Phase 1 Study of the Safety and Immunogenicity of Na-APR-1 (M74)/Alhydrogel® Co-administered With Na-GST-1/Alhydrogel® in Brazilian Adults
Phase: PHASE1
Conditions: Hookworm Infection, Hookworm Disease
Sponsor: Baylor College of Medicine

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PHASE 1 STUDY OF THE SAFETY AND IMMUNOGENICITY 
OF NA-APR-1 (M74)/ALHYDROGEL® CO-ADMINISTERED 
WITH NA-GST-1/ALHYDROGEL® IN BRAZILIAN ADULTS 
 DMID Protocol Number: 15-0012 
 
DMID Funding Mechanism:  NIAID Clinical Trial Implementation Cooperative 
Agreement (U01) 
Pharmaceutical Support Provided by: Not Applicable 
Other Identifying Numbers: IND Sponsor Protocol Number SVI-DBL-01 
IND Sponsor: Albert B. Sabin Vaccine Institute 
Principal Investigator: David Diemert, MD FRCP(C) 
Co-Principal Investigator: Rodrigo Correa-Oliveira, PhD 
DMID Scientific Lead: Gregory Dye, MD 
DMID Medical Monitor: Soju Chang, MD 
 
DMID Regulatory Affairs Specialist: Blossom Smith 
Version Number: 5 
22 July 2015 
 

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STATEMENT OF COMPLIANCE 
The study will be carried out in accordance with Good Clinical Practice (GCP) as required by the 
following: 
• 
United States (US) Code of Federal Regulations (CFR) applicable to clinical studies 
(45 CFR Part 46; 21 CFR Part 50, 21 CFR Part 56, and 21 CFR Part 312) 
• 
ICH E6; 62 Federal Register 25691 (1997)  
• 
Brazilian Resolution Nº 466/12 (replacing Resolution Nº 196/96) on Research Involving 
Human Subjects 
• 
NIH Clinical Terms of Award 
 
All key personnel (all individuals responsible for the design and conduct of this study) have 
completed Human Subjects Protection Training. 
 

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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 US and Brazilian federal regulations and ICH guidelines. 
 
Site Investigator: 
 
Signed: 
 
Date: 
 
 
Name: David Diemert, MD 
Title: Principal Investigator 
 
 
 
 
Site Investigator: 
 
Signed: 
 
Date: 
 
 
Name: Rodrigo Correa-Oliveira, PhD 
Title: Co-Principal Investigator 
 
 
 

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TABLE OF CONTENTS 
Statement of Compliance .............................................................................................................. ii 
Signature Page ............................................................................................................................. iii 
Table of Contents ......................................................................................................................... iv 
List of Abbreviations .................................................................................................................... vii 
Protocol Summary ........................................................................................................................ ix 
1 
Key Roles ................................................................................................................................ 1 
2 
Background Information and Scientific Rationale .................................................................... 3 
2.1 
Background Information ............................................................................................... 3 
2.1.1 
Hookworm Infection and Justification for Vaccine Development .................. 3 
2.1.2 
Study Site ...................................................................................................... 4 
2.1.3 
Prior Clinical Experience with Hookworm Vaccines ...................................... 5 
2.1.4 
The Na-GST-1/Alhydrogel® Hookworm Vaccine ........................................... 7 
2.1.5 
Immunogenicity Studies with Na-GST-1 ..................................................... 10 
2.1.6 
Clinical Experience with Na-GST-1/Alhydrogel® Hookworm Vaccine ......... 11 
2.1.7 
The Na-APR-1 (M74)/Alhydrogel® Hookworm Vaccine .............................. 12 
2.1.8 
Studies of Human Immune Responses to Na-APR-1 in Endemic Areas .... 13 
2.1.9 
Clinical Experience with the Na-APR-1 (M74)/Alhydrogel® Hookworm 
Vaccine ....................................................................................................... 13 
2.1.10 Clinical Experience with Aluminum-Based Adjuvants ................................. 14 
2.1.11 Clinical Experience with GLA-AF ................................................................ 14 
2.2 
Rationale .................................................................................................................... 15 
2.2.1 
Rationale for the Study ............................................................................... 15 
2.2.2 
Rationale for Doses and Dose Schedule to be Studied .............................. 15 
2.2.3 
Clinical Development Plan for the Human Hookworm Vaccine .................. 16 
2.3 
Potential Risks and Benefits ...................................................................................... 17 
2.3.1 
Potential Risks ............................................................................................ 17 
2.3.2 
Procedures Taken to Minimize Risks .......................................................... 18 
2.3.3 
Known Potential Benefits ............................................................................ 18 
3 
Objectives .............................................................................................................................. 20 
3.1 
Study Objectives ........................................................................................................ 20 
3.2 
Study Outcome Measures ......................................................................................... 21 
3.2.1 
Primary Outcome Measure ......................................................................... 21 
3.2.2 
Secondary Outcome Measures .................................................................. 21 
3.2.3 
Tertiary Outcome Measures ........................................................................ 21 
3.2.4 
Exploratory Outcome Measures ................................................................. 22 
4 
Study Design ......................................................................................................................... 23 
4.1 
Substudies (if applicable) ........................................................................................... 25 
5 
Study Enrollment and Withdrawal ......................................................................................... 26 
5.1 
Subject Inclusion Criteria ........................................................................................... 26 
5.2 
Subject Exclusion Criteria .......................................................................................... 26 

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5.3 
Treatment Assignment Procedures ........................................................................... 28 
5.3.1 
Randomization Procedures ......................................................................... 28 
5.3.2 
Masking Procedures ................................................................................... 28 
5.3.3 
Reasons for Withdrawal and Discontinuation of Vaccinations .................... 29 
5.3.4 
Handling of Withdrawals ............................................................................. 31 
5.3.5 
Termination of Study ................................................................................... 31 
6 
Study InterventionS/Investigational ProductS ....................................................................... 32 
6.1 
Study Product Description ......................................................................................... 32 
6.1.1 
Acquisition ................................................................................................... 32 
6.1.2 
Formulation, Packaging, and Labeling ........................................................ 32 
6.1.3 
Product Storage and Stability ..................................................................... 33 
6.2 
Dosage, Preparation and Administration of Study Intervention/Investigational 
Product ................................................................................................................... 33 
6.3 
Modification of Study Intervention/Investigational Product for a Participant .............. 34 
6.4 
Accountability Procedures for the Study Intervention/Investigational Product(s) ...... 34 
6.5 
Assessment of Subject Compliance with Study Intervention/Investigational 
Product ................................................................................................................... 34 
6.6 
Concomitant Medications/Treatments ....................................................................... 34 
7 
Study Schedule ..................................................................................................................... 36 
7.1 
Screening ................................................................................................................... 36 
7.2 
Enrollment/Baseline ................................................................................................... 37 
7.3 
Follow-up ................................................................................................................... 37 
7.4 
Final Study Visit ......................................................................................................... 40 
7.5 
Early Termination Visit ............................................................................................... 40 
7.6 
Unscheduled Visit ...................................................................................................... 40 
8 
Study Procedures/Evaluations .............................................................................................. 42 
8.1 
Clinical Evaluations .................................................................................................... 42 
8.2 
Laboratory Evaluations .............................................................................................. 42 
8.2.1 
Clinical Laboratory Evaluations ................................................................... 42 
8.2.2 
Special Assays or Procedures .................................................................... 43 
8.2.3 
Specimen Preparation, Handling, and Shipping ......................................... 45 
9 
Assessment of Safety ............................................................................................................ 46 
9.1 
Specification of Safety Parameters ............................................................................ 46 
9.2 
Methods and Timing for Assessing, Recording, and Analyzing Safety Parameters .. 46 
9.2.1 
Adverse Events ........................................................................................... 46 
9.2.2 
Reactogenicity ............................................................................................ 47 
9.2.3 
Serious Adverse Events .............................................................................. 51 
9.2.4 
Procedures to be Followed in the Event of Abnormal Laboratory Test 
Values or Abnormal Clinical Findings ......................................................... 51 
9.3 
Reporting Procedures ................................................................................................ 51 
9.3.1 
Serious Adverse Events .............................................................................. 51 
9.3.2 
Regulatory Reporting for Studies Conducted Under DMID-Sponsored 
IND .............................................................................................................. 52 

