Randomized, Controlled, Phase 1 Study to Assess Safety and Immunogenicity of Co-administered Hookworm Vaccine Candidates Na-APR-1 (M74)/Alhydrogel® and Na-GST-1/ Alhydrogel® in Gabonese Children
Phase: PHASE1
Conditions: Hookworm Disease, Hookworm Infection
Sponsor: Baylor College of Medicine

Randomized, controlled, Phase 1 Study to assess safety and immunogenicity of co-administered Hookworm vaccine 
candidates Na-APR-1 (M74) and Na-GST-1 in Gabonese Children 
Version 3.0, 08 Aug 2018
 
 
 
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RANDOMIZED, CONTROLLED, PHASE 1 STUDY TO ASSESS 
SAFETY AND IMMUNOGENICITY OF CO-ADMINISTERED NA-
APR-1 (M74)/ALHYDROGEL® AND NA-GST-1/ALHYDROGEL® 
HOOKWORM VACCINES IN GABONESE CHILDREN 
Protocol Number: HV-002 
 
IND Sponsor: Baylor College of Medicine 
Principal Investigator: Ayola Akim Adegnika, MD, PhD, MSc 
 
 
 
Final Version Number: 3.0  
Including Amendment #2 
08 August 2018 
 

Randomized, controlled, Phase 1 Study to assess safety and immunogenicity of co-administered Hookworm vaccine 
candidates Na-APR-1 (M74) and Na-GST-1 in Gabonese Children 
Version 3.0, 08 Aug 2018
 
 
 
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STATEMENT OF COMPLIANCE 
 
The study will be carried out in accordance with Good Clinical Practice (GCP) as required by the 
International Conference on Harmonisation efficacy guideline E6.  
 
All key personnel (all individuals responsible for the design and conduct of this study) have 
completed ethical training in the protection of the participants of clinical trials. 
 
 

Randomized, controlled, Phase 1 Study to assess safety and immunogenicity of co-administered Hookworm vaccine 
candidates Na-APR-1 (M74) and Na-GST-1 in Gabonese Children 
Version 3.0, 08 Aug 2018
 
 
 
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SIGNATURE PAGE 
The signature below constitutes the approval of this protocol and the attachments, and provides 
the necessary assurances that this trial will be conducted according to all stipulations of the 
protocol, including all statements regarding confidentiality, and according to local legal and 
regulatory requirements and applicable ICH guidelines. 
 
Site Investigator: 
 
 
 
 
Signed: 
 
Date: 
 
 
Name: Ayola Akim Adegnika, MD, PhD, MSc 
Title: Principal Investigator 
 
 
 
 
Signed: 
 
Date: 
 
 
Name: Maria Elena Bottazzi, PhD 
Title: Sponsor representative  
 
 
 
 

Randomized, controlled, Phase 1 Study to assess safety and immunogenicity of co-administered Hookworm vaccine 
candidates Na-APR-1 (M74) and Na-GST-1 in Gabonese Children 
Version 3.0, 08 Aug 2018
 
 
 
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TABLE OF CONTENTS 
Statement of Compliance ............................................................................................................... ii 
Signature Page ............................................................................................................................. iii 
Table of Contents .......................................................................................................................... iv 
List of Abbreviations ..................................................................................................................... vii 
Protocol Summary ........................................................................................................................ xi 
1 
Background Information and Scientific Rationale ................................................................ 1 
1.1 
Background Information ........................................................................................... 1 
1.1.1 
Hookworm Infection and Justification for Vaccine Development .................. 1 
1.1.2 
Study Site ..................................................................................................... 2 
1.1.3 
Prior Clinical Experience with Hookworm Vaccines ..................................... 3 
1.1.4 
The Na-GST-1/Alhydrogel® Hookworm Vaccine .......................................... 4 
1.1.5 
Immunogenicity Studies with Na-GST-1 ....................................................... 8 
1.1.6 
Clinical Experience with Na-GST-1/Alhydrogel® Hookworm Vaccine .......... 9 
1.1.7 
The Na-APR-1 (M74)/Alhydrogel® Hookworm Vaccine .............................. 10 
1.1.8 
Studies of Human Immune Responses to Na-APR-1 in Endemic Areas ... 11 
1.1.9 
Clinical Experience with the Na-APR-1 (M74)/Alhydrogel® Hookworm 
Vaccine ....................................................................................................... 11 
1.1.1 
Clinical Experience with Na-APR-1 (M74)/Alhydrogel® Co-Administered 
with Na-GST-1/Alhydrogel® ........................................................................ 12 
1.1.2 
Clinical Experience with Aluminum-Based Adjuvants ................................ 13 
1.1.3 
Clinical Experience with GLA-AF ................................................................ 13 
1.2 
Rationale ................................................................................................................ 14 
1.2.1 
Rationale for the Study ............................................................................... 14 
1.2.2 
Rationale for Doses and Dose Schedule to be Studied ............................. 14 
1.2.3 
Clinical Development Plan for the Human Hookworm Vaccine .................. 15 
1.3 
Potential Risks and Benefits ................................................................................... 15 
1.3.1 
Potential Risks ............................................................................................ 15 
1.3.2 
Precautions Taken to Minimize Risks ......................................................... 16 
1.3.3 
Known Potential Benefits ............................................................................ 16 
2 
Objectives .......................................................................................................................... 18 
2.1 
Study Objectives .................................................................................................... 18 
2.2 
Study Outcome Measures ...................................................................................... 19 
2.2.1 
Primary Outcome Measures ....................................................................... 19 
2.2.2 
Secondary Outcome Measure .................................................................... 19 
2.2.3 
Tertiary Outcome Measures ....................................................................... 19 
2.2.4 
Exploratory Outcome Measures ................................................................. 20 
3 
Study Design ...................................................................................................................... 21 
4 
Study Enrollment and Withdrawal ...................................................................................... 23 
4.1 
Subject Inclusion Criteria ........................................................................................ 23 
4.2 
Subject Exclusion Criteria ...................................................................................... 23 
4.3 
Vaccine Assignment Procedures ........................................................................... 24 

Randomized, controlled, Phase 1 Study to assess safety and immunogenicity of co-administered Hookworm vaccine 
candidates Na-APR-1 (M74) and Na-GST-1 in Gabonese Children 
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4.3.1 
Randomization Procedures ........................................................................ 24 
4.3.2 
Allocation of vaccination schedule and vaccine randomization .................. 25 
4.3.3 
Masking Procedures ................................................................................... 25 
4.3.4 
Reasons for Withdrawal and Discontinuation of Vaccinations ................... 26 
4.3.5 
Handling of Withdrawals ............................................................................. 27 
4.3.6 
Termination of Study .................................................................................. 28 
5 
Study InterventionS/Investigational ProductS .................................................................... 29 
5.1 
Study Product Description ...................................................................................... 29 
5.1.1 
Acquisition .................................................................................................. 29 
5.1.2 
Formulation, Packaging, and Labeling ....................................................... 29 
5.1.3 
Product Storage and Stability ..................................................................... 30 
5.2 
Dosage, Preparation and Administration of Study Intervention/Investigational 
Product ................................................................................................................... 30 
5.3 
Modification of Study Intervention/Investigational Product for a Participant .......... 31 
5.4 
Accountability Procedures for the Study Intervention/Investigational Product(s) ... 31 
5.5 
Concomitant Medications/Treatments .................................................................... 31 
6 
Study Schedule .................................................................................................................. 33 
6.1 
Screening ............................................................................................................... 33 
6.2 
Enrollment/Baseline ............................................................................................... 34 
6.3 
Follow-up ................................................................................................................ 34 
6.4 
Final Study Visit ...................................................................................................... 37 
6.5 
Early Termination Visit ........................................................................................... 37 
6.6 
Unscheduled Visit ................................................................................................... 38 
7 
Study Procedures/Evaluations ........................................................................................... 39 
7.1 
Clinical Evaluations ................................................................................................ 39 
7.2 
Laboratory Evaluations ........................................................................................... 39 
7.2.1 
Clinical Laboratory Evaluations .................................................................. 39 
7.2.2 
Special Assays or Procedures .................................................................... 40 
7.2.3 
Specimen Preparation, Handling, and Shipping ......................................... 42 
8 
Assessment of Safety ........................................................................................................ 43 
8.1 
Specification of Safety Parameters ........................................................................ 43 
8.2 
Methods and Timing for Assessing, Recording, and Analyzing Safety 
Parameters ............................................................................................................. 43 
8.2.1 
Adverse Events .......................................................................................... 43 
8.2.2 
Reactogenicity ............................................................................................ 44 
8.2.3 
Serious Adverse Events ............................................................................. 47 
8.2.4 
Procedures to be Followed in the Event of Abnormal Laboratory Test 
Values or Abnormal Clinical Findings ......................................................... 48 
8.3 
Reporting Procedures ............................................................................................ 48 
8.3.1 
Serious Adverse Events ............................................................................. 48 
8.3.2 
Regulatory Reporting .................................................................................. 48 
8.3.3 
Adverse Events of Special Interest (AESI) ................................................. 49 
8.3.4 
Medically-Attended Adverse Events (MAAEs) ........................................... 49 

Randomized, controlled, Phase 1 Study to assess safety and immunogenicity of co-administered Hookworm vaccine 
candidates Na-APR-1 (M74) and Na-GST-1 in Gabonese Children 
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8.4 
Type and Duration of Follow-up of Subjects after Adverse Events ........................ 49 
8.5 
Halting Rules .......................................................................................................... 50 
8.6 
Safety Oversight ..................................................................................................... 51 
8.6.1 
Independent Medical Monitor (IMM) ........................................................... 51 
8.6.2 
Safety Monitoring Committee (SMC) .......................................................... 51 
9 
Clinical Monitoring .............................................................................................................. 53 
9.1 
Site Monitoring Plan ............................................................................................... 53 
10 
Statistical Considerations ................................................................................................... 54 
10.1 
Sample Size Considerations .................................................................................. 54 
10.2 
Planned Interim Analyses ....................................................................................... 55 
10.2.1 Safety Review ............................................................................................. 55 
10.2.2 Immunogenicity Review .............................................................................. 55 
10.3 
Final Analysis Plan ................................................................................................. 55 
11 
Source Documents and Access to Source Data/Documents ............................................. 60 
12 
Quality Control and Quality Assurance .............................................................................. 61 
13 
Ethics/Protection of Human Subjects ................................................................................. 62 
13.1 
Ethical Standards ................................................................................................... 62 
13.2 
Institutional Review Boards .................................................................................... 62 
13.3 
Informed Consent and Assent Process .................................................................. 62 
13.4 
Subject Confidentiality ............................................................................................ 63 
13.5 
Study Discontinuation ............................................................................................. 63 
13.6 
Future Use of Stored Specimens ........................................................................... 63 
14 
Data Handling and Record Keeping .................................................................................. 65 
14.1 
Data Management Responsibilities ........................................................................ 65 
14.2 
Data Capture Methods ........................................................................................... 65 
14.3 
Types of Data ......................................................................................................... 65 
14.4 
Timing/Reports ....................................................................................................... 65 
14.5 
Study Records Retention ....................................................................................... 66 
14.6 
Protocol Deviations ................................................................................................ 66 
15 
Publication Policy ............................................................................................................... 67 
16 
Literature References ........................................................................................................ 68 
Supplements/Appendices ........................................................................................................... 71 
Appendix A1: Schedule of Events for groups 1A, 2A, 3A ........................................................... 72 
Appendix A2: Schedule of Events for groups 1B, 2B, 3B ........................................................... 73 
 

Randomized, controlled, Phase 1 Study to assess safety and immunogenicity of co-administered Hookworm vaccine 
candidates Na-APR-1 (M74) and Na-GST-1 in Gabonese Children 
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LIST OF ABBREVIATIONS 
AE 
Adverse Event 
AESI 
Adverse Event of Special Interest 
AIGHD 
Amsterdam Institute for Global Health and Development 
APR-1 
Aspartic Protease-1 
CBC 
Complete Blood Count 
CERMEL 
Centre de Recherches Médicales de Lambaréné 
cGMP 
Current Good Manufacturing Practices 
CIOMS 
Council for International Organizations of Medical Sciences 
CRF 
Case Report Form 
ELISA 
Enzyme linked immunosorbent assay 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
GLA-AF 
Gluco-Pyranosylphospho-Lipid A Aqueous Formulation 
GMP 
Good Manufacturing Practice 
GST-1 
Glutathione S-Transferase 
HBsAg 
Hepatitis B surface antigen 
HBV 
Hepatitis B virus 
hCG 
Human choriogonadotropin 
HCV 
Hepatitis C virus 
HHVI 
Human Hookworm Vaccine Initiative 
HIV 
Human Immunodeficiency Virus 
IB 
Investigator’s Brochure 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation 
IEC 
Independent or Institutional Ethics Committee 
IM 
Intramuscular 
IND 
Investigational New Drug Application 
IP 
Investigational Product 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
MAAE 
Medically-Attended Adverse Event 
MedDRA® 
Medical Dictionary for Regulatory Activities 
MBC 
Memory B Cell 
MOP 
Manual of Procedures 
N 
Number (typically refers to subjects) 
Na 
Necator americanus 
PBMC 
Peripheral Blood Mononuclear Cell 
PI 
Principal Investigator 
QA 
Quality Assurance 
QC 
Quality Control 
SAE 
Serious Adverse Event 
SMC 
Safety Monitoring Committee 
SOP 
Standard Operating Procedure 
SVI 
BCM 
Sabin Vaccine Institute (Albert B Sabin Vaccine Institute; Sabin) 
Baylor college of medicine   
US 
United States 

Randomized, controlled, Phase 1 Study to assess safety and immunogenicity of co-administered Hookworm vaccine 
candidates Na-APR-1 (M74) and Na-GST-1 in Gabonese Children 
Version 3.0, 08 Aug 2018
 
 
 
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WBC 
White Blood Cell 
WHO 
World Health Organization 
 
 
 
 
 

Randomized, controlled, Phase 1 Study to assess safety and immunogenicity of co-administered Hookworm vaccine 
candidates Na-APR-1 (M74) and Na-GST-1 in Gabonese Children 
Version 3.0, 08 Aug 2018
 
 
 
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KEY ROLES  
 
 
Principal Investigator:  
 
 
 
 
Clinical Investigators: 
 
 
 
 
 
Regulatory Affairs 
Contact: 
 
 
(Site) Medical monitor:
 
 
 
 
Individuals: 
 
Ayola Akim Adegnika, MD, PhD. MSc 
Centre de Recherches Médicales de Lambaréné (CERMEL) 
Hôpital Albert Schweitzer, BP: 118  
Lambaréné, Gabon 
Tel: 00241 07406464  
Email: aadegnika@yahoo.fr  
 
 
 
 
 
Martin Grobusch, MD, PhD (Work package leader) 
David Diemert, MD 
Bertrand Lell, MD, MSc 
Sophia de Vries, MD, MSc 
Jeannot Zinsou, MD, MSc 
Yabo Josiane Honkpehedji, MD. MSc   
Jean-Claude Dejon Agobé, MD, MSc 
Romeo Adegbite, MD, MSc  
Jean Ronald Edoa, MD, MSc 
 
 
Maria Elena Bottazzi, PhD 
Baylor college of medicine  
Houston, Texas, USA   
 
 
 
Sèmèvo Fany Quenum, MD  
Albert Schweitzer Hospital 
Lambaréné, 
Gabon                                                                                             
 
 
 
Institutions: 
Clinical trial sponsor: 
 
Clinical trial site:  
 
 
Clinical Laboratory:  
 
 
 
Baylor College of Medicine  
Houston, Texas, USA 
 
 
Centre de Recherches Médicales de Lambaréné (CERMEL)   
Hôpital Albert Schweitzer, BP: 118  
Lambaréné, Gabon 
 
Clinical Laboratory - CERMEL 
Hôpital Albert Schweitzer, BP: 118  
Lambaréné, Gabon  
 
 

Randomized, controlled, Phase 1 Study to assess safety and immunogenicity of co-administered Hookworm vaccine 
candidates Na-APR-1 (M74) and Na-GST-1 in Gabonese Children 
Version 3.0, 08 Aug 2018
 
 
 
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Immunology 
Laboratories:  
 
 
 
 
 
 
 
 
 
Clinical Trial Monitoring 
and Data Management:  
 
 
 
Clinical Immunology Laboratory 
Dept. of Microbiology, Immunology & Tropical Medicine 
George Washington University (GWU) 
2300 I Street NW 
Ross Hall  
Washington, DC 20037 
 
 
Leiden Immunoparasitology Group 
Department of Parasitology  
Immunoparasite Laboratory 
Leiden University Medical Center 
Albinusdreef 2, 2333ZA 
 
P.O. Box 9600   
2300 RC Leiden 
The Netherlands  
 
 
 
Amsterdam Institute of Global Health and Development 
AHTC Building, Tower C4 
Paasheuvelweg 25 
1105 BP Amsterdam 
The Netherlands 
 
 
 

Randomized, controlled, Phase 1 Study to assess safety and immunogenicity of co-administered Hookworm vaccine 
candidates Na-APR-1 (M74) and Na-GST-1 in Gabonese Children 
Version 3.0, 08 Aug 2018
 
 
 
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PROTOCOL SUMMARY 
Title: 
Randomized, controlled, Phase 1 Study to assess safety and 
immunogenicity 
of 
co-administered 
Hookworm 
vaccine 
candidates 
Na-APR-1 
(M74)/Alhydrogel® 
and 
Na-GST-1/ 
Alhydrogel® in Gabonese Children  
Phase: 
1 
Population: 
Healthy male and female children aged 6-10 years, inclusive. 
Number of Sites: 
1 (Centre de Recherches Médicales de Lambaréné, Gabon) 
Study Duration: 
20 months 
Subject Participation 
Duration: 
15 months 
Description of 
Interventions: 
a) The 
Na-APR-1 
(M74) 
candidate 
vaccine 
contains 
recombinant Na-APR-1 (M74) adsorbed onto aluminum 
hydroxide gel (Alhydrogel®) and suspended in a solution 
containing 10mM imidazole, 150mM sodium chloride and 
0.3% Empigen BB, with pH 7.4 ± 0.1. The final concentrations 
of Na-APR-1 (M74) and Alhydrogel® in the drug product are 
0.1mg/ml and 0.8mg/ml, respectively. Doses of 10, 30 and 
100 µg Na-APR-1 (M74) will be delivered IM to the deltoid by 
injecting different volumes of the 0.1mg/ml preparation. 
b) The Na-GST-1 candidate vaccine contains the recombinant 
Na-GST-1 adsorbed onto Alhydrogel® and suspended in a 
solution containing 10mM imidazole and 10% glucose. The 
final concentrations of Na-GST-1 and Alhydrogel® in the drug 
product are 0.1mg/ml and 0.8mg/ml, respectively. Only one 
dose of Na-GST-1 will be tested (100µg), at a volume of 1.0ml 
delivered IM to the deltoid. 
c) Gluco-Pyranosylphospho-Lipid 
A 
Aqueous 
Formulation 
(GLA-AF) is a Toll-like Receptor-4 agonist. Point-of-injection 
formulations with this immunostimulant will be prepared 
immediately prior to vaccination by adding an appropriate 
volume of GLA-AF to Na-APR-1 (M74) and withdrawing an 
appropriate volume to administer the desired amount of Na-
APR-1 (M74) plus 5µg GLA-AF IM to the deltoid.   
d) The ENGERIX-B hepatitis B vaccine will serve as the 
comparator vaccine. Each 0.5 ml dose contains 20 µg 
recombinant hepatitis B surface protein expressed by 

Randomized, controlled, Phase 1 Study to assess safety and immunogenicity of co-administered Hookworm vaccine 
candidates Na-APR-1 (M74) and Na-GST-1 in Gabonese Children 
Version 3.0, 08 Aug 2018
 
 
 
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Saccharomyces cerevisiae and adsorbed to aluminum 
hydroxide will be injected IM to the deltoid. 
e) To maintain the study blind by ensuring that all subjects 
receive two injections at each vaccination point, sterile normal 
saline (0.9%) for injection will be co-administered to those 
subjects randomized to receive Hepatitis B vaccine. 
Objectives: 
 
Primary: 
Safety 
1. To evaluate the safety and reactogenicity of three different 
dose concentrations of Na-APR-1 (M74)/Alhydrogel® co-
administered with Na-GST-1/Alhydrogel® in healthy 
Gabonese children.  
 
Secondary: 
Immunogenicity 
1. To determine the doses of co-administered Na-APR-1 
(M74) and Na-GST-1 that result in the highest levels of 
IgG antibody approximately 14 days after the third 
vaccination.  
 
Tertiary: 
Immunogenicity 
1. To assess and compare the duration of the antibody 
responses of Na-GST-1 and Na-APR-1 (M74). 
2. To assess the distribution of IgG subclass responses to 
Na-GST-1 and Na-APR-1 (M74). 
 
Exploratory: 
1. To assess the cellular immune responses to the Na-GST-
1 and Na-APR-1 (M74) antigens following immunization.  
2. To assess the impact of co-administered Na-GST-1 and 
Na-APR-1 (M74) on the production of memory B cells 
specific for each antigen. 
3. To assess the production of memory B cells specific for 
each vaccine antigen compared to metabolomic changes 
before and after vaccination 
4. To characterize the metabolite composition in urine and 
serum during the course of immunization and assess the 
changes on metabolite profiles related to the co-
administration of Na-GST-1 and Na-APR-1 (M74). 

