- gm/dL 
 
12.0 – 13.0 
10.0 - 11.9 
8.0 - 9.9 
< 8.0 
WBC Increase -
cell/mm3 
 
10,800 – 15,000 
15,001 – 20,000 
20,001 – 25, 000 
> 25,000 
WBC Decrease - 
cell/mm3 
 
2,500-3500 
1,500 – 2499 
1,000 – 1,499 
< 1,000 
Lymphocytes 
Decrease - cell/mm3 
 
750 – 1,000 
500 – 749 
250 – 499 
< 250 
Neutrophils 
Decrease - cell/mm3 
 
1000-1499 
750-999 
500 – 749 
< 500 
Platelets Decreased - 
cell/mm3 
 
125000-140000 
100000-124000 
25,000 – 99000 
< 25,000 


PfSPZ-CVac PYR #2                Version 10.0    Nov. 12, 2019      Page 164 of 169 
 
 
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