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9.3.3 
Regulatory Reporting for Studies Not Conducted Under 
DMID-Sponsored IND ................................................................................. 52 
9.3.4 
Adverse Events of Special Interest (AESI) ................................................. 52 
9.3.5 
Medically-Attended Adverse Events (MAAEs) ............................................ 53 
9.3.6 
Reporting of Pregnancy .............................................................................. 53 
9.4 
Type and Duration of Follow-up of Subjects after Adverse Events ........................... 53 
9.5 
Halting Rules ............................................................................................................. 54 
9.6 
Safety Oversight ........................................................................................................ 55 
9.6.1 
Independent Safety Monitor (ISM) .............................................................. 55 
9.6.2 
Safety Monitoring Committee (SMC) .......................................................... 55 
10 
Clinical Monitoring ............................................................................................................... 56 
10.1 
Site Monitoring Plan ................................................................................................. 56 
11 
Statistical Considerations .................................................................................................... 57 
11.1 
Study Hypotheses .................................................................................................... 57 
11.2 
Sample Size Considerations .................................................................................... 57 
11.3 
Planned Interim Analyses ........................................................................................ 58 
11.3.1 Safety Review ............................................................................................. 58 
11.3.2 Immunogenicity Review .............................................................................. 59 
11.4 
Final Analysis Plan .................................................................................................. 59 
12 
Source Documents and Access to Source Data/Documents .............................................. 66 
13 
Quality Control and Quality Assurance ................................................................................ 67 
14 
Ethics/Protection of Human Subjects .................................................................................. 68 
14.1 
Ethical Standard ...................................................................................................... 68 
14.2 
Institutional Review Boards ..................................................................................... 68 
14.3 
Informed Consent Process ...................................................................................... 68 
14.3.1 Informed Consent/Assent Process (in Case of a Minor) ............................. 69 
14.4 
Exclusion of Women, Minorities, and Children (Special Populations) ..................... 69 
14.5 
Subject Confidentiality ............................................................................................. 70 
14.6 
Study Discontinuation .............................................................................................. 70 
14.7 
Future Use of Stored Specimens ............................................................................. 70 
15 
Data Handling and Record Keeping .................................................................................... 71 
15.1 
Data Management Responsibilities ......................................................................... 71 
15.2 
Data Capture Methods ............................................................................................. 71 
15.3 
Types of Data .......................................................................................................... 71 
15.4 
Timing/Reports ........................................................................................................ 71 
15.5 
Study Records Retention ......................................................................................... 72 
15.6 
Protocol Deviations .................................................................................................. 72 
16 
Publication Policy ................................................................................................................ 73 
17 
Literature References .......................................................................................................... 74 
 
Supplements/Appendices ............................................................................................................ 78 
Appendix A: Schedule of Events ................................................................................................. 79 
 

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LIST OF ABBREVIATIONS 
AE 
Adverse Event 
AESI 
Adverse Event of Special Interest 
APR-1 
Aspartic Protease-1 
CBC 
Complete Blood Count 
CPqRR 
Centro de Pesquisas René Rachou 
cGMP 
Current Good Manufacturing Practices 
CIOMS 
Council for International Organizations of Medical Sciences 
CRF 
Case Report Form 
DMID 
Division of Microbiology and Infectious Diseases, NIAID, NIH 
ELISA 
Enzyme linked immunosorbent assay 
FDA 
Food and Drug Administration 
FIOCRUZ 
Foundation Oswaldo Cruz 
FWA 
Federalwide Assurance 
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 
NIAID 
National Institute of Allergy and Infectious Diseases, NIH, DHHS 
NIH 
National Institutes of Health 
PBMC 
Peripheral Blood Mononuclear Cell 
PI 
Principal Investigator 
QA 
Quality Assurance 
QC 
Quality Control 
SAE 
Serious Adverse Event 

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SMC 
Safety Monitoring Committee 
SOP 
Standard Operating Procedure 
SVI 
Sabin Vaccine Institute (Albert B Sabin Vaccine Institute; Sabin)  
US 
United States 
WBC 
White Blood Cell 
WHO 
World Health Organization 
 
 
 

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PROTOCOL SUMMARY 
Title: 
Phase 1 Study of the Safety and Immunogenicity of Na-APR-1 
(M74)/Alhydrogel® Co-administered with Na-GST-1/Alhydrogel® 
in Brazilian Adults 
Phase: 
1 
Population: 
Healthy male and non-pregnant female volunteers aged 18-45 
years, inclusive. 
Number of Sites: 
1 (Americaninhas Vaccine Center, Minas Gerais, Brazil) 
Study Duration: 
19 months 
Subject Participation 
Duration: 
13 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.   
d) To maintain the study blind by ensuring that all subjects 
receive two injections at each vaccination point, sterile 

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normal saline (0.9%) for injection will be co-administered to 
those subjects randomized to receive Na-APR-1 (M74) 
without co-administration of Na-GST-1. 
Objectives: 
 
 
Primary: 
Safety 
1. To evaluate the safety and reactogenicity of three 
different dose concentrations of Na-APR-1 (M74) 
adjuvanted with Alhydrogel® or Alhydrogel® plus GLA-AF 
and administered either with or without Na-GST-1, in 
healthy Brazilian adults. 
 
Secondary: 
Immunogenicity 
1. To assess the impact of co-administering Na-APR-1 
(M74) and Na-GST-1 on antibody production by 
measuring the antibody levels to each antigen on study 
Day 126.  
 
Tertiary: 
Immunogenicity 
1. To assess the impact of co-administration of Na-GST-1 
on the duration of the antibody responses to Na-APR-1 
(M74), and vice versa.  
2. To assess the impact of co-administration of Na-GST-1 
on the distribution of IgG subclass responses to Na-
APR-1 (M74) and vice versa.  
3. To assess the impact of co-administration of Na-GST-1 
and Na-APR-1 (M74) on the functional capacity of 
vaccine-induced antibodies to neutralize the in vitro 
activity of the native enzymes (Na-GST-1 and wild type 
Na-APR-1).  
4. To assess the impact of co-administration of Na-GST-1 
and Na-APR-1 (M74) on the affinity of the antibody 
interactions with both recombinant antigens. 
5. To assess the impact of co-administration of Na-GST-1 
and Na-APR-1 (M74) on the production of memory B 
cells specific for each antigen. 
 
Description of Study 
Design: 
Double blind, randomized, controlled, dose-escalation Phase 1 
clinical trial in hookworm exposed adults living in the area of 
Americaninhas, Minas Gerais, Brazil. Subjects will receive three 

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doses of the assigned vaccine delivered intramuscularly on 
approximately Days 0, 56, and 112.  
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 an indirect enzyme-linked 
immunosorbent assay (ELISA) and also by ImmunoCAP, on 
serum obtained prior to each study vaccination and at time 
points after each vaccination (see Appendix A); antibody affinity 
by Surface Plasmon Resonance; functional activity of vaccine-
induced antibodies via in vitro enzyme neutralization assays; 
and, 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 6 groups of 10: 
The first 20 subjects will be entered into Groups 1 and 2 through 
open sequential enrollment. 
• 
Group 1 double-blind IP allocation (n=10):  
o 5 subjects will receive 10µg Na-APR-1 (M74) 
delivered by IM injection in the deltoid muscle, with 
sterile saline administered to the alternate arm. 
o 5 subjects will receive 10µg Na-APR-1 (M74) 
delivered by IM injection in the deltoid muscle, with 
100µg Na-GST-1 administered to the alternate arm. 
• 
Group 2 double-blind IP allocation (n=10):  
o 5 subjects will receive 10µg Na-APR-1 (M74) plus 
5µg GLA-AF delivered by IM injection in the deltoid 
muscle, with sterile saline administered to the 
alternate arm.  
o 5 subjects will receive 10µg Na-APR-1 (M74) plus 
5µg GLA-AF delivered by IM injection in the deltoid 