Randomized, controlled, Phase 1 Study to assess safety and immunogenicity of co-administered Hookworm vaccine 
candidates Na-APR-1 (M74) and Na-GST-1 in Gabonese Children 
Version 3.0, 08 Aug 2018
 
 
 
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Description of Study 
Design: 
Double blind, randomized, controlled, dose-escalation Phase 1 
clinical trial in hookworm-exposed children aged 6 to 10 years 
living in the area of Lambaréné, Gabon. Children will receive 
three doses of the assigned vaccine(s) delivered intramuscularly 
(deltoid) on approximately Days 0, 56, and 112 or 180.  
Safety will be measured from the time of each study vaccination 
(Day 0) through 14 days after each study vaccination by the 
occurrence of solicited injection site and systemic reactogenicity 
events.  
Unsolicited non-serious adverse events (AEs) will be collected 
from the time of the first study vaccination through approximately 
1 month after each study vaccination.  New-onset chronic medical 
conditions and Serious Adverse Events (SAEs) will be collected 
from the time of the first study vaccination through approximately 
9 months after the third study vaccination (final visit). Clinical 
laboratory evaluations for safety will be performed on venous 
blood collected approximately 14 days after each vaccination. 
Immunogenicity testing will include IgG antibody responses to 
each vaccine antigen, by a qualified indirect enzyme-linked 
immunosorbent assay (ELISA), on serum or plasma obtained 
prior to each study vaccination and at time points after each 
vaccination (see Appendix A); the functional activity of vaccine-
induced antibodies will be assessed by in vitro enzyme 
neutralization assays; the induction of B cell memory will be 
measured by antigen-specific memory B cell responses.   
Recruitment and enrollment into the study will occur on an 
ongoing basis, with each group being recruited and vaccinated in 
sequence. 
60 subjects will be enrolled into 3 groups of 20: 
The first 20 subjects will be assembled and enrolled into Group 
1: 
• 
Group 1 double-blind IP allocation (n=20):  
o 16 subjects will receive 10 µg Na-APR-1 (M74) plus 5 
µg GLA-AF delivered by IM injection in the deltoid 
muscle, with 10 µg Na-GST-1 administered IM in the 
alternate arm. 
§ 
8 will be vaccinated according to a 0,2,4-month 
schedule 
§ 
8 will be vaccinated according to a 0,2,6-month 
schedule 

Randomized, controlled, Phase 1 Study to assess safety and immunogenicity of co-administered Hookworm vaccine 
candidates Na-APR-1 (M74) and Na-GST-1 in Gabonese Children 
Version 3.0, 08 Aug 2018
 
 
 
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o 4 subjects will receive hepatitis B vaccine comparator: 
§ 
2 will be vaccinated according to a 0,2,4-month 
schedule 
§ 
2 will be vaccinated according to a 0,2,6-month 
schedule 
 
• 
Group 2 double-blind IP allocation (n=20):  
o 16 subjects will receive 30 µg Na-APR-1 (M74) plus 5 
µg GLA-AF delivered by IM injection in the deltoid 
muscle, with 30µg Na-GST-1 administered IM in the 
alternate arm. 
§ 
8 will be vaccinated according to a 0,2,4-month 
schedule 
§ 
8 will be vaccinated according to a 0,2,6-month 
schedule 
o 4 subjects will receive hepatitis B vaccine comparator: 
§ 
2 will be vaccinated according to a 0,2,4-month 
schedule 
§ 
2 will be vaccinated according to a 0,2,6-month 
schedule 
 
• 
Group 3 double-blind IP allocation (n=20):  
o 16 subjects will receive 100 µg Na-APR-1 (M74) plus 
5 µg GLA-AF delivered by IM injection in the deltoid 
muscle, with 100 µg Na-GST-1 administered IM in the 
alternate arm. 
§ 
8 will be vaccinated according to a 0,2,4-month 
schedule 
§ 
8 will be vaccinated according to a 0,2,6-month 
schedule 
o 4 subjects will receive hepatitis B vaccine comparator: 
§ 
2 will be vaccinated according to a 0,2,4-month 
schedule 
§ 
2 will be vaccinated according to a 0,2,6-month 
schedule 
 
Estimated Time to 
Complete Enrollment: 
 
3 months 
 

Randomized, controlled, Phase 1 Study to assess safety and immunogenicity of co-administered Hookworm vaccine 
candidates Na-APR-1 (M74) and Na-GST-1 in Gabonese Children 
Version 3.0, 08 Aug 2018
 
 
 
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Schematic of Study Design: 
Group Allocation: Open sequential enrollment   
Within Group IP Allocation: Double-blind randomized assignment   - - - - - - - - 
 
 
N=60
Group 1
10µg Na-APR-
1
+ 
10µg Na-GST-
1
(n=20)
Group 1a 
(n=10) 
10µg Na-APR-1                          
and 10µg Na-GST-1 at 0, 2, 4 
months (n=8)
HBV and Sterile Saline at 0, 
2, 4 months (n=2)
Group 1b
(n=10)
10µg Na-APR-1                          
and 10µg Na-GST-1 at 0, 2, 6 
months (n=8)
HBV and Sterile Saline at 0, 2, 
6 months (n=2)
Group 2
30µg Na-APR-
1
+ 
30µg Na-GST-
1
(n=20)
Group 2a 
(n=10)
30µg Na-APR-1                          
and 30µg Na-GST-1 at 0, 2, 4 
months (n=8)
HBV and Sterile Saline at 0, 2, 
4 months (n=2)
Group 2b
(n=10)
30µg Na-APR-1                          
and 30µg Na-GST-1 at 0, 2, 6 
months (n=8)
HBV and Sterile Saline at 0, 2, 
6 months (n=2)
Group 3
100µg Na-APR-
1
+ 
100µg Na-GST-
1
(n=20)
Group 3a 
(n=10)
100µg Na-APR-1                          
and 100µg Na-GST-1 at 0, 2, 
4 months (n=8)
HBV and Sterile Saline at 0, 2, 
4 months (n=2)
Group 3b 
(n=10)
100µg Na-APR-1                          
and 100µg Na-GST-1 at 0, 2, 
6 months (n=8)
HBV and Sterile Saline at 0, 
2, 6 months (n=2)

Randomized, controlled, Phase 1 Study to assess safety and immunogenicity of co-administered Hookworm vaccine 
candidates Na-APR-1 (M74) and Na-GST-1 in Gabonese Children 
Version 3.0, 08 Aug 2018 
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1 
1 
BACKGROUND INFORMATION AND SCIENTIFIC 
RATIONALE 
1.1 
Background Information 
1.1.1 Hookworm Infection and Justification for Vaccine Development 
There is an urgent need for new tools to control human hookworm infection and to reduce its 
burden of disease in developing countries. This is especially important for children and women of 
reproductive age who represent populations that are highly vulnerable to the effects of hookworm 
disease. Up to 65,000 deaths annually have been attributed to human hookworm infection (1). 
However, the mortality figures pale in comparison to global disease burden estimates that suggest 
that hookworm may account for the loss of up to 22 million Disability Adjusted Life Years annually 
(2), mainly due to the direct and indirect effects of acute and chronic anemia.  
 
Human hookworm infection is a soil-transmitted helminth infection caused by the nematode 
parasites Necator americanus and Ancylostoma duodenale. It is one of the most common chronic 
infections of humans, affecting over 400 million people in the developing nations of the tropics 
(2). The largest number of cases occurs in impoverished rural areas of sub-Saharan Africa, 
Southeast Asia, China, and the tropical regions of the Americas. Approximately 3.2 billion people 
are at risk for hookworm infection in these areas.  N. americanus is the most common hookworm 
worldwide, whereas A. duodenale is more geographically restricted (3).  
 
Hookworm transmission occurs when skin comes into contact with infective third-stage larvae 
(L3) in fecally contaminated soil. The L3 have the ability to penetrate the skin, usually of the 
hands, feet, arms, buttocks and legs. The L3 invade human tissues and enter the gastrointestinal 
tract where they molt to the adult stage approximately 5-9 weeks following initial host entry. Adult 
hookworms are approximately 1 cm long parasites that cause host injury by attaching to the 
mucosa and submucosa of the small intestine to produce intestinal blood loss. There is a direct 
relationship between hookworm intensity (as determined by fecal egg counts) and host blood loss; 
typically the presence of between 40 and 160 adult hookworms in the intestine results in blood 
loss sufficient to cause anemia and malnutrition. The term “hookworm disease” refers primarily to 
the iron deficiency anemia and protein losses that occur in moderate and heavy infections (4). 
When host iron stores become depleted, there is a direct correlation between hookworm intensity 
and reduced host hemoglobin, serum ferritin, and protoporphyrin.  Because of their low iron 
stores, children and women of reproductive age are the populations considered the most 
vulnerable to hookworm-associated blood loss (4-11).   
 
In children, chronic hookworm infection and the resultant iron deficiency anemia have been shown 
to impair physical and intellectual development (3, 12, 13).  Preschool children are particularly 
vulnerable to the effects of hookworm anemia and disease (8).  In addition to its health impact on 
children, hookworm infection also affects adults. Unlike other soil-transmitted helminth infections 
such as ascariasis and trichuriasis, in which the highest intensity infections occur almost 
exclusively in school-aged children, it has been shown that high-intensity hookworm infections 
may also occur in adults (14-16).   
 

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The primary approach to hookworm control worldwide has been the frequent and periodic mass 
administration of benzimidazole anthelminthics to school-aged children living in high-prevalence 
areas. In 2001, the World Health Assembly adopted Resolution 54.19, which urges member 
states to provide regular anthelminthic treatment to high-risk groups with the target of regular 
treatment of at least 75% of all at-risk school-aged children. However, cure rates for a single dose 
of a benzimidazole are sub-optimal, particularly for mebendazole (17-20). These concerns have 
prompted interest in developing alternative tools for hookworm control (3, 21, 22). Vaccination to 
prevent the anemia associated with moderate and heavy intensity hookworm infection would 
alleviate the public health deficiencies of drug treatment alone. 
 
The feasibility of developing a hookworm vaccine is based on the previous success of using live, 
irradiated hookworm larvae (L3 stage) as a vaccine for canine hookworm infection. This provided 
the experimental basis for the commercial development of a canine hookworm vaccine, which 
was marketed in the United States during the early 1970s. However, it is not realistic to develop 
a live L3 vaccine for humans due to multiple reasons including high production costs, challenging 
storage requirements, a short shelf life, and a lack of sterilizing immunity. 
 
Alternatively, the strategy being pursued is to identify key hookworm proteins to which protective 
immune responses are directed in the animal models for this infection (namely the canine model) 
and to produce these as recombinant proteins that could then be used as vaccine antigens. This 
effort focused initially on identifying antigens expressed by the invading larvae (L3). In addition, a 
separate strategy has been to identify targets of the adult stage of the hookworm lifecycle; since 
hookworms attach onto the intestinal lumen and ingest host blood, antibodies could also be 
ingested that if directed against key hookworm proteins, would interfere with their function, 
ultimately resulting in the death or reduced fecundity of the worm.   
1.1.2 Study Site 
The study will be conducted in Lambaréné, Gabon, a city located in the Province of Moyen 
Ogooué, 240 km from Libreville, the capital of Gabon.  There are approximately 35,000 people 
living in the urban municipal center of Lambaréné, with another 50,000 living in the surrounding 
rural areas. The area is located in the Central Africa rain forest setting at around 0°42’05S of 
latitude and 10°14’25E of longitude. The temperature average over the year is around 27°C. 
There are two annual rainy seasons (September to December and March to June) and two dry 
seasons (July to August and January to February). The city is bisected by the main river delta of 
the Ogooué. The majority of inhabitants in the urban area have tap water or access to supply 
(public wells) in the urban area, but in the rural areas, the water is supplied directly from small 
rivers. Latrines are common in the urbanised areas but not in rural areas. Income is generated 
mainly from farming, fishing and services. The urban part has two main hospitals and five 
pharmacies (“dispensaires”).  Although rural areas are also served by several dispensaries, the 
quality of care varies. Plasmodium falciparum malaria, schistosomiasis, filariasis (Loa loa and 
Mansonella perstans), as well as soil-transmitted helminths including hookworm, Ascaris 
lumbricoides, Strongiloides stercoralis and Trichuris trichiura are endemic in the area.   
 
Lambaréné has been chosen as a site for testing hookworm vaccines based on prevalence 
surveys conducted over the past few years, and the good working relationship that has been 
established between the research staff, the local health authorities, and the community.  A recent 
survey using real time PCR yielded that 51% of 210 study participants assessed were found to 

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be positive for hookworm. The species identification yielded exclusively Necator americanus.  
(Adegnika et al working group) 
1.1.3 Prior Clinical Experience with Hookworm Vaccines 
 
The first hookworm vaccine to be tested in humans was the Na-ASP-2 (Ancylostoma Secreted 
Protein-2 of N. americanus) Hookworm Vaccine, consisting of recombinant Na-ASP-2 expressed 
in Pichia pastoris and adsorbed to aluminum hydroxide gel (Alhydrogel®). Na-ASP-2 is an 
excretory/secretory product produced by infective N. americanus larvae upon penetration of 
human skin. In animal models, vaccination with this recombinant antigen was shown to result in 
reduced worm burdens after challenge infection. Accordingly, a Phase 1 clinical trial of several 
different dose concentrations of the vaccine was conducted in healthy, hookworm-naïve adults 
living in the United States, which showed the formulation to be safe, well tolerated and 
immunogenic (23).   
 
However, upon testing the vaccine in adults who had previously been infected with hookworm in 
Americaninhas, several volunteers in the lowest dose cohort to be vaccinated developed 
generalized urticaria within 2 hours of immunization (24). Due to these immediate-type 
hypersensitivity reactions, vaccinations in this study were halted. Subsequent investigations 
revealed that the volunteers who developed urticaria upon their first dose of Na-ASP-2 had 
elevated levels of baseline (i.e., pre-vaccination) IgE to the vaccine antigen. Subsequently, a sero-
epidemiological survey was conducted in an endemic region of Brazil; this study revealed that 
even in young children, a significant proportion of individuals have detectable levels of IgE to this 
protein, likely due to previous infection with N. americanus (Figure 1). In addition, similar findings 
were demonstrated for other larval proteins that were being considered as vaccine candidates.  
 
 
 

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Figure 1: Anti-Na-ASP-2 IgE levels in residents of a hookworm-endemic region of 
northeastern Minas Gerais state, Brazil. Levels are shown for individuals of various age 
groups, in addition to 9 participants of the Phase 1 trial of Na-ASP-2 in Brazil and the 
participants of the Phase 1 trial of Na-ASP-2 that was conducted in hookworm-naïve adults in 
the United States (24).  
 
Due to these cumulative data, clinical development of the Na-ASP-2 and other larval-stage 
antigens as candidate vaccines was halted. Instead, the current strategy is to develop antigens 
expressed during the adult stage of the hookworm life cycle that play a role in digesting the host 
hemoglobin that is used by the worm as an energy source. These antigens do not induce specific 
IgE antibodies during natural infection, and hence have a low likelihood for inducing allergic 
reactions upon vaccination.   
1.1.4 The Na-GST-1/Alhydrogel® Hookworm Vaccine   
 
The nutritional and metabolic requirements of the adult hookworm living in the human intestine 
are dependent upon degradation of host hemoglobin that has been ingested by the worm. N. 
americanus hookworms depend on host hemoglobin for survival (25). Following hemolysis, adult 
hookworms use an ordered cascade of hemoglobinases to cleave hemoglobin into smaller 
molecules (25-30). Aspartic protease-1 of N. americanus (Na-APR-1) is responsible for initiating 
the proteolytic cascade in hookworms, as described below (Section 1.1.7). After hemoglobin 
digestion, the freed heme generates toxic oxygen radicals that can be bound and detoxified by 
molecules such as glutathione S-transferase-1 (GST-1) (31-33). GST-1 of N. americanus (Na-
GST-1) is a critical enzyme that plays a role in parasite blood feeding; used as a vaccine, we 
hypothesize that the antigen will induce anti-enzyme neutralizing antibodies that will interfere with 
parasite blood-feeding and cause parasite death or reduce worm fecundity.   

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Na-GST-1 is a 24-kDa protein with peroxidase enzymatic activity that catalyzes the conjugation 
of reduced glutathione to a variety of electrophiles (31-33). This hookworm protein belongs to the 
Nu class of nematode GSTs, which also includes GSTs from the blood-feeding parasite of 
ruminants, Haemonchus contortus, and the rodent nematode Heligomosoides polygyrus. This 
class is characterized by diminished peroxidase activity relative to other classes of GSTs, but 
elevated binding capacity for heme and related products (31, 33-36). X-ray crystallography of Na-
GST-1 demonstrates that the protein can form homodimers in solution, which create atypically 
large binding cavities accessible to a diversity of ligands, including heme (Figure 2) (33). Na-
GST-1 binds heme at high affinity in vitro (31, 36). Because both heme and hematin contain 
oxidative iron, these molecules are potent generators of toxic reactive oxygen species that could 
potentially damage parasite macromolecules. In vivo, hookworm GSTs may therefore bind and 
detoxify the heme and hematin byproducts generated during the blood degradation process.   
 
Figure 2: Three-dimensional surface plot of Na-GST-1. The path to the binding cavity is 
indicated by the red arrow (33). 
 
 
 
Based on their putative role in hookworm blood feeding, both Na-GST-1 and its orthologue from 
the canine hookworm Ancylostoma caninum (Ac-GST-1) were tested as experimental vaccines 
in laboratory animal models of infection. In dogs, vaccination with recombinant Ac-GST-1 resulted 
in high levels of antigen-specific antibody (Figure 3); following challenge with A. caninum infective 
larvae, significantly lower host worm burdens and fecal egg counts were observed compared to 
control animals vaccinated only with adjuvant (31). In hamsters, vaccination with recombinant Ac-
GST-1 also resulted in substantially lower worm burdens (51-54%) following heterologous 
challenge with N. americanus infective larvae compared to controls, as did vaccination with 
recombinant Na-GST-1 followed by homologous larval challenge (31, 32, 37).  Because of these 
encouraging preclinical results, Na-GST-1 was manufactured according to current good 
manufacturing practices (cGMP) and formulated on Alhydrogel® in preparation for clinical trials. 
 
 
 
 
Figure 3: Geometric mean titers of the IgG1 and IgG2 antibody responses of 
vaccinated dogs against recombinant Ac-GST-1 formulated with GlaxoSmithKline’s 

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AS03 adjuvant. Vaccination time points (1, 2, 3, and 4) and challenge day (C) are marked 
with arrows (31). 
 
 
 
 
Most importantly, extensive studies have been conducted to test for sensitization to the Na-GST-
1 protein in individuals living in a hookworm endemic area who have been repeatedly exposed 
and infected with N. americanus. As shown in Figure 4, over 1000 individuals of all ages from a 
hookworm endemic area of Brazil have been tested for serum IgE antibodies to Na-GST-1 using 
an indirect ELISA. In addition, a subset of these serum samples stratified by age and infection 
status (n = 179) underwent confirmatory testing at the Johns Hopkins Dermatology, Allergy and 
Clinical Immunology Reference Laboratory (Baltimore, Maryland) using a custom ImmunoCAP 
assay. The ImmunoCAP method is considered the standard for measuring specific IgE to antigens 
in serum. This confirmatory testing demonstrated that none of the samples had Na-GST-1 IgE 
values above the clinical cut-off of 0.35 kUA/L (Figure 5). Therefore, the likelihood of inducing 
immediate-type hypersensitivity reactions by vaccinating individuals living in hookworm-endemic 
areas with Na-GST-1 is low and likely not more than that associated with any new vaccine antigen 
entering clinical trials. The situation with Na-GST-1 is therefore very different from that of Na-
ASP-2 in that repeated infection with hookworm does not induce an IgE response to the antigen, 
most likely due to the fact that it is a protein found in the digestive tract of adult hookworms and 
is therefore relatively hidden from the human immune system. This lack of antigen-specific IgE in 
people living in an area of high transmission has served as a major justification for advancing 
development of Na-GST-1 as a candidate vaccine antigen. 
 
 
 
Figure 4: Anti-Na-GST-1 IgE levels in (A) adults and children and (B) young children 
aged 1-10 years (n=128) living in a hookworm-endemic area of Brazil. IgE levels (optical 
density at 492 nm) were measured by indirect ELISA. 

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A. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
B. 
 
 
 
 
 
Figure 5: Anti-Na-GST-1 IgE levels in a subset of adults and children (n=179) living in 
a hookworm-endemic area of Brazil.  IgE levels (kUA/L) were measured by custom 
ImmunoCAP.  
SAA-2
ASP-2
GST-1
APR-1
0.000
1.000
2.000
3.000
4.000
IgE
OD 492
GST-1
0.000
1.000
2.000
3.000
4.000
OD 492 nm

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Na-GST-1 has been successfully manufactured and tested in the laboratory and in animals with 
both Alhydrogel® and Alhydrogel® plus GLA-AF. Na-GST-1 has been shown to be pure, potent, 
and stable in both of these two formulations.  
 
The Na-GST-1 vaccine formulation to be tested in this study consists of the 24-kDa recombinant 
protein Na-GST-1, adsorbed to an adjuvant, Alhydrogel® (aluminum hydroxide suspension) with 
or without the addition of Gluco-Pyranosylphospho-Lipid A Aqueous Formulation (GLA-AF).  The 
GLA-AF will be added to the Alhydrogel® formulation within 24 hours of immunization. The active 
ingredient is the recombinant Na-GST-1 protein that is derived by fermentation of Pichia pastoris 
yeast cells genetically engineered to express Na-GST-1.     
1.1.5 Immunogenicity Studies with Na-GST-1 
 
Several preclinical animal studies have been conducted in both mice and rats to assess the 
immunogenicity of Na-GST-1 in combination with different adjuvants. First, a study conducted in 
Sprague-Dawley Rats demonstrated that the addition of an adjuvant to recombinant Na-GST-1 
was necessary, since administration of the recombinant protein without an adjuvant resulted in 
minimal specific antibody responses.   
 
A second study was conducted in BALB/c mice to assess the effect of co-administering CPG 
10104 with recombinant Na-GST-1/Alhydrogel® (Figure 6). In this study, mice were vaccinated 
with Na-GST-1/Alhydrogel® at antigen doses ranging from of 0.01 to 31.25 µg Na-GST-1 with or 
without CPG 10104 (5 µg) or CPG 10105 (5 µg). CPG 10105 is a CpG oligodeoxynucleotide 
sequence that is similar to CPG 10104 but that is not being proposed to be tested in the study 
described in this protocol. Mice were vaccinated twice intramuscularly at a 3-week interval, with 
blood collected for anti-Na-GST-1 IgG ELISA two weeks after the second immunization. This 
study demonstrated a large, highly significant increase in IgG specific for Na-GST-1 in the group 

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administered Na-GST-1/Alhydrogel®/CPG 10104 compared to that administered only Na-GST-
1/Alhydrogel® as shown in Figure 6.     
 
Figure 6: Geometric mean anti-Na-GST-1 IgG antibody units 2 weeks after the 2nd 
Immunization of BALB/c mice with Na-GST-1/Alhydrogel® with or without co-
administration of CPG 10104 or CPG 10105. 
 
 
1.1.6 Clinical 
Experience 
with 
Na-GST-1/Alhydrogel® 
Hookworm 
Vaccine   
 
The Na-GST-1/Alhydrogel® hookworm vaccine has been tested in adults in two Phase 1 clinical 
trials in humans in the United States (n=40), one in Brazil (n=102), and one in Gabon (n=32), the 
latter when coadministered with Na-APR-1/Alhydrogel®. To date, no significant reactogenicity or 
safety issues have been observed in any of these studies. In study SVI-11-01 in healthy, 
hookworm-naïve American adults conducted between 2012 and 2014 in Washington, DC, 40 
volunteers received three vaccinations with up to 100 µg Na-GST-1/Alhydrogel® administered 
with or without up to 5 µg GLA-AF (NCT01385189). Mild to moderate injection site pain and 
tenderness were observed in a minority of study subjects; other common adverse events have 
included mild to moderate headache and nausea. The vaccine induced antigen-specific IgG 
antibody responses in a dose-dependent manner, although the addition of GLA-AF did not 
appreciably increase these responses over Na-GST-1/Alhydrogel® alone. 
 
The trial in Brazil (SVI-10-01) tested Na-GST-1/Alhydrogel® in both hookworm-unexposed (n=36) 
and hookworm-exposed (n=66) healthy adults (NCT01261130). This study was initiated in Belo 

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Horizonte, a large urban area in the Brazilian state of Minas Gerais, in healthy, hookworm-
unexposed adults (n=36) who received up to 100 µg Na-GST-1/Alhydrogel® with or without 2.5 
µg GLA-AF. After no significant adverse events were observed in these volunteers, vaccinations 
were begun at the hookworm-endemic region of Americaninhas in Minas Gerais; this second part 
of the trial was a randomized, controlled, double-blind study in which subjects received up to 100 
µg Na-GST-1/Alhydrogel® with or without 2.5 µg GLA-AF (n=60), or the recombinant hepatitis B 
vaccine (n=6). All volunteers in this study received three intramuscular injections at 0, 2, and 4 
months and are being followed for 12 months after the final vaccination. In this study, Na-GST-
1/Alhydrogel® (with or without GLA-AF) appeared to be safe and well tolerated in both hookworm-
unexposed and hookworm-exposed adults. No vaccine-related SAEs have occurred and the 
vaccine appears to be well tolerated, with mild-to-moderate injection site pain and tenderness 
being the most common vaccine-related adverse events. Also, it is important to note that all 
volunteers who were screened for study SVI-10-01 were tested for IgE antibodies to Na-GST-1: 
none of these individuals in either Belo Horizonte or Americaninhas had detectable IgE antibodies 
to the vaccine antigen, as determined by ELISA.  As in SVI-11-01, Na-GST-1/Alhydrogel® induced 
antigen-specific IgG responses in a dose-dependent fashion, but the addition of GLA-AF did not 
improve these responses. Due to the lack of improvement in antibody responses with the addition 
of GLA-AF to Na-GST-1/Alhydrogel®, the regulatory sponsor of the vaccine has recommended 
that GLA-AF should not be tested in combination with this product in future clinical trials. 
 
1.1.7 The Na-APR-1 (M74)/Alhydrogel® Hookworm Vaccine   
 
As described above, hookworms acquire their nutrition by ingesting blood, lysing the erythrocytes, 
and digesting the hemoglobin and serum proteins in the gut of the adult worm via a proteolytic 
cascade (28).  Hookworm hemoglobinases have provided efficacy as recombinant subunit 
vaccines in animal models of hookworm disease, resulting in significant reductions in the intensity 
of infection and, most importantly, in protection against blood loss (29-31, 36). The Na-GST-1 and 
Na-APR-1 proteins are both components of the blood-feeding pathway of N. americanus and 
were selected for clinical development based on their protective efficacy in animal trials. Na-APR-
1 is responsible for initiating the proteolytic cascade in N. americanus and catalytically active 
recombinant APR-1 has been found to confer protection in canine and hamster models of human 
hookworm disease (28, 30, 36). Vaccinated animals are thought to be protected as a result of 
induced antibodies that neutralize the catalytic activity of Ac-APR-1 in the gut of the worm (30).  
 
To improve the stability of the recombinant enzyme, two aspartic acid residues were mutated to 
alanines to make Na-APR-1mut, which is the same as Na-APR-1 (M74), a catalytically inactivated 
mutant protein. Dogs vaccinated with recombinant Na-APR-1mut were protected against blood loss 
and pathology; importantly, animals that were vaccinated with Na-APR-1mut generated antibodies 
that neutralized the catalytic activity of wild type Na-APR-1 (29).  Despite successfully producing 
large quantities of refolded Na-APR-1 and Na-APR-1mut in Escherichia coli, the scalability of 
protein expression was hampered by issues of yield and aggregation (29). Therefore, as an 
alternative strategy, recombinant Na-APR-1 (M74) is derived by infiltration of Agrobacterium 
tumefaciens strain GV3101, genetically engineered to express Na-APR-1 (M74) in Nicotiana 
benthamiana tobacco plants. 

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The Na-APR-1 (M74)/Alhydrogel® vaccine is composed of the 42.18-kDa Na-APR-1 (M74) 
recombinant protein (0.1 mg/ml of the recombinant protein in 10 mM imidazole, 150 mM sodium 
chloride and 0.3% Empigen BB, with pH 7.4 ± 0.1), together with 0.8 mg/ml Alhydrogel®.  
1.1.8 Studies of Human Immune Responses to Na-APR-1 in Endemic 
Areas   
 
To assess presence of antibodies against Na-APR-1mut in humans resident in N. americanus 
endemic areas, sera from 648 hookworm-infected individuals from Minas Gerais state, Brazil, 
were screened for anti-APR-1mut antibody responses. Sixty-two percent showed detectable IgG 
responses (42% had IgG1, 0.6% IgG2, 15% IgG3, 53% IgG4) and only 0.6% had IgE antibodies 
to Na-APR-1mut (Table 1). There was no association between the presence of antibody and current 
infection status (positive or negative), intensity of infection (EPG), or age.  
Table 1: Anti-Na-APR-1mut IgG, IgG subclasses (IgG1-4), IgE, and IgA Antibodies in 
Brazilian Individuals Living in a Hookworm-Endemic Area. 
ELISA method 
IgG1 
IgG2 
IgG3 
IgG4 
Total IgG 
IgE 
IgA 
Seropositive [% 
(n)]a 
42 (270) 
0.6 (4) 
15 (97) 
53 (343) 
62 (401) 
0.6 (4) 
3 (22) 
Levels in μg/mlb 
0.195 ± 
0.104 
−c 
0.123 ± 
0.066 
0.203 ± 
0.136 
0.592 ± 
0.518 
− 
− 
 
a Positivity as indicated by a reactivity threshold, which was determined from a frequency distribution from the upper 95% CI 
of the frequency distribution from log10 transformed OD values from a panel of sera from Brazilian volunteers not resident in 
an N. americanus transmission area (n=56) and a panel of control sera from U.S. volunteers (24).  
b Values are means ± SD. Amounts were not calculated for samples in which <25 individuals were positive for the antibody. 
c Heterologous interpolation of antibody levels to Na-APR-1mut refers to a calibration method in which the standard curve uses 
a set of different reagents from those used to measure the analyte of interest; in this case, Na-APR-1mut-specific antibody 
ELISA was simultaneously performed with purified human IgG, IgG1-4 and IgE bound to the solid phase followed by the 
appropriate 2nd antibody. The antibody values are determined by interpolating the OD of the sample values onto the 
heterologous  standard curve. 
 
The presence of IgG1 to Na-APR-1 in sera from humans resident in hookworm endemic areas 
resolves one of the most critical obstacles of gut antigens as targets for vaccines against blood-
feeding helminths. Many consider antigens in the gut membrane of blood-feeding nematodes as 
“hidden,” i.e., not continuously presented to the immune system (34). The presence of circulating 
IgG1 in individuals in Necator endemic areas indicates that natural infection induces a host 
immune response to Na-APR-1 and that further immunization could augment this existing immune 
response. Importantly, the immune response to Na-APR-1 is neither of the IgG4 or IgE isotype 
as seen with other helminth antigens. This might be the result of expression of Na-APR-1 in the 
host gastrointestinal tract, compared to other organs through which hookworms pass, such as the 
skin or lung, and bias the immune response toward a T-helper type 2-cytokine profile.  Based on 
the above studies, it is unlikely that Na-APR-1 (M74) will elicit an IgG4 or an IgE antibody 
response upon vaccination of individuals in high transmission areas, making this vaccine a highly 
viable hookworm vaccine candidate. 
1.1.9 Clinical 
Experience 
with 
the 
Na-APR-1 
(M74)/Alhydrogel® 
Hookworm Vaccine 
 

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The Na-APR-1 (M74)/Alhydrogel® Hookworm Vaccine has been tested in healthy, hookworm-
unexposed adults in a Phase 1 clinical trial taking place in Washington, DC.  In this study (SVI-
12-01), 40 volunteers were vaccinated with either 30 µg or 100 µg Na-APR-1 (M74)/Alhydrogel® 
with or without 2.5 or 5 µg GLA-AF (NCT01717950). Three intramuscular vaccinations were 
administered to each volunteer according to a 0, 2, and 4-month schedule. The study was 
completed in July 2015. No vaccine-related SAEs were observed, and related AEs were limited 
mostly to mild-to-moderate injection site pain and tenderness. Immunogenicity results from this 
study demonstrated that Na-APR-1 (M74)/Alhydrogel® induces antigen-specific IgG responses in 
a dose-dependent manner, and that the addition of 5 µg GLA-AF noticeably improved these 
responses (Figure 7).  
 
Figure 7. IgG responses in study SVI-12-01 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1.1.1 Clinical Experience with Na-APR-1 (M74)/Alhydrogel® Co-Administered 
with Na-GST-1/Alhydrogel® 
The current two candidate hookworm vaccines described above are being tested together in a 
Phase 1 clinical trial in Lambaréné, Gabon, at the same site as proposed in this protocol.  In this 
study, HV001 (NCT02126462), 32 healthy, hookworm-exposed adults were vaccinated with 30 
µg or 100 µg of both Na-GST-1/Alhydrogel® and Na-APR-1 (M74)/Alhydrogel® (n = 24) versus the 
comparator Hepatitis B Vaccine (n = 8).  Both Na-GST-1/Alhydrogel® and Na-APR-1 
(M74)/Alhydrogel® were mixed with 5 µg GLA-AF immediately prior to injection.  Each study 
subject received three sets of intramuscular injections (one in each deltoid muscle) according to 
a 0, 1, and 6-month schedule. Vaccinations in this study were completed in September 2015. No 
vaccine-related SAEs were observed, and related AEs were limited mostly to mild-to-moderate 
injection site pain and tenderness, headache, nausea, and myalgia. Twelve subjects had 19 
severe (grade 3) adverse events, of which one was an episode of high (severe) fever that was 
possibly related to vaccination while the rest were not considered related to vaccination.  It should 
be noted that the study is yet to be unblinded and therefore it is not possible to state whether the 
AEs occurred in subjects who received the hookworm vaccines vs. the comparator hepatitis B 
vaccine.   
Geometric Mean Fold Increase 
(95% Confidence Intervals) 

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1.1.2 Clinical Experience with Aluminum-Based Adjuvants 
 
Several licensed vaccines contain aluminum-based adjuvants, including the recombinant 
hepatitis B vaccine, the tetanus toxoid vaccine, and the diphtheria-tetanus toxoids vaccine.(38, 
39) For these aluminum hydroxide-adsorbed vaccines, local reactions such as pain, tenderness, 
and swelling are experienced in between 7.6% and 16.7% of volunteers in studies that included 
over 1,200 healthy adults. Fever is seen in 3.2% to 9.3%, headache in 4.1%, and other systemic 
symptoms such as fatigue, malaise, nausea, and diarrhea at lower frequencies.  Urticaria has 
been reported in 0.1% of individuals vaccinated with the hepatitis B vaccine.   
1.1.3 Clinical Experience with GLA-AF 
 
Both Na-GST-1/Alhydrogel® and Na-APR-1 (M74)/Alhydrogel® are being tested in combination 
with the Toll-like Receptor 4 (TLR4) agonist, Gluco-Pyranosylphospho-Lipid A in Aqueous 
Formulation (GLA-AF, Infectious Diseases Research Institute [IDRI], Seattle, WA).  GLA-AF 
contains a synthetic monophosphoryl lipid A (MPL) molecule that has TLR4 agonist activity. MPL 
is itself derived from the lipopolysaccharide (LPS) of Salmonella minnesota, a natural TLR4 
agonist that is pyrogenic and can induce toxic shock. LPS, and more specifically, its lipid A 
component, has long been known for its strong adjuvant effects; however, its high toxicity has 
precluded its use in a vaccine formulation. Ribi et al showed that the monophosphorylated form 
of lipid A retains its adjuvant function and almost completely loses its endotoxin effects (40).  
 
There have been many clinical trials involving thousands of subjects in which MPL or a derivative 
have been administered as vaccine adjuvants to adults and children, including vaccines for 
human papillomavirus, malaria (41-43), leishmaniasis (44), and hepatitis B (45).  In general, these 
trials have demonstrated that administering MPL to humans is safe and well tolerated; when 
compared to formulations of vaccine that do not contain MPL, those adjuvanted with MPL may 
result in a minor increase in the incidence and/or severity of local injection site reactions. However, 
the addition of MPL also often results in a much improved specific antibody response to the 
vaccine antigen(s).   
 
Of note, MPL is one of the components of the licensed Cervarix® vaccine (GlaxoSmithKline, 
Research Triangle Park, NC) for the prevention of cervical cancer due to human papillomavirus 
serotypes 16 and 18. The adjuvant for this vaccine consists of MPL adsorbed to aluminum 
hydroxide salt and is therefore similar to the combination of GLA-AF and Alhydrogel® that we 
propose testing in combination with Na-GST-1 and Na-APR-1 (M74) in the study described in this 
protocol. The Cervarix® vaccine has been shown to have a very favorable safety profile after 
having been tested in tens of thousands of healthy individuals (46, 47). 
 
As mentioned previously, up to 5 µg of GLA-AF has been administered to a small number of 
human 
volunteers 
in 
combination 
with 
both 
Na-GST-1/Alhydrogel® 
and 
Na-APR-1 
(M74)/Alhydrogel®. In addition, an oil-in-water emulsion of GLA (GLA-SE) has been used in 
combination with the Fluzone® trivalent killed influenza vaccine in a Phase 1 trial. In this study, 
doses up to 2.5 µg of GLA-SE were safe and well-tolerated and significantly enhanced influenza-
specific antibody responses (48). 

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1.2 
Rationale 
1.2.1 Rationale for the Study 
 
A product that combines Na-GST-1 and Na-APR-1 (M74) shows promise as an effective bivalent 
hookworm vaccine because vaccination of laboratory animals with recombinant Na-GST-1 or Na-
APR-1 results in significant protection from challenge infections (29, 37). Therefore, vaccination 
of humans with recombinant Na-GST-1 or Na-APR-1 (M74) holds promise for inducing protection 
against this infection, particularly the moderate and heavy intensity infections that are associated 
with clinical sequelae such as intestinal blood loss and iron-deficiency anemia. The Sabin Vaccine 
Institute has sponsored a series of Phase 1 trials of the Na-GST-1 and Na-APR-1 (M74) candidate 
antigens in healthy adult volunteers from hookworm endemic areas (Brazil) and non-endemic 
areas (USA). Given the results of preclinical testing in laboratory animals, it is thought that a 
vaccine based on a single antigen will not be sufficient to provide adequate protection from 
disease (49). Instead, the goal is to develop a multi-component vaccine containing at least two 
recombinant proteins targeting different steps in the hookworm digestion of host hemoglobin.  As 
such, a necessary step in clinical development is to test whether combining Na-GST-1 and Na-
APR-1 (M74) results in an increased safety risk or reduced immunogenicity to either antigen, 
compared to when they are administered individually. 
 
As a necessary first step, Na-GST-1 and Na-APR-1 (M74) were co-administered to healthy adults 
in Gabon, as described above in Section 1.1.1.  However, given that the main target for an 
eventual vaccine will be children living in hookworm-endemic areas, pediatric Phase 1 trials are 
required to evaluate safety and immunogenicity in the target population prior to conducting larger-
scale Phase 2 and 3 clinical trials.  
1.2.2 Rationale for Doses and Dose Schedule to be Studied  
Na-APR-1 (M74)/Alhydrogel® and Na-GST-1/Alhydrogel® (with or without GLA-AF) have been 
tested at the same field site as that proposed in the study described in this protocol, in healthy 
hookworm-exposed adults without any observed safety concerns.  Therefore, in the study 
described in this protocol, these two vaccine products will be administered in escalating doses, to 
school-age children who live in an area where hookworm is endemic.  Doses of 10, 30 and 100 
μg of Na-APR-1 (M74) and Na-GST-1/Alhydrogel® were selected for evaluation based on 
preclinical studies and to maintain an equal fold-increase in the amount of antigen. The 10 μg 
dose was also selected as the lowest dose for clinical trials due to the difficulty in precisely 
dispensing lower volumes (both vaccine antigens have been formulated at a protein concentration 
of 100μg/ml).  Selection of the maximum 100 μg doses of Na-GST-1 and Na-APR-1 (M74) is 
based on the safety and immunogenicity data from the Phase 1 studies of these antigens that 
have been conducted in adults, in which this was the maximum dose tested.  
To date, up to 100 µg Na-APR-1 (M74)/Alhydrogel® administered with 5 µg GLA-AF has been 
very well tolerated in healthy adults, with no observed vaccine-related Serious Adverse Events 
and only mild to moderate local injection site pain and tenderness being the most commonly 
reported adverse events. However, the addition of GLA-AF to Na-GST-1/Alhydrogel did not 
significantly increase IgG levels to the Na-GST-1 antigen.  Therefore, GLA-AF will not be tested 
further in combination with Na-GST-1/Alhydrogel. 

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For the study proposed in this clinical protocol, each child will be vaccinated three times, on Days 
0, 56, and 112 or 180. On each vaccination day each child will receive two injections, one injection 
per arm. This vaccination schedule was selected to coordinate with the Expanded Program on 
Immunization as the human hookworm vaccine is intended to target children, including eventually 
infants. Two different vaccination schedules are being tested (i.e., 0, 2, 4 months vs. 0, 2, 6 
months) to determine the optimal immunogenicity.  
 
Local subcutaneous nodules, believed to be granulomatous reactions to aluminum hydroxide, 
have been observed with use of aluminum hydroxide-based adjuvants. Thus, most aluminum 
hydroxide-adsorbed vaccines are injected intramuscularly (IM) rather than subcutaneously, and 
intramuscular injection in the deltoid muscle will be used for administration of all investigational 
products in the study.  
1.2.3 Clinical Development Plan for the Human Hookworm Vaccine 
The target population for the human hookworm vaccine is children 10 years of age or younger 
living in hookworm endemic areas, since this is the age group that is most at risk of disease from 
this infection. Prior to conducting clinical trials in this age group, however, both Na-GST-1 and 
Na-APR-1 (M74) are currently being tested in healthy adults living in areas of high hookworm 
prevalence. Since co-administration of the two antigens in adults has not resulted in any 
significant safety concerns, testing is proceeding to Phase 1 testing in children living in hookworm 
endemic areas of Africa (Gabon, as described in this protocol) and Brazil. After safety and 
immunogenicity of the co-administered products is shown in children, testing of a co-formulated 
product that contains both Na-GST-1 and Na-APR-1 (M74) will be started, first in adults and then 
in children. The dose concentrations and components of the co-formulated product will be 
determined through the cumulative results of the study described in this protocol and the previous 
Phase 1 trials of these vaccine antigens.  
 
The series of Phase 1 trials in adults and children described above will culminate in a large Phase 
2 trial in which the co-formulated antigens will be administered to children living in endemic areas. 
The hookworm vaccine will be compared to a licensed comparator vaccine to evaluate the impact 
of mean fecal egg counts as well as a variety of clinical and parasitological endpoints. Assuming 
that an impact on infection is shown in the Phase 2 trial, that there are no new safety issues that 
arise due to co-formulation of the antigens, and that combining them into one product does not 
adversely affect the immunogenicity of either, this product will be tested in a pivotal multi-center 
Phase 3 trial in children. The primary endpoint of the Phase 3 trial will be the incidence of 
moderate and heavy hookworm infection (as determined by fecal egg counts) following 
administration of an anthelminthic and vaccination.  
1.3 
Potential Risks and Benefits 
1.3.1 Potential Risks 
 
Risks to subjects are those associated with venipuncture, intramuscular injection of the vaccines, 
possible reactions to the vaccines, and breach of confidentiality.  Risks occasionally associated 

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with venipuncture include pain and bruising at the site of venipuncture, lightheadedness, and 
syncope (rarely). Intramuscular (IM) injection also may cause transient discomfort and fainting.   
 
Possible local vaccine reactions include pain, swelling, erythema, induration, transient limitation 
of limb movement, lymphadenopathy, or pruritus at the injection site. Local subcutaneous 
nodules, believed to be granulomatous reactions to aluminum hydroxide, have been observed 
with use of aluminum hydroxide-based adjuvants, although these have not been observed with 
either Na-APR-1 (M74)/Alhydrogel® or Na-GST-1/Alhydrogel® in the currently ongoing trials of 
these products.  Regardless, most aluminum hydroxide-adsorbed vaccines are injected 
intramuscularly rather than subcutaneously.  Systemic reactions such as fever, headache, 
malaise, myalgia, and joint pain, may also possibly occur. Immediate hypersensitivity reactions 
including urticaria, anaphylaxis, or other IgE-mediated responses are possible as with any 
vaccine. As with any investigational vaccine, 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.  
 
Phase 1 trials of Na-APR-1 (M74)/Alhydrogel® plus GLA-AF have demonstrated similar 
reactogenicity and safety to the antigen administered without GLA-AF. However, in a study of 
an influenza vaccine administered with GLA-SE (a formulation similar to GLA-AF), the 
frequency and intensity of local reactions were greater than when the vaccine was administered 
without GLA-SE (50). 
1.3.2   Precautions Taken to Minimize Risks 
 
In order to minimize the risk to subjects, all children will be monitored closely during their 
participation in this study. The study vaccines and GLA-AF have been produced according to 
current Good Manufacturing Practices (GMP) and will be administered by experienced 
investigators with drugs and equipment available for the treatment of anaphylaxis and other 
potential adverse reactions. All vaccine doses will be given by intramuscular injection to minimize 
injection site reactions such as pain.  
 