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muscle, with 100µg Na-GST-1 administered to the 
alternate arm. 
The next 20 subjects will be entered into Groups 3 and 4 
through open sequential enrollment. 
• 
Group 3 double-blind IP allocation (n=10):  
o 5 subjects will receive 30µg Na-APR-1 (M74) 
delivered by IM injection in the deltoid muscle, with 
sterile saline administered to the alternate arm.  
o 5 subjects will receive 30µg Na-APR-1 (M74) 
delivered by IM injection in the deltoid muscle, with 
100µg Na-GST-1 administered to the alternate arm. 
• 
Group 4 double-blind IP allocation (n=10):  
o 5 subjects will receive 30µg Na-APR-1 (M74) plus 
5µg GLA-AF delivered by IM injection in the deltoid 
muscle, with sterile saline administered to the 
alternate arm.  
o 5 subjects will receive 30µg Na-APR-1 (M74) plus 
5µg GLA-AF delivered by IM injection in the deltoid 
muscle, with 100µg Na-GST-1 administered to the 
alternate arm. 
The final 20 subjects will be entered into Groups 5 and 6 
through open sequential enrollment. 
• 
Group 5 double-blind IP allocation (n=10):  
o 5 subjects will receive 100µg Na-APR-1 (M74) 
delivered by IM injection in the deltoid muscle, with 
sterile saline administered to the alternate arm.  
o 5 subjects will receive 100µg Na-APR-1 (M74) 
delivered by IM injection in the deltoid muscle, with 
100µg Na-GST-1 administered to the alternate arm. 
• 
Group 6 double-blind IP allocation (n=10):  
o 5 subjects will receive 100µg Na-APR-1 (M74) plus 
5µg GLA-AF delivered by IM injection in the deltoid 
muscle, with sterile saline administered to the 
alternate arm.  
o 5 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 to the 
alternate arm. 
 
Estimated Time to 
Complete Enrollment: 
3 months 
 

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Schematic of Study Design: 
Group Allocation: Open sequential enrollment   
Within Group IP Allocation: Double-blind randomized assignment   - - - - - - - - 
 
 
N=60
Groups 1 & 2
10µg Na-APR-1 
(n=20)
Group 1 
(n=10) 
10µg Na-APR-1                       
and Sterile Saline
(n=5)
10µg Na-APR-1                       
and 100µg Na-GST-1 (n=5)
Group 2
(n=10)
10µg Na-APR-1 + GLA-AF        
and Sterile Saline
(n=5)
10µg Na-APR-1 + GLA-AF         
and 100µg Na-GST-1
(n=5)
Groups 3 & 4
30µg Na-APR-1 
(n=20)
Group 3 
(n=10)
30µg Na-APR-1                       
and Sterile Saline
(n=5)
30µg Na-APR-1                       
and 100µg Na-GST-1
(n=5)
Group 4   
(n=10)
30µg Na-APR-1 + GLA-AF        
and Sterile Saline
(n=5)
30µg Na-APR-1 + GLA-AF         
and 100µg Na-GST-1
(n=5)
Groups 5 & 6 
100µg Na-APR-1 
(n=20)
Group 5 
(n=10)
100µg Na-APR-1                      
and Sterile Saline
(n=5)
100µg Na-APR-1                      
and 100µg Na-GST-1
(n=5)
Group 6 
(n=10)
100µg Na-APR-1 + GLA-AF        
and Sterile Saline
(n=5)
100µg Na-APR-1 + GLA-AF         
and 100µg Na-GST-1
(n=5)

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1 
KEY ROLES 
Individuals: 
 
 
 
 
 
Principal Investigator:  
David Diemert, MD FRCP(C), Associate Professor  
George Washington University 
Department of Microbiology, Immunology, and Tropical 
Medicine (GW MITM) 
Washington, DC, USA 
Tel: 202-994-2909  
Fax: 202-994-2913 
Email: ddiemert@gwu.edu 
 
Co-Principal Investigator: 
Rodrgio Correa-Oliveira, PhD 
Centro de Pesquisas René Rachou (CPqRR) - FIOCRUZ 
Av. Augusto de Lima 1715, Belo Horizonte,  
Minas Gerais, Brazil 
 
DMID Medical Monitor: Soju Chang, MD 
Local Independent Safety Monitor:  
Christiane Cimini, MD  
Téofilo Otoni, Minas Gerais, Brazil 
E-mail: christiane.cimini@ufvjm.edu.br 
Institutions: 
 
 
Study Site:  
Americaninhas Vaccine Center 
Americaninhas, Municipality of Novo Oriente 
Minas Gerais, Brazil 
Contact Person: Renata Diniz  
Phone Number: +55 (33) 3532-7015 
E-mail: diniz@cpqrr.fiocruz.br 
 
Clinical Laboratory:  
Laboratório Carlos Chagas  
Rua Sete de Setembro, 2000 – Centro 
CEP 35010 - 172 
Governador Valadares, Minas Gerais, Brazil 
Contact Person: Thiago Mourão 
Tel: +55 (33) 3271-2525 
Fax Number: 55 (33) 3212-5555 
E-mail: thiagomourao@chagasgv.com.br 

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Immunology Laboratories:  
Laboratório de Imunologia Celular e Molecular 
Centro de Pesquisas René Rachou (CPqRR) - FIOCRUZ 
Av. Augusto de Lima 1715, Belo Horizonte,  
Minas Gerais, Brazil 
Contact Person: Fernanda Gambogi 
Tel: +55 (31) 3349-7715 
Fax: +55 (31) 3295-3115 
E-mail: gambogi@cpqrr.fiocruz.br 
 
Clinical Immunology Laboratory 
Dept. of Microbiology, Immunology and Tropical Medicine 
School of Medicine and Health Sciences 
The George Washington University 
2300 Eye Street NW, Ross 512 
Washington, DC  20037 
Contact Person: Jill Brelsford 
Tel: (202) 994-1409 
Fax: (202) 994-2913 
E-mail: jillb85@gwu.edu 
 

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2 
BACKGROUND INFORMATION AND SCIENTIFIC 
RATIONALE 
2.1 
Background Information 
2.1.1 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). With the exception of malaria, hookworm is the most important 
parasitic disease of humans. 
 
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 up to 740 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 

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occur almost exclusively in school-aged children, it has been shown that high-intensity 
hookworm infections may also occur in adults (14-16).   
 
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.   
2.1.2 Study Site 
The trial described in this protocol is being conducted to evaluate the safety and immunogenicity 
of two adult stage N. americanus candidate vaccine antigens, and will be conducted in 
Americaninhas, a town located in the Municipality of Novo Oriente de Minas, 500 km northwest 
of Belo Horizonte, the capital of the Brazilian state of Minas Gerais. The Brazilian Fundação 
National de Saúde (National Health Foundation) estimates that there are approximately 1000 
people living in the urban municipal center of Americaninhas, with another 4000 living in the 
surrounding rural areas. The area is hilly and characterized by a tropical altitude climate, with an 
average temperature of 24°C, and experiences a long rainy season between November and 
March; annual rainfall is 1300-2000 mm. The majority of inhabitants are involved in rural 
subsistence farming, growing mainly coffee, manioc and beans. Cattle ranching is another 
important source of income. Houses are predominantly made from concrete or from a 
combination of wood and mud and have either tiles or iron sheets for roofing. Only 
approximately 50% of these homes have a latrine and people commonly collect their water from 
local springs. There is only one government-run health clinic in the area staffed by one 
physician (part-time) and two auxiliary health workers who are paid by the municipality.  
 