Maintenance of Confidentiality 
Participants will be asked to provide personal health information. All attempts will be made to 
keep this information confidential within the limits of the law. However, there remains the unlikely 
risk that unauthorized persons will see the subjects’ personal health information. All records will 
be kept in a locked file cabinet or maintained in a locked room at the study site. Electronic files 
will be password protected. Only people who are involved in the conduct, oversight, monitoring, 
or auditing of this study will be allowed access to the personal health information that is collected.  
Any publications from this study will not use information that will identify subjects by name.   
 
1.3.3 Known Potential Benefits 
 
Children who are enrolled in the study may not receive any direct benefit from participation. It is 
hoped that information gained in this study will contribute to the development of a safe and 
effective hookworm vaccine. All children will undergo a stool examination for ova and parasites 
at screening and on Day 140 and Day 380 study visits. Those found to have a helminth infection 

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at either of these study visits will be offered appropriate treatment at the conclusion of the study, 
free-of-charge. Free medical treatment will be provided to all vaccinated subjects during the active 
immunization phase and the follow-up period. If the investigators judge that a subject requires 
hospitalization, transportation will be arranged and the medical management of the subject will 
be monitored by a study physician and the local Independent Safety Monitor.  Medical care for 
ailments not related to vaccination will not extend beyond the study period, but will be referred to 
the nearest government-run health clinic. Medical care for ailments related to vaccination will 
extend at least until the condition has resolved or stabilized (if a chronic condition). 

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2 
OBJECTIVES 
2.1 
Study Objectives 
 
Primary: 
Safety 
1. To evaluate the safety and reactogenicity of three different dose concentrations of Na-
APR-1 (M74)/Alhydrogel® co-administered with Na-GST-1/Alhydrogel® in healthy 
Gabonese children.  
 
Secondary: 
Immunogenicity 
2. To determine the doses of co-administered Na-APR-1 (M74) and Na-GST-1 that result in 
the highest levels of IgG against these vaccine antigens approximately 14 days after the 
third vaccination.  
 
Tertiary: 
Immunogenicity 
1. To assess and compare the duration of antibody responses to Na-GST-1 and Na-APR-1 
(M74).  
2. To assess the level of IgG1-4 subclass responses to Na-APR-1 (M74) and Na-GST-1.  
 
Exploratory: 
Immunogenicity 
1. To assess the cellular immune responses to the Na-GST-1 and Na-APR-1 (M74) antigens 
following immunization.  
2. To assess the impact of co-administered Na-GST-1 and Na-APR-1 (M74) on the 
production of memory B cells specific for each antigen. 
3. To assess the production of memory B cells specific for each vaccine antigen compared 
to metabolomic changes before and after vaccination. 
4. To characterize the metabolite composition in urine and serum during the course of 
immunization and assess the changes on metabolite profiles related to each dose of co-
administration of Na-GST-1 and Na-APR-1 (M74). 
 
 

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2.2 
Study Outcome Measures 
2.2.1 Primary Outcome Measures 
Safety 
The following parameters will be evaluated for each dose of Na-APR-1 (M74) co-administered 
with Na-GST-1: 
1. Frequency of solicited injection site and systemic reactogenicity, graded by severity, on 
the day of each study vaccination through 14 days after each study vaccination. 
2. Frequency of study vaccine-related serious adverse events from the time of the first study 
vaccination through approximately 9 months after the last study vaccination. 
3. Frequency of clinical safety laboratory adverse events.  
4. Frequency of unsolicited adverse events, graded by severity, from the time of each study 
vaccination through approximately 1 month after each study vaccination.  
5. Frequency of new-onset chronic medical conditions through approximately 9 months after 
the third study vaccination. 
6. Frequency of Adverse Events of Special Interest through approximately 9 months after 
the third study vaccination. 
2.2.2 Secondary Outcome Measure 
Immunogenicity 
The following parameter will be evaluated for each dose of Na-APR-1 (M74) co-administered with 
Na-GST-1: 
1. The IgG level as measured by a qualified indirect enzyme-linked immunosorbent assay 
(ELISA) for each vaccine antigen approximately 14 days after the third vaccination. 
 
2.2.3 Tertiary Outcome Measures 
Immunogenicity 
The following parameters will be evaluated for each dose of Na-APR-1 (M74) co-administered 
with Na-GST-1: 
1. The IgG antibody response as measured by a qualified indirect ELISA at approximately 7, 
14, and 28 days after each vaccination, and approximately 3 and 6 months after the third 
dose. 
2. The IgG subclass levels (IgG1, IgG3, and IgG4) against the vaccine antigens by indirect 
ELISA approximately 14 days after the third vaccination. 

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3. The IgG1-4 subclass levels as measured by indirect ELISA on each day of vaccination, 
approximately 28 days later, and approximately 3 and 6 months after the final vaccination. 
2.2.4 Exploratory Outcome Measures 
The following parameters will be evaluated for each dose of Na-APR-1 (M74) co-administered 
with Na-GST-1: 
 
1. Cellular immune response will be measured by a qualified indirect ELISA at approximately 
on days of vaccination, and then 7, 14, and 28 days after each vaccination, and 
approximately 3 and 6 months after the third dose. 
2. Metabolomic profiles before vaccination, on days of vaccination, approximately 7 and 28 
days following each vaccination, and 6 months after the third dose. 
 

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3 
STUDY DESIGN 
The study will be conducted as a randomized, placebo-controlled, double-blind Phase 1 dose-
escalating clinical trial in healthy children between the ages of 6 and 10 years, inclusive, living in 
the hookworm-endemic area of Lambaréné, Gabon.  The study is designed to evaluate the safety, 
reactogenicity, and immunogenicity of Na-GST-1/Alhydrogel® co-administered with Na-APR-1 
(M74)/Alhydrogel®, as compared to the hepatitis B vaccine, in this age group.   
 
Prior to beginning study-related activities, the consent and cooperation of representative 
community members will be sought, after which parents of potential pediatric participants will be 
invited to learn about the study and consent to the participation of their child. After a parent or 
legal representative provides written informed consent for the child’s participation and passes the 
informed consent comprehension questionnaire, the child will undergo eligibility screening, 
including a complete medical history, physical examination, hematology testing, liver and renal 
function testing, HIV antibody testing, Hepatitis B and C serology, and urinalysis. In addition, a 
fecal examination will be performed for ova and parasites, and urine will be examined for S. 
haematobium ova. All clinically significant abnormalities will be reviewed with parents/legal 
guardians and referral of the child for follow-up care will be provided. After screening, those 
children determined to be eligible, based on the inclusion and exclusion criteria, will be invited to 
participate in the study.   
 
Sixty children will be progressively enrolled into 1 of 3 Groups over a projected 3-month enrollment 
period, with each child followed for 9 months after the final injection (i.e., until Study Day 380 for 
those receiving vaccinations according to a 0,2,4-month schedule and until Study Day 448 for 
those receiving vaccinations according to a 0,2,6-month schedule). Group enrollment will be done 
in an open sequential fashion, whereas within each Group, investigational product (IP) 
assignment and vaccination will be done in a randomized double-blind fashion. 
 
60 subjects will be enrolled into 3 groups of 20: 
The first 20 subjects will be recruited and enrolled into Group 1: 
• 
Group 1 double-blind IP allocation (n=20):  
o 16 subjects will receive 10µg Na-APR-1 (M74) plus 5µg GLA-AF delivered by IM 
injection in the deltoid muscle, with 10µg Na-GST-1 administered IM in the alternate 
arm. 
§ 
8 will be vaccinated according to a 0,2,4-month schedule 
§ 
8 will be vaccinated according to a 0,2,6-month schedule 
o 4 subjects will receive hepatitis B vaccine comparator: 
§ 
2 will be vaccinated according to a 0,2,4-month schedule 
§ 
2 will be vaccinated according to a 0,2,6-month schedule 
 
• 
Group 2 double-blind IP allocation (n=20):  
o 16 subjects will receive 30µg Na-APR-1 (M74) plus 5µg GLA-AF delivered by IM 
injection in the deltoid muscle, with 30µg Na-GST-1 administered IM in the alternate 
arm. 
§ 
8 will be vaccinated according to a 0,2,4-month schedule 

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§ 
8 will be vaccinated according to a 0,2,6-month schedule 
o 4 subjects will receive hepatitis B vaccine comparator: 
§ 
2 will be vaccinated according to a 0,2,4-month schedule 
§ 
2 will be vaccinated according to a 0,2,6-month schedule 
 
• 
Group 3 double-blind IP allocation (n=20):  
o 16 subjects will receive 100µg Na-APR-1 (M74) plus 5µg GLA-AF delivered by IM 
injection in the deltoid muscle, with 100µg Na-GST-1 administered IM in the alternate 
arm. 
§ 
8 will be vaccinated according to a 0,2,4-month schedule 
§ 
8 will be vaccinated according to a 0,2,6-month schedule 
o 4 subjects will receive hepatitis B vaccine comparator: 
§ 
2 will be vaccinated according to a 0,2,4-month schedule 
§ 
2 will be vaccinated according to a 0,2,6-month schedule 
 
 
As with other aluminum hydroxide-adsorbed vaccines, hypersensitivity reactions would be 
expected to occur within the first 24 hours after receipt of either of the two vaccines, and other 
severe local or systemic reactions within 72 hours of vaccination. Children will therefore be 
observed for immediate reactions following each vaccination for at least 1 hour, and will have a 
clinical assessment either in their home or at the study clinic on Days 1, 3, 7, and 14 following 
each vaccination. See Appendix A for the schedule of clinical and laboratory evaluations. 
In order to review safety data at each dose of Na-APR-1 (M74)/Alhydrogel® prior to testing it in 
combination with the point-of injection addition of GLA-AF, and to review safety data at each dose 
of Na-APR-1 (M74)/Alhydrogel® prior to dose escalation, Groups will be enrolled and vaccinated 
in a staggered fashion. Conference calls between the investigators and the Safety Monitoring 
Committee (SMC) will be scheduled within the week prior to beginning vaccinations in Groups 2 
and 3. A cumulative safety report will be submitted to the SMC before beginning vaccinations in 
these Groups that will include safety data from at least the first 14 days after vaccination of all 
subjects in the preceding Group. Written approval (via fax or email) to proceed to vaccinations of 
Groups 2 and 3 must be obtained from the SMC.  
The SMC and Independent Medical Monitor will have access to the randomization code for the 
study, as they may wish to review the data in an unblinded fashion should significant safety 
questions arise prior to the final unblinding.  The trial will not proceed to the next dose Group if 
any of the stopping criteria listed in Section 8.5 are met or, in the clinical judgment of the SMC or 
the Independent Medical Monitor the next higher dose would pose an unacceptable safety risk to 
the subjects. 

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4 
STUDY ENROLLMENT AND WITHDRAWAL 
Only children who meet all of the inclusion and none of the exclusion criteria will be eligible for 
enrollment into this study. No exemptions will be granted. 
 
A total of 60 children will be enrolled. The study population will be enrolled from in and around 
Lambaréné.  
 
Parents/legal guardians who agree to the participation of their child will first provide written 
informed consent and take a true/false comprehension questionnaire. The questionnaire will be 
administered orally in the case of illiterate volunteers, and for both written and oral questionnaires 
study staff will use incorrect answers to identify aspects of the study that require clarification and 
focus on those areas of the informed consent form for further review with the volunteer. All 
questionnaire questions must be answered correctly, and the informed consent form signed, prior 
to study screening and enrollment. Parents or legal guardians who are unable to read will place 
an imprint of their finger in the place of a signature; in addition, an independent witness, who is 
not a member of the study team, will sign the consent form to attest that the informed consent 
form was read to the parent/legal guardian, their questions were answered, and the parent/legal 
guardian answered all comprehension questions correctly. The original signed informed consent 
form for each parent/guardian will be maintained as part of that child’s study records.  A copy of 
the informed consent form will be provided to every parent/guardian.   
 
Screening can occur up to 90 days prior to enrollment; enrollment/randomization, and 
administration of first dose of study products will occur on the same day. 
4.1 
Subject Inclusion Criteria 
 
1. Males or females between 6 and 10 years, inclusive, who are long-term residents of the study 
area. 
2. Good general health as determined by means of the screening procedure. 
3. Assumed availability for the duration of the trial (up to 15 months). 
4. Willingness of parent or legal guardian for child to participate in the study as evidenced by 
signing the informed consent document in combination with the child assent form. 
5. Negative for hookworm during screening, or if found to be infected with hookworm, has 
completed a course of three doses of albendazole. 
4.2 
Subject Exclusion Criteria 
 
1. Inability of parent/legal guardian to correctly answer all questions on the informed consent 
comprehension questionnaire.  
2. Evidence of clinically significant neurologic, cardiac, pulmonary, hepatic, rheumatologic, 
autoimmune, diabetes, or renal disease by history, physical examination, and/or laboratory 
studies. 
3. Known or suspected immunodeficiency. 
4. Laboratory evidence of liver disease (alanine aminotransferase [ALT] greater than 1.25-times 
the upper reference limit). 

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5. Laboratory evidence of renal disease (serum creatinine greater than 1.25-times the upper 
reference limit, or more than 1+ protein, or more than trace blood on urine dipstick testing with 
the exception of greater than trace blood detected in females during menses). 
6. Laboratory evidence of hematologic disease (absolute leukocyte count <4500/mm3; absolute 
leukocyte count >13.0 x 103/mm3; hemoglobin <9.5 g/dl; or, platelet count <140,000/mm3). 
7. Other condition that in the opinion of the investigator could jeopardize the safety or rights of a 
child participating in the trial or would render the child unable to comply with the protocol. 
8. Participation in another investigational vaccine or drug trial within 30 days of starting this study 
or for the duration of the study. 
9. History of a severe allergic reaction or anaphylaxis. 
10. Severe asthma as defined by the need for daily use of inhalers or emergency room/clinic visit 
or hospitalization within 6 months of the child’s planned first vaccination in the study. 
11. Positive for HCV.  
12. Positive for HBsAg. 
13. Positive for HIV infection.  
14. Use of corticosteroids (excluding topical or nasal) or immunosuppressive drugs within 30 days 
of starting this study or expect to use for the duration of the study. 
15. Receipt of a live vaccine within past 4 weeks or a killed vaccine within past 2 weeks prior to 
entry into the study. 
16. History of a surgical splenectomy. 
17. Receipt of blood products within the 6 months prior to entry into the study.  
18. Previous receipt of a primary series (three doses according to a 0, 1, and 6 -12 month 
schedule) of the hepatitis B vaccine. 
4.3 
Vaccine Assignment Procedures 
4.3.1 Randomization Procedures 
 
Parents/guardians of eligible children will be asked to bring them to the clinic on the scheduled 
day of enrollment into the study (Day 0). If required, transportation to the clinic will be provided 
free-of-charge. One or 2 additional eligible children above the number to be enrolled into a given 
Group may be scheduled as Day 0 visit alternates, if possible. After undergoing a clinical 
interview and physical examination to ensure that they remain eligible for participation in the 
study, that they have had blood collected for safety clinical laboratory and baseline 
immunogenicity assessments, children will be randomized and enrolled in the order that they 
present for vaccination.  
 
Within each group, randomization will be done through use of a randomization code, furnished to 
the study vaccine manager by the data management center (AIGHD). Access to the 
randomization list will be exclusively limited to the study vaccine manager and assistant. Between 
vaccination days, the randomization list will be stored in a locked cabinet. The study vaccine 
manager and assistant will be unblinded, but will not be involved in further evaluation of study 
subjects or assessment of adverse events. Children will not be considered enrolled in the study 
until they have received their first dose of vaccine. In the event that a child is randomized but not 
enrolled on the day of first vaccination, they will be replaced with an eligible alternate. Enrolled 
children that leave the study for any reason following first vaccination will not be replaced. Any 
alternates not vaccinated will be invited to participate as members of the next Group. Any Group 

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3 alternates not vaccinated, and therefore not enrolled in the study, will be offered vaccination 
with a non-study, licensed vaccine such as the tetanus toxoid or influenza vaccine. 
 
4.3.2 Allocation of vaccination schedule and vaccine randomization 
Within each cohort the first 10 participants will receive the 0,2,4-month vaccination schedule and 
the last 10 will receive the 0,2,6-month schedule. The participants will be randomized to either 
the experimental group, receiving Na-APR-1(M74) plus 5 µg GLA-AF co-administered with Na-
GST-1, or randomized to the control group who will receive HBV co-administered with sterile 
saline.  In addition, there will be a randomization to assign left/right arm vaccination for the first 
injection, the arms will be alternated for the second and third sets of vaccinations. The 
Randomization list will thus only be used on Day 0 for a particular participant. 
The vaccination manager assigns the Study ID when the vaccines are being prepared for that 
particular participant. The Study ID is written onto the syringes and then the vaccinator writes 
the Study ID onto the CRF. 
 
 
4.3.3 Masking Procedures 
 
Due to the staggered, dose-escalation design of the trial, it will not be possible to blind to Na-
APR-1 (M74)/Alhydrogel® or Na-GST-1/Alhydrogel® dose assignment. That is, those subjects 
enrolled into Groups 1 and 2 will receive either 10μg Na-APR-1 (M74)/Alhydrogel® administered 
to one arm and 10μg Na-GST-1/Alhydrogel® administered to the alternate arm, or the hepatitis B 
vaccine in one arm and sterile saline administered to the alternate arm. Subjects enrolled into 
Groups 3 and 4 will receive either 30μg Na-APR-1 (M74)/Alhydrogel® administered to one arm 
and 30μg Na-GST-1/Alhydrogel® administered to the alternate arm, or the hepatitis B vaccine in 
one arm and sterile saline administered to the alternate arm, and so on.  
 
Investigators and subjects will be blinded to the within-Group random allocation to receive co-
administered Na-APR-1 (M74)/Alhydrogel® and Na-GST-1/Alhydrogel® or co-administered 
hepatitis B vaccine and sterile saline until all subjects have completed their Day 208 visit, the 
safety and secondary immunogenicity outcomes (i.e., anti-Na-GST-1 and anti-Na-APR-1 [M74] 
IgG antibody results by ELISA) have been monitored and entered into the database, and the 
database has been “soft-locked” for interim analysis. Procedures to maintain blinding include 
administering two injections to all subjects at each vaccination point. For this, sterile saline will be 
co-administered to those subjects randomized to receive the hepatitis B vaccine.  
 
The study vaccine manager and his assistant will prepare all investigational product doses 
(vaccine or sterile saline placebo) in a separate room, and will hand filled syringes to the 
vaccinator(s). Since the 10 and 30μg doses of the Na-APR-1 (M74) and Na-GST-1 formulations 
are of different volumes than that of hepatitis B vaccine, the contents of all syringes will be 
disguised using opaque tape. As a further precaution, the vaccinator(s) will not be involved in 
assessments of reactogenicity or adverse events. 

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Investigators will be unblinded after all subjects have had their study Day 208 visit (i.e., 1 month 
after the final vaccination for those children randomized to receive vaccines on the 0, 2, and 6-
month schedule), the safety and secondary immunogenicity outcomes (i.e., anti-Na-GST-1 and 
anti-Na-APR-1 (M74) IgG antibody results by ELISA) have been monitored and entered into the 
database, and the database has been “soft-locked” for interim analysis. After this point, the study 
will be single-blinded (i.e., subjects will remain blinded to what products they received). The 
principal justification for unblinding the investigators before the final study visit has been 
completed is that the safety and immunogenicity data acquired during the vaccination phase of 
the study will be crucial in guiding the human hookworm vaccine clinical development plan, and 
we feel that to make an informed decision as to whether or not to proceed to pediatric trials, we 
must first assess the unblinded data in a timely fashion.   
4.3.4 Reasons for Withdrawal and Discontinuation of Vaccinations 
Parents/guardians will be free to withdraw their child from the study at any time and for any 
reason, without penalty. Children who have received vaccine, regardless of the number of doses 
received, or who developed an AE or SAE will be strongly encouraged to remain in the study to 
be followed for safety purposes.  
A child may withdraw or be withdrawn from the study for any of the following reasons: 
• 
Withdrawal of consent of the parent or legal guardian. 
• 
Withdrawal of assent of the child. 
• 
As deemed necessary by the Principal Investigator or appropriate co-investigator for 
noncompliance or other reasons. 
• 
Subject lost to follow-up. 
• 
Early termination of the study. 
The second or third study vaccination may not be administered to a child if any of the following 
criteria are met (although the child will remain in the study for safety follow-up visits): 
• 
Medical condition for which continued participation, in the opinion of the Principal 
Investigator or appropriate co-investigator, would pose a risk to the child or would be likely 
to confound interpretation of the results. 
• 
Presence of signs or symptoms that could confound or confuse assessment of study 
vaccine reactogenicity. For children with injection site or systemic signs or symptoms, or 
with an acute illness, including a tympanic temperature greater than 38.0°C, the study 
vaccination should/may be postponed/deferred until signs, symptoms, or acute illness 
have resolved and if within the acceptable protocol-specified window for that visit. If 
outside this window, the Sponsor must first approve the second or third study vaccination 
and the documentation of approval should be filed in the child’s chart. 