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Americaninhas has been chosen as a site for testing hookworm vaccines based on prevalence 
surveys conducted over the past several years, and the good working relationship that has been 
established between the research staff, the local health authorities, and the community.  
Starting in 2004, epidemiological studies have been conducted by the study team in the town of 
Americaninhas and surrounding areas in order to identify the populations at greatest risk for 
heavy hookworm infection and to conduct post-treatment hookworm re-infection studies in order 
to inform the sample size calculations for Phase 1 and Phase 2 studies in the region. These 
studies have been challenging due to a number of environmental obstacles including the 
isolation of the region, the hilly terrain, and the large distances between dwellings. Geographic 
Information System/Remote Sensing identified high levels of vegetation as a critical feature of 
the infection sites. Overall, the force of infection in Minas Gerais is high. It was estimated that 
post-treatment hookworm infection occurs in approximately 300 per 1,000 individuals per year, 
which represents a level of transmission that is comparable to other areas considered to have 
intense or high transmission, such as in East Africa. The overall prevalence of N. americanus 
hookworm infection is approximately 68 percent, with approximately one-half of the population 
also infected with Ascaris lumbricoides and with Schistosoma mansoni (23). 
 
Over the past 8 years, a research team based at the Centro de Pesquisas René Rachou 
(CPqRR) in Belo Horizonte, Minas Gerais, Brazil, has collaborated with researchers at the 
George Washington University (GWU) and the Sabin Vaccine Institute to develop a major 
outpatient clinical trial facility in Americaninhas. CPqRR is a member institution of the Fundação 
Oswaldo Cruz (FIOCRUZ), which is the principal research organization of the Brazilian federal 
Ministry of Health.  CPqRR, with funding from the Sabin Vaccine Institute and the Brazilian 
federal Ministry of Health, has invested in dedicated clinical facilities for conducting clinical trials 
of investigational products in the town of Americaninhas. At the field site in Americaninhas, the 
CPqRR has built a vaccine-testing center in the town for the conduct of vaccine and other 
clinical trials.  To date, two Phase 1 hookworm vaccine trials have successfully been conducted 
at this site. 
 
The vaccine-testing center in Americaninhas is equipped with basic medical instrumentation and 
supplies to stabilize and treat acute medical problems.  The clinic has an automatic generator 
back-up electrical supply, in case of loss of municipal power. This clinic consists of an 
observation room, a waiting room, three clinical examination rooms, a small clinical pharmacy 
with temperature-monitored refrigerator, a small laboratory and phlebotomy room, a room for 
treating acute medical problems such as immediate allergic reactions following vaccination, and 
a secure document storage room. The facilities in Americaninhas have access to emergency 
care and accommodations in the event of serious or other adverse events that require such 
management. Americaninhas is located approximately a one-hour drive from the nearest town 
with a hospital. 
2.1.3 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 

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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 (24).   
 
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 (25). 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.  
 
Figure 1: Anti-Na-ASP-2 IgE antibody levels in residents of a hookworm-endemic 
region of northeastern Minas Gerais state, Brazil. Antibody levels were measured by 
ELISA; each dot represents an individual. Levels are shown for individuals of various age 
groups, in addition to the 9 participants of the Phase 1 trial of Na-ASP-2 in Brazil and the 
participants of the first Phase 1 trial of Na-ASP-2 that was conducted in hookworm-naïve 
adults in the United States (25).  
 
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 

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specific IgE antibodies during natural infection, and hence have a low likelihood for inducing 
allergic reactions upon vaccination.   
2.1.4 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 (26). Following hemolysis, 
adult hookworms use an ordered cascade of hemoglobinases to cleave hemoglobin into smaller 
molecules (26-31). Aspartic protease-1 of N. americanus (Na-APR-1) is responsible for initiating 
the proteolytic cascade in hookworms, as described below (Section 2.1.7). 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) (32-34). 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.   
 
Na-GST-1 is a 24-kDa protein with peroxidase enzymatic activity that catalyzes the conjugation 
of reduced glutathione to a variety of electrophiles (32-34). 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 (32, 34-37). 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) 
(34). Na-GST-1 binds heme at high affinity in vitro (32, 37). 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 (34). 
 
 
 
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 animals 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. 

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caninum infective larvae, significantly lower host worm burdens and fecal egg counts were 
observed compared to control animals vaccinated only with adjuvant (32).  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 (32, 33, 38).  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 
AS03 adjuvant. Vaccination time points (1, 2, 3, and 4) and challenge day (C) are marked 
with arrows (32). 
 
 
 
 
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 

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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 ELISA. 
A. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
B. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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
IgE against GST-1 in pediatric population (n=128), 1-10 years old.
Non-Infected
Infected
0.100
0.150
0.200
0.250
0.300
(n=67)
(n=61)
OD (490 nm)

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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.  
 
 
 
 
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.     
2.1.5 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 8). 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 
0.100
0.150
0.200
0.250
0.300
0
50
100
150
Na-GST-1 IgE (kUA/l) 
Na-GST-1 IgE (kUA/l) 

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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 
administered Na-GST-1/Alhydrogel®/CPG 10104 compared to that administered only Na-GST-
1/Alhydrogel® as shown in Figure 8.     
Figure 8: 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. 
 
2.1.6 Clinical Experience with Na-GST-1/Alhydrogel® Hookworm 
Vaccine   
 
The Na-GST-1/Alhydrogel® hookworm vaccine has been tested in two Phase 1 clinical trials in 
humans in both the United States (n=40) and Brazil (n=102). So far, no significant reactogenicity 
or safety issues have been observed in these two studies. In study SVI-11-01 in healthy, 
hookworm-naïve American adults conducted 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. Vaccinations were completed in this study in early January 2014; interim 
immunogenicity results are currently pending. 
 
The trial in Brazil (SVI-10-01) has tested Na-GST-1/Alhydrogel® in both hookworm-unexposed 
(n=36) and hookworm-exposed (n=66) healthy adults (NCT01261130). This study was initiated 

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in Belo 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. 
Vaccinations were completed in August 2013 and preliminary immunogenicity results will be 
available mid 2014. In this study, the Na-GST-1/Alhydrogel® (with or without GLA-AF) appears 
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. 
2.1.7 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 (29).  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 (30-32, 37). 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 (29, 31, 37). 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 (31).  
 
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 (30).  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 (30). 
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. 
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®.  

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2.1.8 Studies of Human Immune Responses to Na-APR-1 in Endemic 
Areas   
 
To assess serologic recognition of Na-APR-1mut by humans resident in areas of high 
transmission for N. americanus, 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 
Dichotomous, 
positive [% (n)]a 
42 (270) 
0.6 (4) 
15 (97) 
53 (343) 
62 (401) 
0.6 (4) 
3 (22) 
Quantitative 
(μg/ml)b 
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 scheme in which the calibration curve 
uses reagents that have a different specificity from those used to measure the analyte of interest; in this case, Na-APR-1mut-
specific antibody ELISA was simultaneously performed alongside a total Ig assay, with the mass values calibrating by 
interpolating the sample values by the standard curve of the total Ig assay. 
 
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 (35). 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. 
2.1.9 Clinical Experience with the Na-APR-1 (M74)/Alhydrogel® 
Hookworm Vaccine 
 
The Na-APR-1 (M74)/Alhydrogel® Hookworm Vaccine is currently being 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 are being 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 

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vaccinations are being given to each volunteer according to a 0, 2, and 4-month schedule. All 
study vaccinations were completed in June 2014, with preliminary immunogenicity results 
available mid 2014. Thus far, there have been no vaccine-related SAEs and related AEs have 
been limited mostly to mild-to-moderate injection site pain and tenderness. 
2.1.10 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.(39, 
40) 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.   
2.1.11 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 (41).  
 
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 (42-44), leishmaniasis (45), and hepatitis B (46).  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 (47, 48). 
 
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 
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doses up to 2.5 µg of GLA-SE were safe and well-tolerated and significantly enhanced 
influenza-specific antibody responses (49). 
2.2 
Rationale 
2.2.1 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 (30, 38). 
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 (50). 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. 
 
Through the study described in this clinical protocol, we will utilize several state-of-the-art 
antibody-profiling technologies (see Section 8.2) to determine the impact of co-administering 
the Na-GST-1 and Na-APR-1 (M74) hookworm vaccines on the immune response to each 
antigen.  In particular, we will compare conventional indirect enzyme-linked immunosorbent 
assay (ELISA) techniques with cutting-edge antibody and memory B cell immune techniques 
from other disciplines (e.g., allergy, biotherapy, biodefense etc.) that offer a more 
comprehensive profiling of the humoral immune response when administering vaccine antigens.  
This will allow assessment of the humoral immune response during co-administration from 
several unique perspectives, including memory B cell development, antibody affinity, and 
antibody function during co-administration and compare them to conventional antibody assays 
(i.e., ELISA). The introduction of these novel assays early in vaccine clinical development will 
enable decision-making on optimal vaccine composition during clinical development, saving 
time, expense, and the required number of human research subjects. 
2.2.2 Rationale for Doses and Dose Schedule to be Studied  
Na-APR-1 (M74)/Alhydrogel® has not yet been administered to individuals living in a hookworm-
endemic area, whereas Na-GST-1/Alhydrogel® (with or without GLA-AF) has 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, Na-APR-1 (M74)/Alhydrogel® will be administered in escalating doses, whereas the 
dose of Na-GST-1/Alhydrogel will be the maximum tolerated dose in the previous studies of this 
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based on preclinical studies and to maintain an equal fold-increase in the amount of antigen. 
The 10μg 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).  As mentioned above, selection of the 100μg dose of Na-GST-1 is 
base on preliminary safety and immunogenicity date from the ongoing Phase 1 study of 10, 30 
and 100μg Na-GST-1 with or without 2.5μg GLA-AF administered to 102 healthy Brazilian 
adults (protocol SVI-10-01), and the ongoing Phase 1 study of 10, 30, and 100μg of Na-GST-1 
with or without 1μg or 5μg GLA-AF administered to 40 healthy US adults (protocol SVI-11-01).  
To date, 100 µg Na-GST-1/Alhydrogel administered with 5 µg GLA-AF has been very well 
tolerated, 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.  
Unfortunately, the addition of GLA-AF (any dose) 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. 
 
For the study proposed in this clinical protocol, each subject will be vaccinated three times, on 
Days 0, 56, and 112. On each vaccination day each subject 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 infants. 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.  
2.2.3 Clinical Development Plan for the Human Hookworm Vaccine 
The target population for the human hookworm vaccine is children less than 10 years of age 
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 being tested in healthy adults living in areas of high hookworm 
prevalence.  
 
The decision to proceed to Phase 1 testing in children will be taken after the investigators, the 
Safety Monitoring Committee (SMC), and the Sponsor have reviewed an interim report of the 
study described in this protocol. This interim report will include the safety and immunogenicity 
data of all subjects following three immunizations. It is anticipated that the interim report will be 
available for review in early 2016. Assuming that co-administration of the two antigens in adults 
does not result in significant safety concerns, testing will proceed to a Phase 1 trial in Brazilian 
children living in a hookworm endemic area. 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.  
 
Besides the vaccine co-administration trial described in this protocol, a second Phase 1 trial to 
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in healthy adults in a hookworm-endemic region of Gabon is also being planned, with 
anticipated initiation in late 2014. A 0, 28 and 180-day (0, 1 and 6 months) vaccine schedule is 
being evaluated in the Gabon study, as compared to the 0, 56, and 112-day (0, 2 and 4 months) 
vaccination schedule evaluated in this protocol. Results from the studies in Gabon and Brazil 
will be compared to evaluate co-administration safety and immune interference in diverse 
hookworm-exposed populations, and to decide on antigen doses, formulations, and vaccination 
schedule to be used in future clinical trials.  
 
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.  
2.3 
Potential Risks and Benefits 
2.3.1 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 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-GST-1/Alhydrogel® plus GLA-AF and Na-APR-1 (M74)/Alhydrogel® plus 
GLA-AF have demonstrated similar reactogenicity and safety to either 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 (51).   

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Female subjects will be cautioned of the unknown risk of the Na-GST-1/Alhydrogel® or Na-APR-
1 (M74)/Alhydrogel® vaccines to the fetus.  Females of childbearing potential, unless surgically 
sterile (i.e., bilateral tubal ligation, bilateral oophorectomy, or complete hysterectomy), at least 
2 years postmenopausal, or practicing abstinence, must use an effective method of avoiding 
pregnancy (including oral, transdermal, or implanted contraceptives, intrauterine device, female 
condom with spermicide, diaphragm with spermicide, cervical cap, or use of a condom with 
spermicide by the sexual partner) from 30 days prior to first vaccination until at least one month 
(defined as 30 days) following the last vaccination.  Female subjects will be counseled by a 
study clinician or nurse, or referred to the local health clinic for evaluation and institution of an 
appropriate contraceptive method. 
2.3.2 Precautions Taken to Minimize Risks 
 
In order to minimize the risk to subjects, all subjects 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. Furthermore, to reduce the likelihood of 
immediate-type allergic reactions to the vaccine formulations, all potential trial subjects will be 
screened for detectable IgE antibodies to Na-GST-1 and Na-APR-1 (M74); individuals with Na-
GST-1 IgE antibodies detectable by the ELISA assay that are confirmed to be >0.35 kUA/L by 
the ImmunoCAP method will be excluded from participation in the study, as will any individuals 
with Na-APR-1 (M74) IgE antibodies above the ELISA reactivity threshold.  
 
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 is a chance 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.   
 
2.3.3 Known Potential Benefits 
 
Subjects may not receive any direct benefit from participation in this study. It is hoped that 
information gained in this study will contribute to the development of a safe and effective 
hookworm vaccine. All subjects will undergo a stool examination for ova and parasites at the 
Day 140 and Day 380 study visits. Subjects found to have a helminth infection 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 
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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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3 
OBJECTIVES 
3.1 
Study Objectives 
 
Primary: 
Safety 
1. To evaluate the safety and reactogenicity of three different dose concentrations of Na-
APR-1 (M74) adjuvanted with Alhydrogel® or Alhydrogel® plus GLA-AF and administered 
either with or without Na-GST-1, in healthy Brazilian adults. 
 
Secondary: 
Immunogenicity 
1. To assess the impact of co-administering Na-APR-1 (M74) and Na-GST-1 on antibody 
production by measuring the antibody levels to each antigen on study Day 126.  
 
Tertiary: 
Immunogenicity 
1. To assess the impact of co-administration of Na-GST-1 on the duration of the antibody 
responses to Na-APR-1 (M74), and vice versa.  
2. To assess the impact of co-administration of Na-GST-1 on the distribution of IgG 
subclass responses to Na-APR-1 (M74) and vice versa.  
3. To assess the impact of co-administration of Na-GST-1 and Na-APR-1 (M74) on the 
functional capacity of vaccine-induced antibodies to neutralize the in vitro activity of the 
native enzymes (Na-GST-1 and wild type Na-APR-1).  
4. To assess the impact of co-administration of Na-GST-1 and Na-APR-1 (M74) on the 
affinity of the antibody interactions with both recombinant antigens. 
5. To assess the impact of co-administration of Na-GST-1 and Na-APR-1 (M74) on the 
production of memory B cells specific for each antigen. 
 
Exploratory: 
Immunogenicity 
1. To assess the cellular immune responses to the Na-GST-1 and Na-APR-1 (M74) 
antigens following immunization. 
 

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3.2 
Study Outcome Measures 
3.2.1 Primary Outcome Measures 
Safety 
The following parameters will be evaluated for each dose and formulation of Na-APR-1 (M74) 
alone or 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. 
3.2.2 Secondary Outcome Measures 
Immunogenicity 
The following parameters will be evaluated for each dose and formulation of Na-APR-1 (M74) 
alone or co-administered with Na-GST-1: 
1. The IgG level by an indirect enzyme-linked immunosorbent assay (ELISA) on 
approximately Day 126. 
2. The IgG level by ImmunoCAP on approximately Day 126. 
 