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• 
Any unresolved or continuing solicited or unsolicited Grade 3 adverse event. An 
unresolved or continuing Grade 1 or Grade 2 adverse event is permissible unless, in the 
opinion of the Principal Investigator or appropriate co-investigator, it would render study 
vaccination unsafe or interfere with the evaluation of responses. 
• 
Grade 3 clinical safety laboratory value that does not decrease to Grade 2 or less prior to 
the second or third study vaccination. Any clinical safety laboratory parameter may be re-
evaluated only once at the clinical laboratory prior to the second or third study vaccination. 
If the clinical safety laboratory value decreases to Grade 2 or less, the child may receive 
the second or third study vaccination. The second or third study vaccination should be 
scheduled to occur within the acceptable protocol-specified window for that visit. If outside 
this window, the Sponsor must first approve the study vaccination and the documentation 
of approval should be filed in the child’s chart. 
• 
Severe or sustained reaction or disability related to the first study vaccination. 
• 
New onset of illness or condition that meets exclusion criteria. 
• 
Child no longer meets eligibility criteria. 
• 
As deemed necessary by the Principal Investigator or appropriate co-investigator for 
noncompliance or other reasons. 
• 
Refusal of further study vaccination by the parent/guardian. 
• 
Withdrawal of consent of the parent or legal guardian. 
• 
Child lost to follow-up. 
• 
Early termination of the study. 
• 
New information becomes available that makes further vaccinations unsafe. 
4.3.5 Handling of Withdrawals 
The primary reason for withdrawal from the study will be recorded on the appropriate data 
collection form. Parents/guardians will be encouraged to have their child complete the Early 
Termination Visit. The Early Termination Visit procedures are listed in Section 7.5. Although 
children are free to withdraw at any time or may be withdrawn by the Principal Investigator or 
appropriate co-investigator at any time, those who receive at least one dose of study vaccines 
will be encouraged to remain in the study for follow-up safety assessments and collection of 
venous blood samples for immunogenicity testing. Every attempt will be made to follow all adverse 
events, including solicited injection site and systemic reactions, serious adverse events, and new-
onset chronic medical conditions ongoing at the time of early withdrawal to resolution. 
In the case of children who fail to appear for a follow-up safety assessment, extensive efforts (i.e., 
three documented contact attempts via in-person visits to the child’s home, made on separate 

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occasions) will be made to locate or recall them, or at least to determine their health status. These 
efforts will be documented in the child’s records. 
 
4.3.6 Termination of Study  
 
If a dose of vaccine is considered significantly reactogenic (see Section 8.5) dose escalation 
and/or additional vaccinations will be suspended until reviewed by the Independent Medical 
Monitor, SMC, and study sponsor. Any recommendation of the Independent Medical Monitor or 
SMC to resume or suspend further injections (either for an individual child or an entire dose 
Group) will be communicated in writing to the sponsor and Principal Investigator. All 
communications from the SMC will subsequently be forwarded by the investigators to the IRB.  

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5 
STUDY INTERVENTIONS/INVESTIGATIONAL 
PRODUCTS 
Investigators will receive the current versions of the Clinical Investigator’s Brochures for Na-GST-
1/Alhydrogel® and Na-APR-1 (M74)/Alhydrogel®, which comprehensively describe all available 
preclinical and human experience with the experimental vaccines. If relevant new information 
becomes available during the course of the trial, the investigators will receive the revised 
Investigator’s Brochure(s). 
5.1 
Study Product Description 
5.1.1 Acquisition 
Vials of Na-GST-1/Alhydrogel®, Na-APR-1 (M74)/Alhydrogel®, GLA-AF, ENGERIX hepatitis B 
vaccine, and sterile saline for injection for this study will be supplied to the study site. Na-GST-
1/Alhydrogel®, Na-APR-1 (M74)/Alhydrogel®, and GLA-AF will be transported to the study site at 
0.5oC to 10oC; temperature recording devices will accompany the vaccines at all times during 
transport to ensure temperature limits have not been violated.  ENGERIX hepatitis B vaccine and 
sterile saline will be purchased at the study site.   
5.1.2 Formulation, Packaging, and Labeling 
5.1.2.1 
Na-APR-1 (M74)/Alhydrogel® 
 
Na-APR-1 (M74)/Alhydrogel® is supplied as a sterile milky-white suspension (when shaken 
slightly). Each 2.0 ml vial contains a 0.1 mg/ml suspension of Na-APR-1 (M74) adsorbed to 
0.8 mg/mL of Alhydrogel® in a solution containing 10 mM imidazole, 150 mM sodium chloride and 
0.3% Empigen BB, with pH 7.4 ± 0.1. The maximum dose that will be administered is 100 μg of 
Na-APR-1 (M74), or 1.0 ml of the final drug product. This volume contains the equivalent of 
approximately 400 μg aluminum. Lower doses of Na-APR-1 (M74) are delivered by injecting 
smaller volumes of the 0.1 mg/ml suspension: for example, 0.3 ml will be injected to deliver 30 μg 
Na-APR-1 (M74). For all doses, the ratio of Na-APR-1 (M74) to Alhydrogel® will therefore remain 
constant: for the 30 and 100 μg doses of Na-APR-1 (M74) the respective amounts of Alhydrogel® 
will be 240 and 800 μg (corresponding to approximately 120 and 400 μg aluminum, respectively). 
Na-APR-1 (M74) protein expression was completed at Fraunhofer Center for Molecular 
Biotechnology (FhCMB/Newark, DE) and purification and vialing at the Walter Reed Army Institute 
of Research (WRAIR/Silver Spring, MD). The product conforms to established requirements of 
purity, sterility, safety, and identity. In this study, each dose of Na-APR-1 (M74)/Alhydrogel® will 
be administered in combination with 5 µg GLA-AF. 
5.1.2.2 
Na-GST-1/Alhydrogel® 
Na-GST-1/Alhydrogel® is supplied as a sterile milky-white suspension (when shaken slightly).  
Each 2.0 ml vial contains 1.35 ml of a 0.1 mg/ml suspension of Na-GST-1 adsorbed to 0.8 mg/ml 
of Alhydrogel® in a buffer consisting of 10% glucose and 10 mM imidazole, pH 7.4.  Glucose acts 
as an excipient and imidazole as the buffer based on evidence that these components specifically 

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enhance the stability and solubility of Na-GST-1. The maximum dose that will be administered is 
100 μg of Na-GST-1, or 1.0 ml of the final drug product. This volume contains the equivalent of 
approximately 400 μg aluminum. Lower doses of Na-GST-1 are delivered by injecting smaller 
volumes of the 0.1 mg/ml suspension: for example, 0.3 ml will be injected to deliver 30 μg Na-
GST-1. For all doses, the ratio of Na-GST-1 to Alhydrogel® will therefore remain constant: for the 
30 and 100 μg doses of Na-GST-1 the respective amounts of Alhydrogel® will be 240 and 800 μg 
(corresponding to approximately 120 and 400 μg aluminum, respectively). Na-GST-1/Alhydrogel® 
was manufactured, formulated and vialed at Aeras Global Vaccine Foundation (Rockville, 
Maryland, USA).   
5.1.2.3 
Glucopyranosyl-Lipid A Aqueous Formulation (GLA-AF) 
GLA-AF will be supplied to the trial site as a 0.5 mL aqueous solution in multi-dose vials containing 
25 µg/mL of GLA without preservative. Appropriate volumes of GLA-AF will be withdrawn from 
the multi-dose vials using a syringe and added to a vial containing Na-APR-1 (M74)/Alhydrogel®, 
or vice versa (described in the Investigator’s Brochure). The mixture must be administered not 
more than 24 hours after mixing the GLA-AF with Na-APR-1 (M74)/Alhydrogel®. 
5.1.2.4 
Saline Placebo 
Sterile isotonic (0.9%) sodium chloride solution (“normal saline”) for injection will be procured by 
the study site.  Normal saline is a clear liquid. In study subjects randomized to receive Hepatitis 
B vaccine, normal saline will be administered intramuscularly as placebo in an equal volume to 
the Hepatitis B vaccine. It will be administered in the deltoid muscle of the arm opposite to the 
one in which the Hepatitis B vaccine is delivered so that all study subjects receive two injections 
on each day of vaccination, thus maintaining the study blind. 
 
5.1.2.5 
Hepatitis B Vaccine 
The Hepatitis B vaccine is a non-infectious subunit viral vaccine derived from hepatitis B surface 
antigen (HBsAg) produced in yeast cells. The antigen is harvested and purified from fermentation 
cultures of a genetically modified strain of yeast containing the gene for HBsAg.  The purified 
protein is then adjuvanted with aluminum hydroxide adjuvant.  The hepatitis B vaccine is a slightly 
turbid sterile suspension for intramuscular injection.  The vaccine is supplied in single-dose vials, 
with each vial containing enough vaccine to deliver one 0.5 mL dose.  Each 0.5 mL dose contains 
10 µg of HBsAg and 250 µg of aluminum as aluminum hydroxide.  The product conforms to 
established requirements for sterility, safety, and identity.   
5.1.3 Product Storage and Stability 
Vaccines, GLA-AF, and placebo will be stored at the site in a refrigerator at 2oC to 8oC until just 
prior to administration and will not be frozen; refrigerator temperature will be monitored 
continuously. All vaccine vials will be stored in the upright position. 
5.2 
Dosage, Preparation and Administration of Study 
Intervention/Investigational Product 
 

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Doses of Na-GST-1/Alhydrogel® will be prepared by withdrawing appropriate volumes into 
syringes of appropriate size. The Na-APR-1 (M74)/Alhydrogel® plus GLA-AF formulations will be 
prepared by adding appropriate volumes of GLA-AF solution to vials of Na-APR-1 
(M74)/Alhydrogel®, or vice versa, within 24 hours of vaccination. Doses of the Hepatitis B vaccine 
and sterile saline control will be prepared by withdrawing 0.5 mL into syringes of appropriate size.  
 
Vaccine doses will be administered by qualified study personnel (a study nurse who will not be 
involved in further assessment of subjects) in the deltoid muscle of the appropriate arm after 
disinfecting the skin with an alcohol swab and allowing it to dry.   
5.3 
Modification of Study Intervention/Investigational Product for a 
Participant 
There will be no dose modifications. If a child’s second and/or third dose of study vaccination is 
deferred, it should be rescheduled to occur within the acceptable protocol-specified window for 
that visit. If this period elapses, the site must obtain prior approval from the Sponsor to administer 
the second and/or third study vaccination and the documentation of approval should be filed in 
the child’s chart. Children who do not receive the second and/or third study vaccination will be 
asked to return for safety assessments and for scheduled venous blood sample collections for 
immunogenicity testing and will be followed for the duration of the study. 
Unblinding can occur upon request of the Independent Medical Monitor or the SMC at any time 
during the study. 
5.4 
Accountability Procedures for the Study 
Intervention/Investigational Product(s) 
 
Study vaccines, GLA-AF, and sterile saline placebo supplies must be received by a designated 
person at the study site, handled and stored safely and properly, and kept in a secure location to 
which only the designated individuals have access. Study-site personnel are responsible for 
maintaining accurate records of the vaccine supplies (i.e., Na-GST-1/Alhydrogel®, Na-APR-1 
(M74)/Alhydrogel®, Hepatitis B vaccine, the sterile saline placebo, and GLA-AF) received, the 
quantities administered to study subjects, and the amounts remaining at the conclusion of the 
study.   
 
After administration of vaccine or GLA-AF doses, the empty or wasted vials will be accounted for 
and stored at the study site until monitoring by the study Sponsor or their designee. At the 
conclusion of the study, all used and unused Na-GST-1/Alhydrogel®, Na-APR-1 (M74) 
/Alhydrogel®, and GLA-AF vials will be returned to the Sponsor or destroyed on site upon direction 
from the Sponsor, or maintained at 2 to 8oC until further notice from the Sponsor regarding their 
disposition.   
5.5 
Concomitant Medications/Treatments 
Administration of any medications, therapies, or vaccines will be recorded on the appropriate data 
collection form. Concomitant medications will include all medications taken within the 30 days 
prior to enrollment, 28 days after each dose of study product, and for new-onset chronic medical 

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conditions through the end of participation in the study for each subject. Prescription and over-
the-counter drugs will be included as well as vitamins and supplements. 
Use of new medication will prompt an evaluation for the presence of a new diagnosis of chronic 
medical disease or chronic medical condition. 
Medications that might interfere with the evaluation of the investigational product should not be 
used unless absolutely necessary. Medications in this category include, but are not limited to, 
glucocorticoids, 
i.e., 
high 
dose 
oral, 
parenteral 
and 
daily 
inhaled 
steroids, 
and 
immunosuppressive or cytotoxic drugs. Other than from participation in this study, subjects should 
not receive experimental agents, including vaccines, for the duration of the study.  
The administration of licensed vaccines, should be delayed until 28 days after the last 
administration of the investigational vaccine. 

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6 
STUDY SCHEDULE 
The following sections provide a detailed listing of the procedures and tests to be performed in 
this study at designated time points. The total volume of blood (approximately 94 mL) to be 
collected from each participating child over the duration of the trial (which is 13-15 months, 
depending on the vaccination schedule) is approximately the volume collected when donating 
one unit of blood and should not compromise the health of the children. See Appendix A for the 
schedule of study procedures. 
6.1 
Screening  
 
The following procedures will be performed upon initial screening (note that all procedures might 
not be performed on the same day):  
  
1. Explain the study, Informed Consent and the child assent form to the child’s parent/legal 
guardian. 
2. Ensure the child’s parent/legal guardian has passed the informed consent comprehension 
questionnaire, has signed the Informed Consent as well as the child assent form and receives 
a signed copy of the Informed Consent. 
3. Elicit a complete medical history, including medication history, for the child. 
4. Administer a physical examination, including vital signs (heart rate, respiratory rate, arterial 
blood pressure, and a tympanic temperature). 
5. Obtain blood for hematology, biochemistry, and serologic tests for HIV and viral hepatitis (B 
and C).  
6. Obtain fecal sample for examination for ova and parasites. 
7. Obtain urine for urine dipstick testing and examination for ova. 
8. Obtain urine for metabolomics study. 
 
Screening steps 3-7 must be performed within 90 days before the planned enrollment into the 
study. Should this screening window be exceeded before the first vaccination, screening 
procedures (not including administration of the informed consent form or comprehension 
questionnaire) may be repeated to ensure continued eligibility for the study (screening procedures 
can be repeated a maximum of one time). Screening laboratory tests (other than serologic tests 
for HIV and viral hepatitis) may be repeated up to one time to confirm any abnormalities.  
 
If the fecal exam performed during screening demonstrates infection with an intestinal parasite 
the child will be treated with an appropriate medication prior to being enrolled in the study and 
before any vaccinations. Children infected with Ascaris lumbricoides, hookworm or Trichuris 
trichiura will be treated with three daily 400 mg doses of albendazole; children infected with S. 
intercalatum or Taenia spp. will be treated with a single 40 mg/kg oral dose of praziquantel; and 
individuals infected with Strongyloides stercoralis will be treated with three 400 mg daily oral 
doses of albendazole. If the urine exam performed during screening demonstrates infection with 
Schistosoma haematobium, infected children will be treated with a single 40 mg/kg oral dose of 
praziquantel. Children who have been treated for any parasitic infections, cannot be enrolled for 
vaccination within 14 days after the last day of treatment.  
 
 

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6.2 
Enrollment/Baseline 
 
Children will not be considered enrolled in the study until they have received their first dose of 
vaccine. In the event that a volunteer is randomized but not enrolled on the day of first vaccination, 
they will be replaced with an eligible alternate. 
 
Study Day 0 (Day of First Vaccination) 
1. Verify that Informed Consent was obtained from the child’s parent/guardian. 
2. Verify that all applicable eligibility criteria have been met. 
3. Perform abbreviated history (including concomitant medications) and physical exam, focusing 
on any acute complaints. 
4. Obtain blood for hematology, biochemistry, anti-Na-GST-1 and anti-Na-APR-1 (M74) humoral 
and cellular immunology assays.   
5. Obtain a urine sample for metabolomics study. 
6. Record vital signs (blood pressure, a tympanic temperature, and heart rate). 
7. Administer the vaccines. 
8. Observe for at least 1 hour after vaccination to evaluate for immediate adverse reactions.  
During the 1-hour post-immunization wait period, study staff will discuss signs and symptoms 
of potential AEs with parent/guardian and child. 
6.3 
Follow-up  
 
Study Day 1 
1. Perform basic history and physical exam (including injection site), emphasizing examination 
of any acute complaints (i.e., AE data collection). 
2. Record vital signs. 
3. Obtain a urine sample for metabolomics study. 
 
Study Day 3 +/- 1 
1. Perform basic history and physical exam (including injection site), emphasizing examination 
of any acute complaints. 
2. Record vital signs.  
3. Obtain a urine sample for metabolomics study. 
 
Study Day 7 +/- 1 
1. Perform basic history and physical exam (including injection site), emphasizing examination 
of any acute complaints. 
2. Record vital signs. 
3. Obtain blood for anti-Na-GST-1 and anti-Na-APR-1 (M74) antibody assays and cellular 
immunology assays. 
4. Obtain a urine sample for metabolomics study. 
 
 
Study Day 14 +/- 2 
1. Perform basic history and physical exam (including injection site), emphasizing examination 
of any acute complaints. 
2. Record vital signs. 

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3. Obtain blood for hematology, biochemistry, anti-Na-GST-1 and anti-Na-APR-1 (M74) humoral 
and cellular immunology assays. 
4. Obtain a urine sample for metabolomics study. 
 
Study Day 28 +/- 4 
1. Perform basic history and physical exam (including injection site), emphasizing examination 
of any acute complaints. 
2. Record vital signs. 
3. Obtain blood for anti-Na-GST-1 and anti-Na-APR-1 (M74) humoral and cellular immunology 
assays. 
4. Obtain a urine sample for metabolomics study. 
 
Study Day 56 +/- 7 (Day of Second Vaccination) 
1. Perform basic history (including concomitant medications) and physical exam (including 
injection site), emphasizing examination of any acute complaints.  
2. Obtain blood for hematology, biochemistry, anti-Na-GST-1 and anti-Na-APR-1 (M74) humoral 
and cellular immunology assays. 
3. Obtain a urine sample for metabolomics study. 
4. Record vital signs. 
5. Administer the vaccine. 
6. Observe for at least 1 hour after vaccination to evaluate for immediate adverse reactions.   
 
Study Day 57 (1 day after Second Vaccination) 
1. Perform basic history and physical exam (including injection site), emphasizing examination 
of any acute complaints. 
2. Record vital signs. 
 
Study Day 59 (3 +/- 1 days after Second Vaccination) 
1. Perform basic history and physical exam (including injection site), emphasizing examination 
of any acute complaints. 
2. Record vital signs. 
3. Obtain a urine sample for metabolomics study. 
 
Study Day 63 (7 +/- 1 days after Second Vaccination) 
1. Perform basic history and physical exam (including injection site), emphasizing examination 
of any acute complaints. 
2. Record vital signs. 
3. Obtain blood for anti-Na-GST-1 and anti-Na-APR-1 (M74) humoral and cellular immunology 
assays. 
4. Obtain a urine sample for metabolomics study. 
 
Study Day 70 (14 +/- 2 days after Second Vaccination) 
1. Perform basic history and physical exam (including injection site), emphasizing examination 
of any acute complaints. 
2. Record vital signs. 
3. Obtain blood for hematology, biochemistry, anti-Na-GST-1 and anti-Na-APR-1 (M74) humoral 
and cellular immunology assays.   
4. Obtain a urine sample for metabolomics study. 

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Study Day 84 (28 +/- 4 days after Second Vaccination) 
1. Perform basic history and physical exam, emphasizing examination of any complaints. 
2. Record vital signs. 
3. Obtain blood for anti-Na-GST-1 and anti-Na-APR-1 (M74) humoral and cellular immunology 
assays. 
4. Obtain a urine sample for metabolomics study. 
 
Study Day 112 +/- 14 [Groups 1a, 2a, & 3a] or 180 [Groups 1b, 2b, & 3b]  
(Day of Third Vaccination) 
1. Perform basic history (including concomitant medications) and physical exam (including 
injection site), emphasizing examination of any acute complaints.  
2. Obtain blood for hematology, biochemistry, anti-Na-GST-1 and anti-Na-APR-1 (M74) humoral 
and cellular immunology assays. 
3. Obtain a urine sample for metabolomics study. 
4. Record vital signs. 
5. Administer the vaccine. 
6. Observe for at least 1 hour after vaccination to evaluate for immediate adverse reactions.   
 
Study Day 113/181 (1 day after Third Vaccination) 
1. Perform basic history and physical exam (including injection site), emphasizing examination 
of any acute complaints. 
2. Record vital signs. 
3. Obtain a urine sample for metabolomics study. 
 
Study Day 115/183 (3 +/- 1 days after Third Vaccination) 
1. Perform basic history and physical exam (including injection site), emphasizing examination 
of any acute complaints. 
2. Record vital signs. 
3. Obtain a urine sample for metabolomics study. 
 
Study Day 119/187 (7 +/- 1 days after Third Vaccination) 
1. Perform basic history and physical exam (including injection site), emphasizing examination 
of any acute complaints. 
2. Record vital signs. 
3. Obtain blood for anti-Na-GST-1 and anti-Na-APR-1 (M74) humoral and cellular immunology 
assays. 
4. Obtain a urine sample for metabolomics study. 
 
Study Day 126/194 (14 +/- 2 days after Third Vaccination) 
1. Perform basic history and physical exam (including injection site), emphasizing examination 
of any acute complaints. 
2. Record vital signs. 
3. Obtain blood for hematology, biochemistry, anti-Na-GST-1 and anti-Na-APR-1 (M74) humoral 
and cellular immunology assays.   
4. Obtain a urine sample for metabolomics study. 
 
 

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Study Day 140 (28 +/-4 days after Third Vaccination) [Groups 1a, 2a, & 3a ONLY] 
1. Perform basic history and physical exam, emphasizing examination of any acute complaints. 
2. Obtain blood for anti-Na-GST-1 and anti-Na-APR-1 (M74) antibody assays and cellular 
immunology assays.  
3. Obtain stool for examination for ova and larvae from parasites. 
4. Obtain a urine sample for metabolomics study. 
 
Study Day 200 (3 months +/-14 days after Third Vaccination) [Groups 1a, 2a, & 3a ONLY] 
1. Perform basic history and physical exam, emphasizing examination of any acute complaints. 
2. Obtain blood for anti-Na-GST-1 and anti-Na-APR-1 (M74) humoral and cellular immunology 
assays. 
3. Obtain a urine sample for metabolomics study. 
 
Study Day 208 (28 +/-4 days after Third Vaccination) [Groups 1b, 2b, & 3b ONLY] 
1. Perform basic history and physical exam, emphasizing examination of any acute complaints. 
2. Obtain blood for anti-Na-GST-1 and anti-Na-APR-1 (M74) humoral and cellular immunology 
assays. 
3. Obtain stool for examination for ova and larvae from parasites. 
4. Obtain a urine sample for metabolomics study. 
 