3.2.3 Tertiary Outcome Measures 
Immunogenicity 
The following parameters will be evaluated for each dose and formulation of Na-APR-1 (M74) 
alone or co-administered with Na-GST-1: 
1. The IgG antibody response, by an indirect enzyme-linked immunosorbent assay (ELISA) 
at approximately 7, 14, and 28 days after each vaccination, and approximately 3, 6, and 
9 months after the third dose. 

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2. The IgG antibody response, by ImmunoCAP at approximately 7, 14, and 28 days after 
each vaccination, and approximately 3, 6, and 9 months after the third dose. 
3. The IgG subclass distribution (IgG1, IgG3, and IgG4) by plate based ImmunoCAP on 
approximately Day 126. 
4. The IgG subclass distribution by plate based ImmunoCAP each day of vaccination, 
approximately 28 days later, and approximately 3 and 6 months after the final 
vaccination. 
5. The functional capacity of vaccine-induced antibodies to neutralize the in vitro activity of 
the native enzymes.  
6. The affinity of the antibody interactions with both recombinant antigens at approximately 
7, 14, and 28 days after each vaccination, and 3, 6, and 9 months after the third dose. 
7. The production of memory B cells specific for each antigen on days of vaccination, 
approximately 28 days following each vaccination, and 6 and 9 months after the third 
dose.  
3.2.4 Exploratory Outcome Measures 
The following parameter will be evaluated for each dose and formulation of Na-APR-1 (M74) 
alone or co-administered with Na-GST-1: 
1. Cellular immune responses following vaccination. 
 

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4 
STUDY DESIGN 
The study will be conducted as a randomized, placebo-controlled, double-blind Phase 1 dose-
escalating clinical trial in healthy adult volunteers living in the hookworm-endemic area of 
Americaninhas, Brazil.  
 
Prior to beginning study-related activities, the consent and cooperation of local municipal 
government leaders and representative community members will be sought, after which 
volunteers will be invited to participate in the study. After providing written informed consent and 
passing the informed consent comprehension questionnaire, volunteers will undergo eligibility 
screening, including a complete medical history, physical examination, hematology testing, 
serum glucose testing, liver and renal function testing, HIV antibody testing, Hepatitis B and C 
serology, and urinalysis; urine pregnancy testing will be performed on all female volunteers. In 
addition, all volunteers will be tested for serum IgE antibodies to Na-APR-1 (M74) and Na-GST-
1, and a fecal examination will be performed for ova and parasites. All clinically significant 
abnormalities will be reviewed with volunteers and referral for follow-up care will be provided. 
After screening, those volunteers determined to be eligible, based on the inclusion and 
exclusion criteria, will be invited to participate in the study.   
 
Sixty volunteers will be progressively enrolled into 1 of 6 Groups over a projected 3-month 
enrollment period, with each subject followed for 13 months post-enrollment (to Study Day 380). 
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. 
 
The first 20 subjects will be entered into Groups 1 and 2  
• 
Group 1 double-blind IP assignment (n=10):  
o 5 subjects will receive 10µg Na-APR-1 (M74) delivered by IM injection in the 
deltoid muscle, with sterile saline administered to the alternate arm. 
o 5 subjects will receive 10µg Na-APR-1 (M74) delivered by IM injection in the 
deltoid muscle, with 100µg Na-GST-1 administered to the alternate arm. 
• 
Group 2 double-blind IP assignment (n=10):  
o 5 subjects will receive 10µg Na-APR-1 (M74) plus 5µg GLA-AF delivered by IM 
injection in the deltoid muscle, with sterile saline administered to the alternate 
arm.  
o 5 subjects will receive 10µg Na-APR-1 (M74) plus 5µg GLA-AF delivered by IM 
injection in the deltoid muscle, with 100µg Na-GST-1 administered to the 
alternate arm. 
 
The next 20 subjects will be entered into Groups 3 and 4  
• 
Group 3 double-blind IP assignment (n=10):  
o 5 subjects will receive 30µg Na-APR-1 (M74) delivered by IM injection in the 
deltoid muscle, with sterile saline administered to the alternate arm.  
o 5 subjects will receive 30µg Na-APR-1 (M74) delivered by IM injection in the 
deltoid muscle, with 100µg Na-GST-1 administered to the alternate arm. 
 

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• 
Group 4 double-blind IP allocation (n=10):  
o 5 subjects will receive 30µg Na-APR-1 (M74) plus 5µg GLA-AF delivered by IM 
injection in the deltoid muscle, with sterile saline administered to the alternate 
arm.  
o 5 subjects will receive 30µg Na-APR-1 (M74) plus 5µg GLA-AF delivered by IM 
injection in the deltoid muscle, with 100µg Na-GST-1 administered to the 
alternate arm. 
 
The final 20 subjects will be entered into Groups 5 and 6  
• 
Group 5 double-blind IP allocation (n=10):  
o 5 subjects will receive 100µg Na-APR-1 (M74) delivered by IM injection in the 
deltoid muscle, with sterile saline administered to the alternate arm.  
o 5 subjects will receive 100µg Na-APR-1 (M74) delivered by IM injection in the 
deltoid muscle, with 100µg Na-GST-1 administered to the alternate arm. 
• 
Group 6 double-blind IP allocation (n=10):  
o 5 subjects will receive 100µg Na-APR-1 (M74) plus 5µg GLA-AF delivered by IM 
injection in the deltoid muscle, with sterile saline administered to the alternate 
arm.  
o 5 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 to the 
alternate arm. 
 
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. Subjects 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 3 and 5. 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 3 and 5 must be obtained from the SMC.  
The SMC and Independent Safety 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 9.5 are met or, in the clinical judgment of the SMC, 
Independent Safety Monitor, and/or DMID Medical Monitor the next higher dose would pose an 
unacceptable safety risk to the subjects. 

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4.1 
Substudies (if applicable) 
Not Applicable. 

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5 
STUDY ENROLLMENT AND WITHDRAWAL 
Only subjects 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 subjects will be enrolled. The study population will be enrolled from in and around 
Americaninhas, a town located in the Municipality of Novo Oriente de Minas, 500 km northwest 
of Belo Horizonte, the capital of the Brazilian state of Minas Gerais. 
 
Volunteers agreeing to participate 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. Volunteers 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 
volunteer, the volunteer’s questions were answered, and the volunteer answered all 
comprehension questions correctly. The original signed informed consent form for each 
volunteer will be maintained as part of that volunteer’s study records.  A copy of the informed 
consent form will be provided to every volunteer.   
 
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. 
5.1 
Subject Inclusion Criteria 
 
1. Males or non-pregnant females between 18 and 45 years, inclusive. 
2. Good general health as determined by means of the screening procedure. 
3. Available for the duration of individual subject study participation (13 months). 
4. Willingness to participate in the study as evidenced by signing the informed consent 
document.  
5.2 
Subject Exclusion Criteria 
 
1. Pregnancy as determined by a positive urine hCG (if female). 
2. Subject unwilling to use reliable contraception (as described in Section 2.3.1) from 
30 days prior to the first immunization and up until one month following the third 
immunization (if female and not surgically sterile, abstinent or at least 2 years post-
menopausal, or determined otherwise by medical evaluation to be sterile). 
3. Currently lactating and breast-feeding (if female). 
4. Inability to correctly answer all questions on the informed consent comprehension 
questionnaire.  