Study Day 268 (3 months +/-14 days after Third Vaccination) [Groups 1b, 2b, & 3b ONLY] 
1. Perform basic history and physical exam, emphasizing examination of any acute complaints. 
2. Obtain blood for anti-Na-GST-1 and anti-Na-APR-1 (M74) humoral and cellular immunology 
assays. 
3. Obtain a urine sample for metabolomics study. 
 
Study Day 290/358 (6 months +/-14 days after Third Vaccination) 
1. Perform basic history and physical exam, emphasizing examination of any acute complaints. 
2. Obtain blood for anti-Na-GST-1 and anti-Na-APR-1 (M74) humoral and cellular immunology 
assays. 
3. Obtain a urine sample for metabolomics study. 
6.4 
Final Study Visit 
 
Study Day 380/448 (9 months +/-21 days after Third Vaccination) 
1. Perform basic history and physical exam, emphasizing examination of any acute complaints. 
2. Obtain stool for examination for ova and larvae from parasites. 
6.5 
Early Termination Visit 
The following activities will be performed at the early termination visit for study participants who 
withdraw, or are withdrawn or terminated from the study: 
• 
Obtain interim medical history by interview of subjects and note any changes since the 
previous visit. 

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• 
All concomitant medications will be recorded on the appropriate data collection form 
(if within 28 days of child’s last vaccination received in the study). 
• 
Information regarding adverse events/Serious Adverse Events will be assessed and 
recorded on the appropriate data collection form (adverse events will be limited to new-
onset chronic medical conditions and SAEs if after 28 days after the third study 
vaccination). 
• 
A targeted physical examination may be, if indicated based on review of interim 
medical history. 
• 
Approximately 2.4 mL of venous blood may be collected for safety labs and performed 
by the clinical laboratory (if prior to the post-vaccination day 14 visit and if not recently 
obtained per study schedule). 
• 
Approximately 6.2 mL of venous blood may be collected for antibody and cellular 
assays (if prior to post-vaccination day 14 visit and if not recently obtained). 
6.6 
Unscheduled Visit  
 
Unscheduled visits may occur at any time during the study. Any of the following activities may be 
performed: 
• 
Review concomitant medications (if started prior to 28 days after a study vaccination). 
• 
Review adverse events (if started prior to 28 days after a study vaccination). 
• 
Review serious adverse events and new-onset chronic medical conditions  
• 
Obtain medical history by interview of children and their parent/guardian and note any 
changes since the previous visit (if indicated). 
• 
A targeted physical examination may be performed, if indicated based on review of 
medical history. 
• 
Examine study vaccination site (if within 14 days after a study vaccination). 
• 
Approximately 2.4 mL of venous blood will be collected for safety labs and performed by 
the study clinical laboratory (if indicated). 
 

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7 
STUDY PROCEDURES/EVALUATIONS 
7.1 
Clinical Evaluations 
 
Children will be monitored for local and systemic adverse events during specific protocol-defined 
post-vaccination periods. As with other aluminum hydroxide-adsorbed vaccines, hypersensitivity 
reactions would be expected to occur within the first 24 hours after receipt of either of the two 
vaccines, and other severe local or systemic reactions within 72 hours of vaccination. Children 
will therefore be observed for immediate reactions following each vaccination for at least 1 hour, 
and will have a clinical assessment either in their home or at the study clinic on Days 1, 3, 7, 14 
and 28 following each vaccination. If required, transportation to and from the clinic will be 
provided. Study clinicians will also stay in or near the study site for the duration of the trial and 
will be available to study subjects at all times. Should the parent/guardian of a child participating 
in the study call on a study clinician to report an adverse event (AE), it will be fully documented in 
their study chart, and discussed with the Principal Investigator.  
 
All AEs will be graded for severity and relationship to study vaccine, captured on the appropriate 
case report form (CRF), and followed to resolution. All Serious Adverse Events (SAEs) will be 
reviewed by a study physician, recorded on the appropriate SAE form, reported according to 
applicable regulations and guidelines, and followed through to resolution or stabilization by a 
study physician. Special attention will also be paid to monitoring for the occurrence of Adverse 
Events of Special Interest (AESIs), which include inflammatory and autoimmune disorders that 
may potentially be related to the use of an immunostimulatory adjuvant (although none have been 
associated with the use of GLA-AF, to date).  
 
A local Independent Medical Monitor will be appointed and a Safety Monitoring Committee (SMC) 
formed to monitor subject safety and to advise the Principal Investigator and co-investigators on 
trial-related medical questions or problems. The SMC will periodically review data on safety and 
enrollment, and will review cumulative safety data for evidence of study related AEs, adherence 
to the protocol, and factors that may affect outcome or study data such as protocol violations and 
losses to follow up. 
7.2 
Laboratory Evaluations 
7.2.1 Clinical Laboratory Evaluations 
Using standard techniques, the following tests will be performed at the CERMEL clinical 
laboratory in Lambaréné, Gabon.  One or more of the laboratory parameters may be repeated at 
any time during the study as determined by the PI, if indicated by an AE. A clinically significant 
abnormal value should be repeated within 14 days if possible and followed up as clinically 
relevant. 

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1. Complete blood count plus white blood cell differential (WBC, absolute neutrophil count 
[ANC], hemoglobin concentration and platelet count)  
2. Serum creatinine 
3. Alanine aminotransferase (ALT) 
4. HBsAg rapid test 
5. HCV rapid test 
6. HIV rapid test 
 
Urine dipstick testing will also be performed at the trial sites using an approved product.  
7.2.2 Special Assays or Procedures 
 
Anti-Na-GST-1 and anti-Na-APR-1 antibody assays (e.g., ELISAs and functional antibody 
assays) will be performed at the Department of Microbiology, Immunology and Tropical Medicine 
of the George Washington University (Washington, DC, USA).  Cellular immunological responses 
and metabolomics studies will be performed at CERMEL and at the University of Leiden (Leiden, 
The Netherlands).  Cryopreservation of sera, plasma and PBMCs will be at -80°C or in liquid 
nitrogen.  
7.2.2.1 
Anti-Na-APR-1 (M74) and Anti-Na-GST-1 Antibody Assays 
 
Antibodies to Na-GST-1 and Na-APR-1 will be measured in serum or plasma of study subjects 
using an indirect ELISA assay.  Antigen-specific IgG and IgE antibody levels will be measured at 
baseline and at several time-points post-vaccination. In addition, antigen-specific IgG subclasses 
(IgG1-4) will be measured by indirect ELISA.  For the ELISA method, microwell plates will be 
coated with purified recombinant Na-GST-1 or Na-APR-1 (M74), blocked, and incubated with 
serial dilutions of a positive “standard reference serum” pool to generate a reference curve from 
which antibody levels of test sera can be interpolated.  After washing, a horseradish peroxidase 
conjugated anti-human antibody will be added and incubated with on the plates, washed again, 
incubated with a chromogenic substrate, and antibody levels measured on an ELISA plate reader.  
7.2.2.2 
Enzyme Neutralization Assays 
In vitro assays have been developed to measure the functional capacity of antibodies induced by 
vaccination with recombinant Na-GST-1 or Na-APR-1 (M74) to inhibit or neutralize the activity of 
the native forms of these proteins. Serum or plasma samples collected from study subjects will 
be tested for their neutralizing capacity at several post-vaccination time points.  These assays will 
be performed at the Clinical Immunology Laboratory of the Department of MITM of the George 
Washington University. 
7.2.2.3 
Cellular Immunology Assays 
Cytokine and chemokine assays will be performed on supernatants from cryopreserved PBMCs 
collected at various time points in the study, by separating PBMCs from whole blood and culturing 
cells with media only, the mitogen phytohemagglutinin, the antigens Na-APR-1 (M74) or Na-GST-

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1, and crude N. americanus antigens extracts (e.g., adult somatic crude antigen extract). 
Cytokines (e.g., IL-2, IL-4, IL-5, IL-10, TNFa, and IFNg) will be quantified using Cytometric Bead 
Analysis kits of supernatants collected from PBMCs stimulated with Na-APR-1 (M74) or Na-GST-
1 antigen for 48 and 72 hours in vitro. Chemokines (e.g., RANTES, MIP1a, MCP-1, and IP-10) 
will be quantified in supernatants using commercially available ELISA kits.  
7.2.2.4 
Parasitology Assays 
 
Fecal samples will be tested for helminth ova or larvae by the Kato Katz fecal thick smear 
technique. 
7.2.2.5 
Metabolomic Assays 
Time-course of metabolic change after vaccination using urine will be performed by 1H-NMR and 
LC-MS metabolomics approaches that use the NMR and mass spectrometry platforms in LUMC.  
Non-invasive urine sampling will be undertaken at regular time points when study participants are 
seen before and after vaccination. 
  
In order to assess the metabolite changes after the administration of anthelmintic drugs, urine 
sampling will be undertaken at screening, for baseline measurements. For children who need 
albendazole or praziquantel treatment for helminthic infections, and are included for vaccination, 
the urine sampling will be repeated the day of vaccination, to measure the metabolite changes. 
Samples will be coded with the child’s screening number. For children who will not be enrolled for 
vaccination, obtained urine samples will be destroyed.  
 
The metabolomics studies are included as exploratory endpoints. Metabolomics involves profiling 
and measuring small molecules that reflect the metabolic response at the cellular, tissue and 
organ level, and can provide insight into the functioning and induction of the immune response. 
Two metabolomic studies in infection models have revealed novel insights into how lipid related 
metabolites play an important role in viral (Wikoff, 2009) and bacterial (Tobin, 2012) infections. 
Importantly, work has shown that the metabolic state of immune cells can have far reaching 
consequences for the development of long-term memory responses (Pearce, 2009). Therefore, 
whole metabolome measurements will be made in urine and both untargeted and pathway 
specific analysis will be applied to identify biomarkers and also provide insight into mechanisms 
of successful vaccination as determined by the neutralizing capacity of antibodies raised to Na-
APR-1 and Na-GST-1. These ancillary cellular and metabolic studies in this Phase 1 trial will 
generate data that will be correlated with i) the development of neutralizing antibodies to Na-GST-
1 and Na-APR-1, in order to understand which processes are important for immunogenicity of the 
vaccine and successful generation of neutralizing antibodies; and, ii) the clinical data obtained in 
order to delineate mechanisms underlying any potential adverse reactions.  
 
 

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7.2.3 Specimen Preparation, Handling, and Shipping 
7.2.3.1 Instructions for Specimen Preparation, Handling, and Storage 
Instructions for specimen preparation, handling, and storage are included in the protocol-specific 
Manual of Procedures, as appropriate. 
7.2.3.2 Specimen Shipment 
Specimen shipment will occur at intervals during the course of the study following all applicable 
International Air Transport Association (IATA) requirements and according to the specifics for 
storage temperature and documentation as detailed in protocol-specific SOPs, as appropriate. 
 
Specimens for immune responses will be shipped from the study site at CERMEL to the George 
Washington University in Washington, DC, and to the LUMC in Leiden, The Netherlands, in a 
blinded manner in batches as they become available.   

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8 
ASSESSMENT OF SAFETY 
8.1 
Specification of Safety Parameters 
 
Safety will be assessed by the frequency and severity of:  
1. Study vaccine-related serious adverse events occurring from the time of the first study 
vaccination through approximately 9 months after the last study vaccination. 
 
2. Solicited Adverse Events – reactogenicity events occurring on the day of each study 
vaccination through 14 days after each study vaccination: 
a) Injection site reactions including erythema (redness), induration (hardness)/swelling, 
pain, and tenderness. 
b) Systemic reactions including fever, myalgia (body aches/muscular pain exclusive of 
the injection site), arthralgia (joint pain exclusive of the injection site), headache, 
urticaria, vomiting, and nausea. 
 
3. Clinical safety laboratory adverse events occurring from the time of each study vaccination 
through approximately 14 days after each set of vaccinations. Parameters to be evaluated 
include WBC, ANC, hemoglobin concentration, and platelet count; ALT; and, creatinine. 
 
4. Unsolicited Adverse Events – non-serious adverse events occurring from the time of teach 
study vaccination through approximately 28 days after each vaccination. 
 
5. New-onset chronic medical conditions occurring from the time of the first study vaccination 
through approximately 9 months after the last study vaccination. 
 
6. Adverse Events of Special Interest occurring from the time of the first study vaccination 
through approximately 9 months after the last study vaccination. 
 
8.2 
Methods and Timing for Assessing, Recording, and Analyzing 
Safety Parameters 
8.2.1 Adverse Events 
An adverse event (AE) includes any noxious, pathological or unintended change in anatomical, 
physiological or metabolic functions as indicated by physical signs, symptoms and/or laboratory-
detected changes occurring in any phase of the clinical study, whether associated with the study 
vaccine or placebo, and whether or not considered vaccination related. This includes an 
exacerbation of pre-existing conditions and intercurrent illnesses.  
 
All AEs will be graded for severity and relationship to study vaccine. A study clinician will be readily 
available for the duration of the trial to assess AEs. Should a parent/guardian or child call on a 
study clinician to report an AE, it will be fully documented in their study chart, and discussed with 
the Principal Investigator. 

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candidates Na-APR-1 (M74) and Na-GST-1 in Gabonese Children 
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All AEs will be assessed by the investigator using the following protocol-defined grading system, 
as or described in Tables 2-5: 
 
Grade 1: 
Mild - No effect on activities of daily living; no medical 
intervention/therapy required 
Grade 2:  
Moderate - Partial limitation in activities of daily living (can complete 
>50% of baseline); no or minimal medical intervention/therapy 
required 
Grade 3:  
Severe - Activities of daily living limited to < 50% of baseline; 
medical evaluation/therapy required 
All AEs will have their possible relationship to study vaccine assessed using the following terms: 
 
Definite:  
Clear-cut temporal association, and no other possible cause. 
Probable: 
Clear-cut temporal association and a potential alternative etiology is not 
apparent. 
Possible: 
Less clear temporal association; other etiologies also possible. 
Unlikely: 
Temporal association between the AE and the vaccine or the nature of the 
event is such that the vaccine is not likely to have had any reasonable 
association with the observed illness/event (cause and effect relationship 
improbable but not impossible).   
Not Related: The AE is completely independent of vaccine administration; and/or evidence 
exists that the event is definitely related to another etiology. 
 
The degree of certainty with which an AE can be attributed to administration of study vaccine will 
be determined by how well the event can be understood in terms of one or more of the following: 
 
1. The event being temporally related with vaccination or reproduced on re-vaccination. 
2. A reaction of similar nature having previously been observed with this type of vaccine and/or 
formulation. 
3. The event having often been reported in the literature for similar types of vaccines. 
8.2.2 Reactogenicity 
Reactogenicity events are AEs that are known to occur with types of vaccine similar in 
composition to those being tested in this study. The following Toxicity Grading Scales will be used 
to grade solicited local (injection site) and systemic (subjective and quantitative) reactions: 
 
 

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Table 2:  Assessment of Solicited or Expected Adverse Event Severity 
Adverse Event 
Grade 
Severity 
Pain/tenderness at injection site 1 
Easily tolerated, does not interfere with activity 
 
2 
Limits use of limb or interferes with daily activity 
 
3 
Prevents daily activity 
Erythema at injection site 
1 
< 10 mm 
 
2 
10 mm – 25 mm 
 
3 
> 25 mm 
Induration/swelling at injection  
1 
< 10 mm 
Site 
2 
10 mm – 25 mm 
 
3 
> 25 mm 
Fever (tympanic) 
1 
38.0°C – 38.4°C 
 
2 
38.5°C – 38.9°C 
 
3 
>39.0°C  
Headache 
1 
Easily tolerated, does not interfere with daily activity  
 
2 
 
Responds to non-narcotic pain reliever or interferes with daily 
activity 
 
3 
Responds to narcotic pain reliever or prevents daily activity 
Nausea 
1 
Easily tolerated, does not interfere with daily activity 
 
2 
Decreased oral intake 
 
3 
Minimal oral intake 
Vomiting 
1 
1-2 episodes in 24 hours  
 
2 
> 2 episodes in 24 hours  
 
3 
Prevents daily activity or requires outpatient IV hydration 
Myalgia 
1 
Easily tolerated, does not interfere with daily activity 
 
2 
Interferes with daily activity 
 
3 
Prevents daily activity 
Arthralgia 
1 
Easily tolerated, does not interfere with activity 
 
2 
Interferes with daily activity 
 
3 
Prevents daily activity 
Urticaria 
1 
Requiring no medications 
 
2 
 
Requiring oral or topical treatment, or IV medication or steroids for 
<24 hours 
 
3 
Requiring IV medication or steroids for >24 hours 
 
Table 3: Assessment of Unsolicited Systemic Adverse Event Severity 
Systemic AE 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Anorexia 
Decreased appetite 
without decreased 
oral intake lasting 
greater than 48 
hours 
Decreased appetite 
associated with 
decreased oral intake 
without significant 
weight loss 
Loss of appetite associated with 
significant weight loss 
Diarrhea 
3 
loose 
stools/24 
hours 
Liquid stools 
Liquid stools > 4x the amount or 
number normal for this child 
Constipation 
Not Applicable 
Hard, dry stools with 
a change in 
frequency 
Resulting in abdominal pain 
Fatigue 
No interference 
w/activity 
Some interference 
w/activity 
Significant, prevents daily activity 

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Arthritis 
Mild pain with 
inflammation, 
erythema or joint 
swelling – but not 
interfering with 
function 
Moderate pain with 
inflammation, 
erythema or joint 
swelling – interfering 
with function, but not 
with activities of daily 
living 
Severe pain with inflammation, 
erythema or joint swelling –and 
interfering with activities of daily living 
 
Vasovagal 
episode 
(associated 
with 
a 
procedure of any kind) 
 
Present without loss 
of consciousness 
Present with 
transient loss of 
consciousness 
Not Applicable 
Cough  
Transient- 
no 
treatment 
Persistent cough; 
treatment responsive 
Paroxysmal cough; uncontrolled with 
treatment 
 
Bronchospasm, 
Acute 
Transient; 
no 
treatment; 
70% - 80% FEV1 
of peak flow 
Requires treatment; 
normalizes 
with 
bronchodilator; 
FEV1 50% - 70% 
(of peak flow) 
No normalization with bronchodilator; 
FEV1 25% - 50% 
of peak flow; or  retractions present 
Dyspnea 
 
Dyspnea on exertion 
Dyspnea with normal 
activity 
Dyspnea at rest 
Hypersensitivity 
Transient flushing or 
pruritus without rash 
Pruritic rash or mild 
urticaria 
Symptomatic bronchospasm, with or 
without urticarial 
 
 
Table 4: Assessment of Basic Body Function Adverse Event Severity 
Vital Signs* 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Tachycardia –  
beats per minute 
115-130 
131-150 
>150 
Bradycardia –  
beats per minute 
55-59 
50-54 
<50 
Hypertension** 
(systolic, mm Hg) 
126-135 
136-145 
>145 
Hypertension** 
(diastolic, mm Hg) 
86-90 
91-96 
>96 
Hypotension 
Symptomatic, oral fluids 
not required 
Symptomatic, corrected 
with oral fluid 
replacement 
Symptomatic, IV fluids 
indicated 
*Subject should be at rest for measurement of vital signs 
**With repeat testing at same visit 
 
 
 
 
 

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Table 5: Assessment of Laboratory Adverse Event Severity 
HEMATOLOGY 
Grade 1 
(Mild) 
Grade 2 
(Moderate) 
Grade 3 
(Severe) 
Hemoglobin 
 
8.2 – 9.2 g/dL  
7.1 – 8.1 g/dL 
<7.0 g/dL 
Platelets 
110,000 
– 
130,000/mm3 
90,000 
– 
109,999/mm3 
<90,000/mm3 
WBCs 
  (increase) 
13,800 
– 
17,000/mm3 
17,001 
– 
20,000/mm3 
<20,000/mm3 
WBCs 
  (decrease) 
2500 – 4200/mm3 
1500 – 2499/mm3 
<1500/mm3 
ANC 
  (decrease) 
750 – 1000/mm3 
500 – 749/mm3 
<500/mm3 
CLINICAL 
CHEMISTRIES 
Grade 1 
Grade 2 
Grade 3 
 
Serum 
creatinine 
 
1.2 – 1.4 mg/dL 
 
1.5 – 1.7 mg/dL 
 
>1.8 
mg/dL 
or 
requires dialysis 
ALT 
 
 
48 – 105 U/L 
 
 
106 – 220 U/L 
 
>220 U/L 
 
 
All local (injection-site) reactions occurring within the first 14 days after vaccination will be 
considered definitely related to vaccination. 
 
8.2.3 Serious Adverse Events 
 
An SAE is an AE, whether considered related to a study vaccine or not, meeting one of the 
following conditions: 
 
1. Death during the period of protocol-defined surveillance 
2. Life threatening: defined as an event that places a subject at immediate risk of death at the 
time of the event and does not refer to an event that hypothetically might have caused death 
were it more severe 
3. Hospitalization during the period of protocol-defined surveillance: defined as at least an 
overnight stay in the hospital or emergency ward for treatment that would have been 
inappropriate if administered in the outpatient setting 
4. Results in a congenital anomaly or birth defect 
5. Results in a persistent or significant disability or incapacity: defined as a substantial disruption 
of the study subject’s ability to carry out normal life functions 
6. Any other important medical event that may not result in death, be life threatening, or require 
hospitalization, may be considered a serious AE when, based upon appropriate medical 
judgment, the event may jeopardize the subject and may require medical or surgical 
intervention to prevent one of the outcomes listed above. 

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8.2.4 Procedures to be Followed in the Event of Abnormal Laboratory Test 
Values or Abnormal Clinical Findings 
The Principal Investigator or appropriate co-investigator is responsible for reporting all AE/SAEs 
that are observed or reported during the study, regardless of the relationship to study products.  
AE/SAEs, abnormal clinical laboratory test values, or abnormal clinical findings will be 
documented, reported, and followed appropriately. 
8.3 
Reporting Procedures 
8.3.1 Serious Adverse Events 
 
All SAEs will be reviewed by a study physician, recorded on the appropriate SAE form, and 
followed through to resolution or stabilization by a study physician. All SAEs will be reported to 
AIGHD within 1 working day of notification of the SAE occurrence to the Principal Investigator, via 
either: 
 
- 
Notification of the SAE via the eDM1 system (automatic updates are then provided to all 
relevant pharmacovigilance staff), OR 
- 
Notification via a scanned PDF version of an SAE form to SAE@hookvac.eu. This 
distribution list will provide automatic distribution to all relevant pharmacovigilance staff. 
 
In addition, the Principal Investigator will report all SAEs that are determined to be definitely or 
probably related to vaccination to the Institutional Review Board (IRB) that approved the study 
(i.e., the local IRB in Gabon) according to the IRB’s reporting timelines and using the IRB-specified 
reporting forms, as well as to the Independent Medical Monitor. 
 
All local and systemic reactions not meeting the criteria for SAE will be captured on the 
appropriate case report form (CRF). These events will be followed to resolution.  
 
8.3.2 Regulatory Reporting  
 
Following notification from the Principal Investigator, the IND sponsor (Sabin Vaccine Institute) 
will report events that are both serious and unexpected that are possibly, probably, or definitely 
related to the vaccine, to the FDA within the required timelines: fatal and life-threatening events 
within 7 calendar days (by phone, fax, or internet) and all other SAEs in writing within 15 calendar 
days. All SAEs not listed as possibly, probably, or definitely related will be reported to the FDA 
and ANVISA at least annually in a summary format. 
 
All Adverse Events of Special Interest (AESIs; see below) will be reported to FDA according to 
the same procedure as for reporting SAEs, and according to the same timelines as described 
above.  
 
1 Electronic Data Management system 

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8.3.3 Adverse Events of Special Interest (AESI) 
Special attention will be paid to monitoring for the occurrence of certain adverse events termed, 
“Adverse Events of Special Interest” or AESIs.  These include inflammatory and autoimmune 
disorders that may potentially be related to the use of an immunostimulatory adjuvant (although 
none have been associated with the use of GLA-AF). The occurrence of the following AESI’s will 
be closely monitored:  
• 
Neuroinflammatory disorders (optic neuritis, multiple sclerosis, demyelinating disease, 
transverse myelitis, Guillain-Barré syndrome, myasthenia gravis, encephalitis, neuritis, Bell’s 
palsy)  
• 
Musculoskeletal disorders (systemic lupus erythematosus, cutaneous lupus, Sjogren’s 
syndrome, scleroderma, dermatomyositis, polymyositis, rheumatoid arthritis, juvenile 
rheumatoid arthritis, polymyalgia, rheumatica, reactive arthritis, psoriatic arthropathy, 
ankylosing spondylitis, spondylarthropathy)  
• 
Gastrointestinal disorders (Crohn’s disease, ulcerative colitis, celiac disease)  
• 
Metabolic diseases (autoimmune thyroiditis, Grave's or Basedow’s disease, Hashimoto 
thyroiditis, insulin-dependent diabetes mellitus, Addison’s disease)  
• 
 Skin disorders (psoriasis, vitiligo, Raynaud’s phenomenon, erythema nodosum, 
autoimmune bullous skin diseases)  
• 
Others 
(autoimmune 
hemolytic 
anemia, 
idiopathic 
thrombocytopenic 
purpura, 
antiphospholipid syndrome, temporal arteritis, Behcet's syndrome, pernicious anemia, 
autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, autoimmune 
glomerulonephritis, autoimmune uveitis, autoimmune cardiomyopathy, sarcoidosis, Stevens-
Johnson syndrome).  
• 
Vasculitides  
8.3.4 Medically-Attended Adverse Events (MAAEs) 
Special attention will also be paid to recording medically-attended adverse events, which are 
defined as any clinical symptom or diagnosis (including local symptoms at the injection site or 
systemic symptoms) for which medical evaluation and/or care is sought from a healthcare 
professional, outside of a regularly scheduled study visit. Note that MAAEs are not the same as 
SAEs. Whether or not an AE is an MAAE will be recorded on the appropriate Case Report Form. 
8.4 
Type and Duration of Follow-up of Subjects after Adverse Events 
AEs will be recorded if they start from the time of the first study vaccination (Day 0) up to 28 days 
after each dose of study vaccine.  AEs limited to new-onset chronic medical conditions will be 
followed through approximately 9 months after the third study vaccination (Day 380/448). 
 

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8.5 
Halting Rules 
 
If a dose of study vaccine is considered significantly reactogenic (see below and Section 8.6.2), 
dose escalation and/or additional vaccinations will be suspended until reviewed by the SMC, 
Independent Medical Monitor, and IND sponsor (SVI). Any recommendation of the Independent 
Safety Monitor and/or SMC to resume or suspend further injections (either for an individual 
subject, an entire Group, or the full study) will be communicated in writing to the sponsor and 
Principal Investigator. All communications from the SMC will subsequently be forwarded by the 
investigators to the IRB.  
 
The following criteria will be used to define significant reactogenicity: 
 
• 
One or more participants experience a protocol defined SAE that is determined to be 
possibly, probably, or definitely related to a study vaccine, OR, 
• 
One or more participants experience a protocol defined AESI that is determined to be 
possibly, probably, or definitely related to a study vaccine, OR, 
• 
One or more participants experience a protocol defined Grade 3 hypersensitivity reaction 
that is possibly, probably or definitely related to a study vaccine, OR, 
• 
Two or more participants experience a protocol defined Grade 2 hypersensitivity reaction 
that is probably or definitely related to a study vaccine, OR, 
• 
Three or more participants in a single Group (i.e., Group 1, Group 2, etc.) experience the 
same objective physical finding or laboratory abnormality of Grade 3 or higher (with the 
exception of isolated Grade 3 erythema or swelling), that is determined to be probably or 
definitely related to a study vaccine, OR, 
• 
Three or more participants in a single Group experience the same Grade 2 or higher safety 
laboratory abnormality or Grade 3 clinical AE that is possibly, probably or definitely related 
to a study vaccine. 
 
The study will be double-blinded until all study subjects have completed their Day 208 visit, the 
safety and secondary immunogenicity outcomes (i.e., anti-Na-GST-1 and anti-Na-APR-1 (M74) 
IgG antibody results by ELISA) have been monitored and entered into the database, and the 
database has been “soft-locked” for interim analysis. After this milestone, the Sponsor and 
investigators will be unblinded.  During the double-blinded part of the study, a study subject’s 
randomization code may be unblinded only for safety purposes. This is unlikely to occur, since 
once a vaccine is administered, knowing which vaccine was given is unlikely to influence the 
medical management of an AE. This procedure is therefore exceptional and any decision to 
unblind will be discussed with the sponsor, the Principal Investigator, the Independent Medical 
Monitor, and the SMC. If deemed necessary for urgent safety reasons, the Independent Medical 
Monitor, in consultation with the SMC (if possible in a timely manner), may unblind a specific 
subject without revealing the study blind to the investigators or the Sponsor. Any unblinding will 
be thoroughly documented. It is to be emphasized that the Independent Medical Monitor may put 
the study on hold at any time and discuss with the SMC. In the event that the investigators come 
to know the study code prior to final unblinding, the Principal Investigator must notify the Sponsor 
immediately. The reasons will be documented by the Principal Investigator and added to the study 
file. 
 

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The decision to completely unblind the study prior to Day 208 or permanently stop the study prior 
to the final study visit of all participating children will take the form of a formal recommendation by 
the SMC to the study Sponsor. The Principal Investigator must then notify the IRB of this decision. 
8.6 
Safety Oversight  
8.6.1 Independent Medical Monitor (IMM) 
 
An independent Medical Monitor will be appointed for oversight of subject safety in this trial. The 
IMM will be a local, qualified physician who will be available to advise the investigators on trial-
related medical questions or problems. The IMM will work with the study team to ensure adequacy 
of adverse event monitoring and reporting. Should the IMM not be available, he/she will 
recommend an alternative to serve as a substitute IMM. 
 
The IMM’s primary responsibility will be to monitor subject safety. The Principal Investigator is 
responsible for ensuring that the IMM is aware of any new safety information that becomes 
available during the course of the trial.   
8.6.2 Safety Monitoring Committee (SMC) 
At least three individuals will be selected to serve as the study Safety Monitoring Committee 
(SMC) to advise the Sponsor and the study investigators on the trial. All SMC members will be 
independent from the Sponsor and study site. The SMC’s primary responsibility will be to monitor 
subject safety. The Principal Investigator is responsible for ensuring that the SMC is aware of all 
new safety information. The SMC will periodically review data on safety and enrollment, and will 
review cumulative safety data for evidence of study related AEs, adherence to the protocol, and 
factors that may affect outcome or study data such as protocol violations and losses to follow up. 
 
Cumulative safety data reports from the trial will be submitted to the SMC before beginning 
vaccinations in Group 2 and Group 3 of the study. These safety data reports will include data from 
at least the first 14 days after first vaccination of all subjects in the prior Group. After the third and 
final vaccination has been administered to all cohorts, additional safety and immunology results 
and reports will be submitted to the SMC as they become available.  
 
Conference calls between the investigators and the SMC will be scheduled at least one week 
prior to beginning vaccinations in Group 2 and Group 3 of the study. If no criteria for halting the 
study are met (see Section 8.5 above) vaccinations will proceed with approval from the SMC.   
 
Written approval (via fax or email) to proceed to vaccinating Group 2 and Group 3 must be 
obtained from the SMC prior to vaccine administration. Both the SMC and IMM will have access 
to the randomization code, as they may wish to review the data in an unblinded fashion should 
significant safety questions arise prior to the final unblinding of the study. 
It is the Principal Investigator’s (or designated agent) responsibility to ensure that the SMC 
reviews the current safety data (grouped by dose cohort), study protocol, and any other requested 
documents at its meetings. Occurrence of an SAE will be reported to the SMC at the same time 

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that it is reported to the IRB. Additionally, any new information that may adversely affect the safety 
of the subjects or the conduct of the study will be submitted to the SMC as it becomes available. 

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9 
CLINICAL MONITORING 
9.1 
Site Monitoring Plan 
The Sponsor (or its designee) will monitor all aspects of the study, with respect to current Good 
Clinical Practices, and for compliance with applicable government regulations. Prior to the start 
of the study, the Principal Investigator will be informed of the frequency of monitoring visits and 
will be given reasonable notification prior to each visit. The objectives of a monitoring visit will be 
to verify the prompt reporting of SAEs, to check the availability of the signed Informed Consent 
and the child assent form for enrolled study subjects, to compare CRFs and spreadsheets with 
source data for completeness and accuracy, to verify compliance with the clinical protocol, and to 
check investigational product accountability. During the monitoring visit, the Principal Investigator 
(and/or designee) and other study personnel should be available to discuss the study. Study 
documents must be available for review throughout the course of the study.   
 

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10 
STATISTICAL CONSIDERATIONS 
10.1 Sample Size Considerations 
 
This Phase 1 trial is not powered to detect statistically significant differences between groups. 
Even though comparative statistics for the safety variables will be computed, the study will have 
low power to detect anything other than very large differences in the incidence of local injection 
site and systemic side effects between the vaccination groups. This is done weighing the need to 
detect any possible untoward reactions against the need to limit the number of volunteers involved 
for safety purposes. The sample size of 60 for this study is within the range commonly used in 
Phase 1 trials for the initial assessment of the safety, tolerance and immunogenicity of 
investigational vaccines.  
The primary objective of the study is to estimate the frequency of vaccine-related adverse events. 
Therefore, rather than providing a statistical power calculation for a specific hypothesis, we 
illustrate below the probability of observing one or more events, such as an adverse event of a 
particular type, for a single vaccine dose (N=20), formulation (N=30), or for all participants (N=60) 
under different assumptions of the true rate at which such events occur in the population. 
 
 
Probability of Observing 1 
or More Events 
N=20 
N=30 
N=60 
"True" (but unknown) Probability of an Event 
9.54% 
13.96% 
25.97% 
0.5% 
1.0% 
18.21% 
26.03% 
45.28% 
3.0% 
45.62% 
59.90% 
83.92% 
5.0% 
64.15% 
78.54% 
95.39% 
10.0% 
87.84% 
95.76% 
99.82% 
As illustrated in the table above, the study will have high probability (>= 80%) of detecting events 
that are commonly occurring in the population. For example, with a sample of 20 participants 
(which is equivalent to the size of each dose group of Na-APR-1 (M74) in this study), there is an 
87.84% probability of observing 1 or more events when the events occur at a rate of 10.0% in the 
population. As the sample size increases, the study is able to detect events that occur at lower 
rates. For example, using a sample size of 30 participants (which is equivalent to the number of 
participants assigned to vaccine co-administration in this study vs. Na-APR-1 (M74) alone), there 
is a 78.54% probability of observing 1 or more events that occur at a rate of 5% in the population. 
Using a sample of size 60 (equivalent to the entire study population) there is an 83.92% probability 
of observing 1 or more events that occur at a rate of 3% in the population. Detection of rare 
events—those that occur at a rate of 1% or less in the population--is not likely in this study. 
 

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10.2 Planned Interim Analyses  
 
See section 14.4 for timing of reports.  
10.2.1 Safety Review  
 
Descriptive and hypothesis-testing approaches will be used to estimate AE rates and to compare 
these rates in the difference doses and formulations of Na-APR-1 (M74) alone or co-administered 
with Na-GST-1. Estimates will be presented with their 95% confidence intervals. Formal statistical 
tests, as outlined below, will be used to compare doses. No formal adjustments for multiple 
comparisons will be made. Statistical tests will use a two-sided significance level of 5%.  
 
The proportion of subjects with at least one injection site AE will be compared by vaccine 
allocation (e.g., Na-APR-1 (M74) co-administered with Na-GST-1 vs. hepatitis B vaccine), by and 
by Na-APR-1 (M74)/Na-GST-1 dose.  
Laboratory results (hematological and clinical chemistry) will be examined for trends over time 
and any clinically significant values for individuals will be reported. 
 
10.2.2 Immunogenicity Review 
The proportion of subjects with detectable anti-Na-GST-1 and anti-Na-APR-1 (M74) antibody 
responses, and the duration of these responses, will be summarized as descriptive measures. 
Geometric mean antibody responses will be compared between  dose groups for each vaccine 
antigen with comparisons made by analysis of variance (ANOVA) tests and pair-wise 
comparisons by contrasts. Antibody responses will be measured at Days 0, 7, 14, 28, 56, 63, 70, 
84, 112/180, 119/187, and 126/194 for interim analysis, with Days 140/208, 200/268, 290/358, 
and 380/448 added for final analysis.  
 
To exploit the multiple measures of antibody within each subject, a longitudinal model may be 
built (if possible) to describe the antibody responses over time. The models will explore if there 
are any differences between those administered Na-APR-1 (M74)/Alhydrogel® co-administered 
Na-GST-1/Alhydrogel® and those administered the hepatitis B vaccine. 
 
Summary statistics will be used to present exploratory study results of the cellular immune 
responses to Na-GST-1 and Na-APR-1 (M74) before and after immunization. To assess the 
changes in lymphocyte proliferative responses and cytokine/chemokine profiles over time, a 
generalized estimating equations model with robust standard errors will be constructed. When 
necessary, data reduction approaches will be used. 
10.3 Final Analysis Plan 
 
The purpose of this trial is to estimate AE rates and patterns of immune response as well as to 
compare these rates and patterns when Na-GST-1/Alhydrogel® and Na-APR-1 (M74)/Alhydrogel® 
are administered separately or when they are co-administered. 
 

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This section briefly describes the statistical methods to be used; a detailed analytical plan will fully 
describe the methods. The analytical plan will discuss the planned approaches to missing data.  
Deviations from the original analytical plan will be thoroughly documented and reported to the 
Sponsor.   
 
Descriptive and hypothesis-testing approaches will be used to meet the protocol objectives as 
stated in Section 3. Estimates will be presented with their 95% confidence intervals. Formal 
statistical tests, as outlined below, will be used to compare doses. Statistical tests will use a two-
sided significance level of 5%.  
 
Primary Objective: To evaluate the safety and reactogenicity of three different dose 
concentrations of Na-APR-1 (M74)/Alhydrogel® co-administered with Na-GST-1/Alhydrogel® in 
healthy Gabonese children. 
 
AEs will be coded according to Medical Dictionary of Regulatory Activities (MedDRAÔ) preferred 
terms. The frequency, severity, and relationship of AEs per each dose of Na-APR-1 
(M74)/Alhydrogel® and Na-GST-1/Alhydrogel® compared to Hepatitis B vaccine will be presented 
in tabular form using the MedDRAÔ coded term and organized by MedDRAÔ System, Organ, 
and Class (SOC) designations.  
 
• 
The frequency of immediate, systemic, and local injection site AEs will be summarized by 
SOC and preferred term. 
• 
Line listings of clinical and laboratory AEs classified as immediate (within the first 60 
minutes), systemic, and local will be displayed in tables stratified by vaccine (i.e., 
hookworm vs. Hepatitis B vaccines) dose of Na-APR-1 (M74)/Alhydrogel® and Na-GST-
1/Alhydrogel®. 
• 
AEs will be summarized by severity and relationship to vaccine by individuals and dose of 
Na-GST-1 and Na-APR-1 (M74). 
• 
The frequency of vaccine-related SAEs will be tallied as well as summarized by body 
system, by vaccine and dose. 
 
Primary Outcome Measures: 
The following summary parameters will be evaluated for each vaccine (hookworm vs. 
Hepatitis B) and dose of Na-APR-1 (M74) co-administered with Na-GST-1: 
1. Frequency of solicited injection site and systemic reactogenicity, graded by severity, 
on the day of each study vaccination through 14 days after each study vaccination. 
2. Frequency of study vaccine-related serious adverse events from the time of the first 
study vaccination through approximately 9 months after the last study vaccination. 
3. Frequency of clinical safety laboratory adverse events.  
4. Frequency of unsolicited adverse events, graded by severity, from the time of each 
study vaccination through approximately 1 month after each study vaccination.  
5. Frequency of new-onset chronic medical conditions through approximately 9 months 
after the third study vaccination. 
6. Frequency of Adverse Events of Special Interest through approximately 9 months after 
the third study vaccination. 
 

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Analysis Plan: 
 
a. The proportion of participants with at least one injection site AE will be compared by 
vaccine and dose cohort. We will test the null hypotheses that the type and number of 
adverse events is the same across all groups by Fisher’s exact test. 
 
b. Laboratory results (hematological and clinical chemistry) will be examined for trends 
over time and any clinically significant values for individuals will be reported. 
 
 
Secondary Objective: To determine the doses of co-administered Na-APR-1 (M74) and Na-GST-
1 that result in the highest levels of IgG antibody approximately 14 days after the third vaccination.  
 
Secondary Outcome Measure: 
 
The following parameters will be evaluated for each dose of Na-APR-1 (M74) co-administered 
with Na-GST-1: 
1. The IgG level by a qualified indirect ELISA approximately 14 days after the third 
vaccination. 
 
Analysis Plan: 
a. The proportion of participants with detectable anti-Na-GST-1 and anti-Na-APR-1 
(M74) responses will be summarized as a descriptive measure.   
b. IgG levels will be displayed graphically by study group using notched box plots. 
c. Geometric mean antibody responses will be compared between vaccine formulation 
and dose groups. Comparisons between groups will be made by a one-way analysis 
of variance (ANOVA) with pair-wise comparisons between groups made by contrasts. 
d. Geometric mean antibody responses for each antigen will be compared using a 
regression model with main effects for the doses of Na-APR-1 (10, 30 or 100μg), and 
Na-GST-1 (10, 30 or 100μg). 
 
 
Tertiary Objective 1: To assess and compare the duration of antibody responses to Na-GST-1 
and Na-APR-1 (M74). 
.  
Tertiary Outcome Measure 1: The IgG antibody response, by indirect enzyme-linked 
immunosorbent assay (ELISA) at approximately 7, 14, and 28 days after each vaccination, 
and approximately 3 and 6 months after the third dose. 
 
Analysis Plan: 
a. The proportion of participants with detectable anti-Na-GST-1 and anti-Na-APR-1 
(M74) responses will be summarized as a descriptive measure.   
b. Antigen-specific IgG levels will be displayed graphically by study group using notched 
box plots at each time point. 
c. Geometric mean antibody responses will be compared between vaccine and dose 
groups at each time point. Comparisons between groups will be made by a one-way 

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analysis of variance (ANOVA) with pair-wise comparisons between groups made by 
contrasts. 
d. Percent change in IgG antibody levels from days of vaccination to days 7,14 and 28 
will be compared between vaccine formulation and dose groups. Values will be 
reported with 95% confidence intervals. 
e. Percent change in IgG antibody levels from vaccination day #1 to #2, and from 
vaccination day #2 to #3 will be compared between vaccine formulation and dose 
groups to assess the sustainability of the response from trough to trough. Values will 
be reported with 95% confidence intervals. 
f. Percent change in IgG antibody levels from the peak following the third vaccination, to 
3 and 6 months after the third vaccination. Values will be reported with 95% confidence 
intervals. 
g. Geometric mean antibody responses for each antigen will be compared using a 
regression model at each time point with main effects for the doses of Na-APR-1 (M74) 
(10, 30 or 100μg) and Na-GST-1 (10, 30 or 100μg) 
h. A longitudinal model will be built to describe the IgG levels over time.  Using a 
longitudinal panel model, differences in antibody isotype levels by dose of Na-APR-1 
(M74) and Na-GST-1 will be explored. This will take account of the correlation between 
measurements on the same participant. 
 
 
Tertiary Objective 2: To assess the distribution of IgG subclass responses to Na-APR-1 (M74) 
and Na-GST-1.  
 
Tertiary Outcome Measure 2: The IgG subclass distribution (IgG1, IgG3, and IgG4) by indirect 
ELISA approximately 14 days after the third vaccination. 
 
Tertiary Outcome Measure 3: The IgG1-4 subclass distribution by ELISA on each day of 
vaccination, approximately 28 days later, and approximately 3 and 6 months after the final 
vaccination. 
 
Analysis Plan: 
a. The proportion of subjects with detectable anti-Na-GST-1 and anti-Na-APR-1 (M74) 
IgG1-4 subclass responses will be summarized at each time point as a descriptive 
measure.   
b. IgG1-4 subclass antibody levels will be displayed graphically by study group using 
notched box plots at each time point.   
c. Geometric mean antibody responses will be compared between vaccine and dose 
groups. Comparisons between groups will be made by a one-way analysis of variance 
(ANOVA) with pair-wise comparisons between groups made by contrasts. 
d. A longitudinal model will be built to describe each of the antibody isotype levels over 
time for each of IgG1-IgG4.  Using a longitudinal panel model, differences in antibody 
isotype levels by dose of Na-APR-1 (M74) and Na-GST-1 will be explored. This will 
take account of the correlation between measurements on the same participant. 
 