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5. Evidence of clinically significant neurologic, cardiac, pulmonary, hepatic, 
rheumatologic, autoimmune, diabetes, or renal disease by history, physical 
examination, and/or laboratory studies (a history of essential hypertension that is well 
controlled by medication will not be considered exclusionary.) 
6. Has a diagnosis of schizophrenia, bipolar disease or other major psychiatric 
condition that would make compliance with study visits/procedures difficult (e.g., 
subject with psychoses or history of suicide attempt or gesture in the 3 years before 
study entry, ongoing risk for suicide). 
7. Known or suspected immunodeficiency. 
8. Laboratory evidence of liver disease (alanine aminotransferase [ALT] greater than 
1.25-times the upper reference limit). 
9. Laboratory evidence of renal disease (serum creatinine greater than 1.25-times the 
upper reference limit, or more than trace protein or blood on urine dipstick testing 
with the exception of greater than trace blood detected in females during menses). 
10. Laboratory evidence of hematologic disease (absolute leukocyte count <3200/mm3; 
absolute leukocyte count >10.8 x 103/mm3; hemoglobin <11.4 g/dl [females] or <12.1 
g/dl [males]; or, platelet count <130,000/mm3). 
11. Serum glucose (random) greater than 1.2-times the upper reference limit. 
12. Other condition that in the opinion of the investigator would jeopardize the safety or 
rights of a volunteer participating in the trial or would render the subject unable to 
comply with the protocol. 
13. Participation in another investigational vaccine or drug trial within 30 days of starting 
this study. 
14. Previous receipt of the Na-GST-1/Alhydrogel® hookworm vaccine. 
15. Volunteer has had medical, occupational, or family problems as a result of alcohol or 
illicit drug use during the past 12 months. 
16. History of a severe allergic reaction or anaphylaxis. 
17. Severe asthma as defined by the need for daily use of inhalers or emergency clinic 
visit or hospitalization within the last 6 months. 
18. Positive test for hepatitis B surface antigen (HBsAg). 
19. Positive confirmatory test for HIV infection. 
20. Positive confirmatory test for hepatitis C virus (HCV) infection. 
21. Use of corticosteroids (excluding topical or nasal) or immunosuppressive drugs 
within 30 days of the volunteer’s expected first vaccination in this study or planned 
use up to one month following the last vaccination. 
22. Receipt of a live vaccine within past 4 weeks or a killed vaccine within past 2 weeks 
prior to the volunteer’s expected first vaccination in the study. 
23. History of a surgical splenectomy. 
24. Receipt of blood products within the past 6 months.  
25. Anti-Na-GST-1 IgE antibody level above 0.35 kUA/L by the ImmunoCAP method. 
26. Anti-Na-APR-1 IgE antibody level above ELISA reactivity threshold. 
 
 

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5.3 
Treatment Assignment Procedures 
5.3.1 Randomization Procedures 
 
Eligible volunteers will be asked to come to the clinic on their 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 volunteers 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, and that females have had a urine pregnancy test performed that is documented 
to be negative, volunteers 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. 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. Volunteers 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. Enrolled 
subjects 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 
6 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. 
5.3.2 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® dose assignment or GLA-AF co-administration assignment. That is, 
those subjects enrolled into Group 1 will receive either 10μg Na-APR-1 (M74)/Alhydrogel® 
administered to one arm and sterile saline administered to the alternate arm, or 10μg Na-APR-1 
(M74)/Alhydrogel® in one arm and 100μg Na-GST-1/Alhydrogel® administered to the alternate 
arm. Subjects enrolled into Group 2 will receive either 10μg Na-APR-1 (M74) /Alhydrogel® plus 
5μg GLA-AF administered to one arm and sterile saline administered to the alternate arm, or 
10μg Na-APR-1 (M74) /Alhydrogel® plus 5μg GLA-AF in one arm and 100μg Na-GST-
1/Alhydrogel® administered to the alternate arm, and so on.  
 
Investigators and subjects will be blinded to the within-Group random allocation to receive co-
administered sterile saline or Na-GST-1/Alhydrogel® until all subjects have completed their Day 
140 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 Na-APR-1 (M74) 
without co-administration of Na-GST-1.  

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The study vaccine manager will also 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) formulations are of different volumes (0.1 and 0.3ml, 
respectively for administration without GLA-AF) than that of Na-GST-1, 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. 
 
Investigators will be unblinded after all subjects have had their study Day 140 visit. 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.   
5.3.3 Reasons for Withdrawal and Discontinuation of Vaccinations 
Subjects are free to withdraw from the study at any time and for any reason. Subjects who have 
received vaccine, regardless of the number of doses received, or who developed an AE or SAE 
will be encouraged to remain in the study to be followed for safety purposes.  
Subjects may voluntarily withdraw their consent for study participation at any time and for any 
reason, without penalty. 
A subject may withdraw or be withdrawn from the study for any of the following reasons: 
• 
Medical disease or condition, or any new clinical findings for which continued 
participation, in the opinion of the Principal Investigator or appropriate co-investigator, 
would compromise the safety of the subject, or would interfere with the subject's 
successful completion of the study, or would interfere with the evaluation of responses. 
• 
Subject no longer meets eligibility criteria. 
• 
As deemed necessary by the Principal Investigator or appropriate co-investigator for 
noncompliance or other reasons. 
• 
Subject withdrawal of consent. 
• 
Subject lost to follow-up. 
• 
Termination of the study. 
• 
New information becomes available that makes further participation unsafe. 
The second or third study vaccination may not be administered to a subject if any of the 
following criteria are met: 

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• 
Medical condition (including pregnancy) for which continued participation, in the opinion 
of the Principal Investigator or appropriate co-investigator, would pose a risk to the 
subject 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 subjects with injection site or systemic signs or symptoms, or 
with an acute illness, including an oral 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 subject’s chart. 
• 
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 central (clinical) laboratory prior to the second or third 
study vaccination. If the clinical safety laboratory value decreases to Grade 2 or less, the 
subject 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 subject’s chart. 
• 
Severe or sustained reaction or disability related to the first study vaccination. 
• 
New onset of illness or condition that meets exclusion criteria. 
• 
Subject no longer meets eligibility criteria. 
• 
As deemed necessary by the Principal Investigator or appropriate co-investigator for 
noncompliance or other reasons. 
• 
Subject refusal of further study vaccination. 
• 
Subject withdrawal of consent. 
• 
Subject lost to follow-up. 
• 
Termination of the study. 
• 
New information becomes available that makes further participation unsafe. 

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5.3.4 Handling of Withdrawals 
The primary reason for withdrawal from the study will be recorded on the appropriate data 
collection form. Subjects will be encouraged to complete the Early Termination Visit. The Early 
Termination Visit procedures are listed in Section 7.5. Although subjects are free to withdraw at 
any time or may be withdrawn by the Principal Investigator or appropriate co-investigator at any 
time, subjects 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 subjects who fail to appear for a follow-up safety assessment, extensive effort 
(i.e., three documented contact attempts via in-person visits to the subject’s home, made on 
separate occasions) will be made to locate or recall them, or at least to determine their health 
status. These efforts will be documented in the subject’s records. 
 
5.3.5 Termination of Study  
 
If a dose of vaccine is considered significantly reactogenic (see Section 9.2.2), dose escalation 
and/or additional vaccinations will be suspended until reviewed by the Independent Safety 
Monitor, SMC, and study sponsor. Any recommendation of the Independent Safety Monitor, 
DMID Medical Monitor, and SMC to resume or suspend further injections (either for an 
individual subject 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 applicable IRBs.  

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6 
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). 
6.1 
Study Product Description 
6.1.1 Acquisition 
Vials of Na-GST-1/Alhydrogel®, Na-APR-1 (M74)/Alhydrogel®, GLA-AF, and sterile saline for 
injection for this study will be supplied to the study site by the Sabin Vaccine Institute. Na-GST-
1/Alhydrogel®, Na-APR-1 (M74)/Alhydrogel®, GLA-AF, and the sterile saline placebo 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.   
6.1.2 Formulation, Packaging, and Labeling 
6.1.2.1 
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. 
 