 

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Exploratory Objective 1: To assess the cellular immune responses to the Na-GST-1 and Na-APR-
1 (M74) antigens following vaccination. 
 
The following assays will be performed to assess the cellular immune responses to vaccination 
with Na-APR-1 (M74) +/- Na-GST-1: 
 
 
a. Lymphocyte proliferative responses to in vitro stimulation with Na-GST-1 or Na-APR-
1 (M74). 
b. Cytokine and chemokine production in vitro in response to stimulation with Na-GST-1 
or Na-APR-1 (M74). 
c. Changes in in vivo PBMC subpopulations as determined by flow cytometry. 
d. A longitudinal panel analysis will examine for trends over time.  
 
Exploratory Objective 2: To assess the impact of co-administered Na-GST-1 and Na-APR-1 (M74) 
on the production of memory B cells specific for each antigen.  
 
a. The percentage of specific memory B cells will be displayed graphically by study group 
at each time point. 
b. Two separate longitudinal panel analyses will test the null hypothesis that average 
percentages for each of memory B cells are the same in groups over time. This will 
take account of the correlation between measurements on the same participant.   
c. Mann-Whitney tests will assess the null hypothesis that cytokine levels are the same 
in the groups at the primary time point two weeks after final vaccination.  
 
Exploratory Objective 3: To assess the production of memory B cells specific for each vaccine 
antigen compared to metabolomic changes before and after vaccination. 
 
Exploratory studies will be performed of changes in the metabolomic profiles in response to 
vaccination using urine samples that will be analyzed using LC-MS. Data will be analyzed using 
multivariate modeling and multilevel data analysis pipeline. 
 
Should the study be terminated early, the investigative team will discuss with the SMC the reason 
for termination and determine which study questions can be addressed in an unbiased manner 
with the available data. The available data will be analyzed and interpreted in light of early 
termination. 
 

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11 
SOURCE DOCUMENTS AND ACCESS TO SOURCE 
DATA/DOCUMENTS 
Complete source documentation (clinical evaluations and test results) will be collected for every 
child participant for the duration of the study, with supplementary documents (laboratory test 
reports, supplementary hospital or medical records, etc.) forming part of the source 
documentation. Case Report Forms (CRFs) will be used to record study-specific data for enrolled 
subjects, and study-specific data may be entered directly onto CRFs: in these cases, the 
documents will be both source and CRF. The Principal Investigator will responsible for the 
accuracy and completeness of the data reported in the CRFs and the source documents. Data 
reported in the CRFs that is derived from source documents should be consistent with source 
documents and the discrepancies should be explained. 
 

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12 
QUALITY CONTROL AND QUALITY ASSURANCE 
The investigational site is responsible for conducting routine quality assurance and quality control 
activities to internally monitor study progress and protocol compliance. The PI will provide direct 
access to all trial-related sites, source data/documents, and reports for the purpose of monitoring 
and auditing by the sponsor, and inspection by local and regulatory authorities. The PI will ensure 
all study personnel are appropriately trained and applicable documentation is maintained on site.  
Clinical monitors will verify that the clinical trial is conducted and data are generated, documented 
(recorded), and reported in compliance with the protocol, ICH/GCP guidelines, and the applicable 
regulatory requirements. Clinical monitoring reports will be submitted to the Sponsor. 
The data management vendor will implement quality control procedures beginning with the data 
entry system and generate data quality control checks that will be run on the database. Any 
missing data or data anomalies will be communicated to the site(s) for clarification and resolution. 
 

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13 
ETHICS/PROTECTION OF HUMAN SUBJECTS 
The study will be conducted according to fundamental ethical principles including: The principle 
of respect for human dignity and the principles of non-exploitation, non-discrimination and non-
instrumentalisation. The principle of individual autonomy (entailing the giving of free and informed 
consent, and respect for privacy and confidentiality of personal data); The principle of justice (the 
equitable distribution of burdens and benefits of research). The principle of beneficence and non-
maleficence, namely with regard to the improvement and protection of health; and, the principle 
of proportionality (including that research methods are necessary to the aims pursued and that 
no alternative more acceptable methods are available). 
13.1 Ethical Standards 
 
The study will be conducted according to: the Declaration of Helsinki (amended in 2008); CIOMS 
(Council for International Organizations of Medical Sciences) International Ethical Guidelines for 
Biomedical Research Involving Human Subjects (2002); International Conference on 
Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use 
(ICH) (1996) Guideline for good clinical practice E6 (R1); and, the US Code of Federal 
Regulations (Protection of Human Subjects [21 CFR 50], Institutional Review Boards [21 CFR 
56], and Obligations of Clinical Investigators [21 CFR 312]).  
13.2 Institutional Review Boards 
 
The investigators will be responsible for obtaining full IRB approvals for the study from the local 
and national IRB in Gabon. Before the start of the study, the appropriate documents (including 
the protocol, Investigator’s Brochures, and informed consent form) will be submitted to the IRBs. 
The IRBs will be informed by the Investigator of any new information that may adversely affect 
the safety of the subjects or the conduct of the study, an annual update and/or request for re-
approval, and when the study has been completed. 
13.3 Informed Consent and Assent Process 
 
The principles of informed consent in the current edition of the Declaration of Helsinki will be 
implemented and informed consent obtained in accordance with ICH guidelines. A community 
meeting will be held to provide volunteers with an overview of the clinical trial and study 
procedures, and to answer questions in an open forum. This group meeting will be followed by 
individual informed consent sessions with each interested parent or legal guardian of a potential 
child participant. During the individual session a member of the study team will read the consent 
form together with the parent/guardian, regardless of whether or not the parent/guardian is 
illiterate. The study team member will explain why the parent/guardian’s child is being invited to 
participate in the study and will clarify all of the parent/guardian’s questions. Throughout the 
informed consent process, parents/guardians will be encouraged to ask questions, and if they 
wish, to discuss the study with family or community members prior to making a decision.  
 

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Parents/guardians agreeing to have their child participate in the study will first provide written 
informed consent and take a true/false comprehension questionnaire. The questionnaire is 
administered orally in the case of illiterate individuals, and for both written and oral questionnaires 
study staff will use incorrect answers to identify aspects of the study that require clarification and 
focus on those areas of the informed consent form for further review with the parent/guardian. All 
questionnaire questions must be answered correctly, and the informed consent form signed, prior 
to study screening and enrollment of the child. Parents/guardians unable to read will place an 
imprint of their finger in the place of a signature; in addition, an independent witness, who is not 
a member of the study team, will sign the consent form to attest that the informed consent form 
was read to the parent/guardian, the parent/guardian’s questions were answered, and the 
parent/guardian answered all comprehension questions correctly. The original signed informed 
consent form for each parent/guardian will be maintained as part of their child’s study records. A 
copy of the informed consent form will be provided to every child’s parent/guardian.   
 
The child assent form will also be introduced to the participants because it is directly addressed 
to the child where procedures related to the study are explained to them in simple language. The 
child will have to assent and sign. However, if the child is unable to read, an independent witness, 
who is not a member of the study team will sign the form.  
 
13.4 Subject Confidentiality 
 
All study-related information will be stored securely at the study site. All information on 
participating children will be stored in locked file cabinets in areas with access limited to study 
staff. All laboratory specimens, reports, study data collection, and administrative forms will be 
identified by coded numbers only, to maintain subject confidentiality. All computer entries will also 
be made using coded number only, and all local databases will be secured with password-
protected access systems. Forms, lists, and any other listings that link subject identification 
numbers to other identifying information will be stored in a separate, locked file in an area with 
limited access. 
Subject study information will not be released without the written permission of the subject, except 
as necessary for monitoring by the sponsor and/or its designee, the FDA or Gabonese regulatory 
authorities. 
13.5 Study Discontinuation 
If the trial is discontinued, children whose parent/guardian signed the informed consent and child 
assent forms, and are randomized and vaccinated will continue to be followed for safety 
assessments. No further study vaccinations will be administered. 
13.6 Future Use of Stored Specimens 
 
Some of the biological samples collected from study participants may be stored at the local site 
or at the MITM at GWU or at LUMC in Leiden. Stored samples may be shared with other 
investigators at other institutions for the purposes of conducting the tests outlined in this study 

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protocol.  Stored samples will not be sold or used directly for production of any commercial 
product. No human genetic tests will be performed on samples. Each sample will be encoded 
(labeled) only with a unique code and a unique tracking number to protect subject’s confidentiality. 
There are no benefits to subjects in the collection, storage and subsequent research use of 
samples. Reports about research done with subject’s samples will NOT be kept in their health 
records.  Any proposed future research using samples collected from study participants will first 
be submitted to the applicable IRBs for approval.  The IRBs will determine if the study participants 
must be re-consented for the use of their samples in such research.   

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14 
DATA HANDLING AND RECORD KEEPING 
14.1 Data Management Responsibilities 
All data collection forms and laboratory reports must be reviewed by the clinical team and data 
entry personnel, who will ensure that they are accurate and complete. Adverse events must be 
recorded on the appropriate data collection form, assessed for severity and relationship to 
vaccination, and reviewed by the Principal Investigator or appropriate co-investigator. 
Data collection is the responsibility of the study personnel at the study site under the supervision 
of the Principal Investigator. During the study, the Principal Investigator must maintain complete 
and accurate documentation for the study. 
The data management vendor for this study will be responsible for data management, quality 
review, analysis, and reporting of the study data. 
14.2 Data Capture Methods 
 
CRFs will be used to record study-specific data for enrolled subjects, and study-specific data may 
be entered directly onto CRFs: in these cases, the documents will be both source and CRF. The 
Principal Investigator will responsible for the accuracy and completeness of the data reported in 
the CRFs and the source documents. Data reported in the CRFs that are derived from source 
documents should be consistent with source documents and the discrepancies should be 
explained.  
 
The information collected on the paper CRFs is entered into an electronic data collection system 
(EDC); the eCRF. The eCRF will be developed by the data management center (AIGHD). All 
users who have access to the EDC system will be trained. The access to the eCRF is password 
controlled. Plausibility checks will be performed according to a data validation plan. 
Inconsistencies in the data will be queried to the investigators via the electronic data collection 
system; answers to queries or changes of the data will directly be documented in the system. 
After all data are entered and all queries are solved, the database will be closed. 
14.3 Types of Data 
Data for this study will include clinical, safety, and outcome measures (e.g., clinical laboratory 
values, reactogenicity, and immunogenicity data). 
14.4 Timing/Reports 
 
In addition to the study-related documentation required by the regulatory authorities, 4 study 
reports will be generated. These include 2 interim SMC safety reports, 1 interim safety and 
immunogenicity study report, and the final study report. The first interim SMC safety report will be 
completed after the safety data from the Day 14 visits of Group 1 have been compiled, for the 
purposes of deciding whether it is safe to precede with enrollment into Group 2 of the study. The 

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candidates Na-APR-1 (M74) and Na-GST-1 in Gabonese Children 
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second interim SMC safety report will be completed after the safety data from the Day 14 visits of 
Group 2 have been compiled, for the purposes of deciding whether it is safe to precede with 
enrollment into Group 3 of the study. The interim study report will be compiled after interim safety 
and primary immunogenicity data (i.e., antigen-specific IgG responses to Na-GST-1 and Na-APR-
1 (M74) as determined by ELISA) from all Day 208 visits is available and the study investigators 
unblinded. This report will serve as the basis for deciding whether to continue with future Phase 
2 testing of the vaccines in children. A final report containing all safety and immunology data will 
be prepared after trial completion. 
14.5 Study Records Retention 
 
Trial-related documents will be maintained by the Investigator for a period of 2 years after final 
marketing approval of the vaccine, or if 2 years have elapsed since the formal discontinuation of 
clinical development of the product. During the study and while archived at the site all study-
related information will be stored securely in locked file cabinets in areas with access limited to 
study staff. All laboratory specimens, reports, study data collection, and administrative forms will 
be identified by coded number only, to maintain subject confidentiality. All computer entries will 
be done using a coded number only, and all local databases will be secured with password-
protected access systems. Forms, lists, and any other listings that link subject ID numbers to 
other identifying information will be stored in a separate, locked file in an area with limited access. 
Subject study information will not be released without the written permission of the subject, except 
as necessary for monitoring, and the FDA or Gabonese regulatory authorities. 
14.6 Protocol Deviations 
No modifications to this protocol will be implemented without prior written IRB approval. This does 
not apply to changes made to reduce discomfort or avert risk to study subjects. Furthermore, in 
the event of a medical emergency, the investigators shall perform any medical procedures that 
are deemed medically appropriate. The Principal Investigator must notify the Sponsor and IRBs 
of all such occurrences. Any change to the protocol will be submitted to the IRB as a protocol 
amendment, and changes not affecting risk to subjects may be expedited, as appropriate. 
Any deviation from the IRB-approved protocol will be documented, including the date and detailed 
description of the deviation and all corrective actions taken. For any deviation determined to have 
potential or known impact on subject safety, a report will be submitted to the IRB according to 
their guidelines and reporting timelines.  

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15 
PUBLICATION POLICY 
It is anticipated that results from this study will be published in peer-reviewed journals. If 
publication is sought, the identity of study subjects or any easily traceable identifiers will not be 
revealed. Authorship issues will be discussed and agreed upon between the Sponsor and 
collaborating partners prior to submission for publication. Additionally, the results of the study will 
be communicated to both study subjects and the community at large. 
The Principal Investigator and all partners on this study will make publicly available any final 
research data resulting from the trial, in a timely fashion following closure of the clinical trial (not 
more than 12 months after the last subject follow-up visit). 
 

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16 
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candidates Na-APR-1 (M74) and Na-GST-1 in Gabonese Children 
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19. 
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21. 
Hotez PJ, Zhan B, Bethony JM, Loukas A, Williamson A, Goud GN, Hawdon JM, et al. 
Progress in the development of a recombinant vaccine for human hookworm disease: the Human 
Hookworm Vaccine Initiative. Int J Parasitol 2003;33:1245-1258. 
22. 
Diemert DJ, Bethony JM, Hotez PJ. Hookworm vaccines. Clin Infect Dis 2008;46:282-288. 
23. 
Bethony JM, Simon G, Diemert DJ, Parenti D, Desrosiers A, Schuck S, Fujiwara R, et al. 
Randomized, placebo-controlled, double-blind trial of the Na-ASP-2 hookworm vaccine in 
unexposed adults. Vaccine 2008;26:2408-2417. 
24. 
Diemert DJ, Pinto AG, Freire J, Jariwala A, Santiago H, Hamilton RG, Periago MV, et al. 
Generalized urticaria induced by the Na-ASP-2 hookworm vaccine: implications for the 
development of vaccines against helminths. J Allergy Clin Immunol 2012;130:169-176 e166. 
25. 
Ranjit N, Zhan B, Hamilton B, Stenzel D, Lowther J, Pearson M, Gorman J, et al. 
Proteolytic degradation of hemoglobin in the intestine of the human hookworm Necator 
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26. 
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27. 
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Hookworm aspartic protease, Na-APR-2, cleaves human hemoglobin and serum proteins in a 
host-specific fashion. J Infect Dis 2003;187:484-494. 
28. 
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29. 
Loukas A, Bethony JM, Mendez S, Fujiwara RT, Goud GN, Ranjit N, Zhan B, et al. 
Vaccination with recombinant aspartic hemoglobinase reduces parasite load and blood loss after 
hookworm infection in dogs. PLoS Med 2005;2:e295. 
30. 
Pearson MS, Bethony JM, Pickering DA, de Oliveira LM, Jariwala A, Santiago H, Miles 
AP, et al. An enzymatically inactivated hemoglobinase from Necator americanus induces 
neutralizing antibodies against multiple hookworm species and protects dogs against 
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31. 
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characterization and vaccine potential of a heme-binding glutathione transferase from the adult 
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32. 
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hookworm Necator americanus. BMC Struct Biol 2007;7:42. 
34. 
van Rossum AJ, Jefferies JR, Rijsewijk FA, LaCourse EJ, Teesdale-Spittle P, Barrett J, 
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candidates Na-APR-1 (M74) and Na-GST-1 in Gabonese Children 
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70 
36. 
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biochemical characterization, and partial protective immunity of the heme-binding glutathione S-
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Xiao S, Zhan B, Xue J, Goud GN, Loukas A, Liu Y, Williamson A, et al. The evaluation of 
recombinant hookworm antigens as vaccines in hamsters (Mesocricetus auratus) challenged with 
human hookworm, Necator americanus. Exp Parasitol 2008;118:32-40. 
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45. 
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Van Belle P, et al. Vaccine adjuvant systems containing monophosphoryl lipid A and QS21 induce 
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46. 
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Behzad H, Huckriede AL, Haynes L, Gentleman B, Coyle K, Wilschut JC, Kollmann TR, 
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49. 
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hookworm infection and intestinal schistosomiasis. Nat Rev Microbiol 2010;8:814-826. 
50. 
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of 
safety 
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of 
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influenza 
hemagglutinin 
(H5/Indonesia/05/2005) formulated with and without a stable oil-in-water emulsion containing 
glucopyranosyl-lipid A (SE+GLA) adjuvant. Vaccine 2013;31:5760-5765. 
 
 

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SUPPLEMENTS/APPENDICES 

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APPENDIX A1: SCHEDULE OF EVENTS FOR GROUPS 1A, 2A, 3A 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1 Completed within 90 days of first vaccination 
2 CBC safety parameters include WBC, absolute neutrophil count, hemoglobin, and platelet count 
 
 
 
Study Days 
Procedures 
Blood 
Volu
me 
Pre
1 
0 
 
1 
 
3 
± 1 
7 
±1 
14 
±2 
28 
±4 
56 
±7 
57 
 
59 
±1 
63 
±1 
70 
±2 
84 
±4 
112 
±14 
113 
 
115 
±1 
119 
±1 
126 
±2 
140 
±4 
200 
±14 
290 
±14 
380 
±21 
Obtain Informed Consent 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Complete History/Physical  
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Interim Clinical Evaluation 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
AE/SAE/AESI Assessment  
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine Dipstick + 
Parasitology 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HCV 
1.5 mL 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HBsAg 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HIV 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
ALT 
1.2 mL X 
X 
 
 
 
X 
 
X 
 
 
 
X 
 
X 
 
 
 
X 
 
 
 
 
Creatinine 
X 
X 
 
 
 
X 
 
X 
 
 
 
X 
 
X 
 
 
 
X 
 
 
 
 
CBC2  
1.2 mL X 
X 
 
 
 
X 
 
X 
 
 
 
X 
 
X 
 
 
 
X 
 
 
 
 
Antibody Assays (serum or 
plasma) 
1.2 mL 
 
X 
 
 
X 
X 
X 
X 
 
 
X 
X 
X 
X 
 
 
X 
X 
X 
X 
X 
 
Cellular Assays 
5 mL 
 
X 
 
 
X 
X 
X 
X 
 
 
X 
X 
X 
X 
 
 
X 
X 
X 
X 
X 
 
Daily Blood  
Volume (mL) 
 
3.9 8.6 
 
 
2.5 
8.6 
6.2 
8.6 
 
 
2.5 
8.6 
6.2 
8.6 
 
 
2.5 
8.6 
6.2 
6.2 
6.2 
 
Total Blood  
Volume (mL) 
 
3.9 12.5 
 
 
15 23.6 29.8 38.4 
 
 
40.9 49.5 55.7 64.3 
 
 
66.8 75.4 81.6 87.8 
94 
94 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine sample for 
metabolomics  
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Stool Examination 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
X  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
VACCINATIONS 
 
 
1 
 
 
 
 
 
2 
 
 
 
 
 
3 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Randomized, controlled, Phase 1 Study to assess safety and immunogenicity of co-administered Hookworm vaccine candidates Na-APR-1 (M74) and Na-GST-1 
in Gabonese Children 
Version 3.0, 08 Aug 2018 
 ____________________________________________________________________________________________  
 
 ____________________________________________________________________________________________  
73 
APPENDIX A2: SCHEDULE OF EVENTS FOR GROUPS 1B, 2B, 3B 
 
 
 
 
 
 
 
Study Days 
Procedures 
Blood 
Volu
me 
Pre
1 
0 
 
1 
 
3 
± 1 
7 
±1 
14 
±2 
28 
±4 
56 
±7 
57 
 
59 
±1 
63 
±1 
70 
±2 
84 
±4 
180 
±14 
181 
 
183 
±1 
187 
±1 
194 
±2 
208 
±4 
268 
±14 
358 
±14 
448 
±21 
Obtain Informed Consent 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Complete History/Physical  
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Interim Clinical Evaluation 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
AE/SAE/AESI Assessment  
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine Dipstick + 
Parasitology 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HCV 
1.5 mL 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HBsAg 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HIV 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
ALT 
1.2 mL X 
X 
 
 
 
X 
 
X 
 
 
 
X 
 
X 
 
 
 
X 
 
 
 
 
Creatinine 
X 
X 
 
 
 
X 
 
X 
 
 
 
X 
 
X 
 
 
 
X 
 
 
 
 
CBC2  
1.2 mL X 
X 
 
 
 
X 
 
X 
 
 
 
X 
 
X 
 
 
 
X 
 
 
 
 
Antibody Assays (serum or 
plasma) 
1.2 mL 
 
X 
 
 
X 
X 
X 
X 
 
 
X 
X 
X 
X 
 
 
X 
X 
X 
X 
X 
 
Cellular Assays 
5 mL 
 
X 
 
 
X 
X 
X 
X 
 
 
X 
X 
X 
X 
 
 
X 
X 
X 
X 
X 
 
Daily Blood  
Volume (mL) 
 
3.9 8.6 
 
 
2.5 
8.6 
6.2 
8.6 
 
 
2.5 
8.6 
6.2 
8.6 
 
 
2.5 
8.6 6.2 
6.2 
6.2 
 
Total Blood  
Volume (mL) 
 
3.9 12.5 
 
 
15 23.6 29.8 38.4 
 
 
40.9 49.5 55.7 64.3 
 
 
66.8 75.4 81.6 87.8 
94 
94 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine sample for 
metabolomics  
 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Stool Examination 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
VACCINATIONS 
 
 
1 
 
 
 
 
 
2 
 
 
 
 
 
3