6.1.2.2 
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 
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Glucose acts as an excipient and imidazole as the buffer based on evidence that these 
components specifically enhance the stability and solubility of Na-GST-1. The dose that will be 
administered in this study is 100 μg of Na-GST-1, or 1.0 ml of the final drug product. This 
volume contains the equivalent of approximately 400μg aluminum. Na-GST-1/Alhydrogel® was 
manufactured, formulated and vialed at Aeras Global Vaccine Foundation (Rockville, Maryland, 
USA).   
6.1.2.3 
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®. 
6.1.2.4 
Saline Placebo 
Sterile isotonic (0.9%) sodium chloride solution (“normal saline”) for injection, U.S. 
Pharmacopeia (USP), will be procured and shipped to the study site at ambient temperature.  
Normal saline is a clear liquid. In study subjects randomized to receive Na-APR-1 (M74) without 
co-administered Na-GST-1, normal saline will be administered intramuscularly as placebo in an 
equal volume to the Na-APR-1 (M74). It will be administered in the deltoid muscle of the arm 
opposite to the one in which the Na-APR-1 (M74) is delivered so that all study subjects receive 
two injections on each day of vaccination, thus maintaining the study blind. 
6.1.3 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. 
6.2 
Dosage, Preparation and Administration of Study 
Intervention/Investigational Product 
 
Doses of Na-APR-1 (M74)/Alhydrogel® and Na-GST-1/Alhydrogel® administered without GLA-
AF 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 sterile saline control will be prepared by withdrawing the 
appropriate volume into syringes of appropriate size.  
 
Vaccine doses will be administered by qualified study personnel (a study nurse or physician) in 
the deltoid muscle of the appropriate arm after disinfecting the skin with an alcohol swab and 
allowing it to dry.   

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6.3 
Modification of Study Intervention/Investigational Product for a 
Participant 
There will be no dose modifications. If a subject’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 subject’s chart. Subjects 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 Safety Monitor or the SMC at any time 
during the study. 
6.4 
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®, 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.   
6.5 
Assessment of Subject Compliance with Study 
Intervention/Investigational Product  
Not Applicable. 
6.6 
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 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. 

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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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7 
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 669 mL) to be 
collected from each volunteer over their 13-month participation in the trial is approximately the 
volume collected when donating one unit of blood and should not compromise the health of trial 
subjects. See Appendix A for the schedule of study procedures. 
7.1 
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 and Informed Consent to the volunteer. 
2. Ensure the subject has passed the informed consent comprehension questionnaire, has 
signed the Informed Consent and receives a signed copy of the Informed Consent. 
3. Elicit a complete medical history, including medication history, and for female subjects, a 
menstrual and contraceptive history and/or history of surgical sterility. 
4. Administer a physical examination, including vital signs (heart rate, respiratory rate, arterial 
blood pressure, and oral temperature). 
5. Obtain blood for hematology, biochemistry, serologic tests for HIV and viral hepatitis (B and 
C), and serological tests for IgE antibodies to Na-APR-1 (M74) and Na-GST-1.  
6. Obtain fecal sample for examination for ova and parasites. 
7. Obtain urine for urine dipstick testing, as well as urine hCG testing in females. 
8. Counsel females to avoid becoming pregnant during the vaccination phase of the study. 
 
Screening steps 3-8 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, viral hepatitis, and antigen specific IgE antibodies) 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 individual will be treated with an appropriate medication prior to being enrolled in the study 
and before any vaccinations. Individuals infected with Ascaris lumbricoides, hookworm or 
Trichuris trichiura will be treated with three daily 400 mg doses of albendazole; individuals 
infected with S. mansoni or Taenia spp. will be treated with a single 60 mg/kg oral dose of 
praziquantel; and, individuals infected with Strongyloides stercoralis will be treated with three 
400 mg daily oral doses of albendazole. 
 
 

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7.2 
Enrollment/Baseline 
 
Volunteers 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. 
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) 
antibody assays, and cellular immunology assays.   
5. For females, obtain a urine sample for hCG testing. Ensure that test is negative before 
proceeding; a positive test will exclude the volunteer from the trial. 
6. Record vital signs (blood pressure, oral temperature, and heart rate). 
7. Administer the vaccine. 
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. 
7.3 
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. 
 
Study Day 3 +/- 1 
1. Perform basic history and physical exam (including injection site), emphasizing examination 
of any acute complaints. 
2. Record vital signs.  
 
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. 
 
Study Day 14 +/- 2 
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) 
antibody assays, and cellular immunology assays. 
 
 

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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) antibody assays and cellular 
immunology assays. 
 
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) 
antibody assays, and cellular immunology assays. 
3. For females, obtain a urine sample for hCG testing.  Ensure that test is negative before 
proceeding; a positive test will exclude the volunteer from further vaccinations. 
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. 
 
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) antibody assays and cellular 
immunology assays. 
 
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) 
antibody assays, and cellular immunology assays.   
 
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) antibody assays and cellular 
assays. 
 
 

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Study Day 112 +/- 14 (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) 
antibody assays, and cellular immunology assays. 
3. For females, obtain a urine sample for hCG testing. Ensure that test is negative before 
proceeding; a positive test will exclude the volunteer from further vaccinations. 
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 (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. 
 
Study Day 115 (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. 
 
Study Day 119 (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) antibody assays and cellular 
immunology assays. 
 
Study Day 126 (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) 
antibody assays, and cellular immunology assays.   
 
Study Day 140 (28 +/-4 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) antibody assays and cellular 
immunology assays.  
3. Obtain stool for examination for ova and parasites. 
 
Study Day 200 (3 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) antibody assays, and cellular 
immunology assays. 
 
Study Day 290 (6 months +/-14 days after Third Vaccination) 
1. Perform basic history and physical exam, emphasizing examination of any acute complaints. 

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2. Obtain blood for anti-Na-GST-1 and anti-Na-APR-1 (M74) antibody assays and cellular 
immunology assays. 
7.4 
Final Study Visit 
 
Study Day 380 (9 months +/-21 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) antibody assays and cellular 
immunology assays. 
3. Obtain stool for examination for ova and parasites.  
7.5 
Early Termination Visit 
The following activities will be performed at the early termination visit for subjects 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. 
• 
All concomitant medications will be recorded on the appropriate data collection form 
(if within 28 days of subject’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 performed by a study clinician licensed to 
make medical diagnoses and listed on the Form FDA 1572 as the Principal 
Investigator or co-investigator, if indicated based on review of interim medical history. 
• 
Approximately 10 mL of venous blood may be collected for safety labs and 
performed by the local clinical laboratory (if prior to Visit 14 and if not recently 
obtained per study schedule). 
• 
Approximately 40 mL of venous blood may be collected for antibody and cellular 
assays (if prior to Visit 14 and if not recently obtained). 
7.6 
Unscheduled Visit  
 
Unscheduled visits may occur at any time during the study. Any of the following activities may 
be performed: 
• 
Review concomitant medications (if prior to 28 days after a study vaccination). 
• 
Review adverse events (if prior to 28 days after a study vaccination). 

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• 
Review serious adverse events and new-onset chronic medical conditions  
• 
Obtain medical history by interview of subjects and note any changes since the previous 
visit (if indicated). 
• 
A targeted physical examination may be performed by a study clinician, if indicated 
based on review of medical history. 
• 
Examine study vaccination site (if within 14 days after a study vaccination). 
• 
Approximately 10 mL of venous blood will be collected for safety labs and performed by 
the study clinical laboratory (if indicated). 
 

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8 
STUDY PROCEDURES/EVALUATIONS 
8.1 
Clinical Evaluations 
 
Subjects 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. Subjects 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 clinical will be provided. A study clinician will also stay in or near Americaninhas for 
the duration of the trial and will be available to study subjects at all times. Should a subject 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 DMID Medical Monitor and 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. 
8.2 
Laboratory Evaluations 
8.2.1 Clinical Laboratory Evaluations 
Using standard techniques, the following tests will be performed at the accredited Carlos 
Chagas clinical laboratory in Governador Valadares, Minas Gerais, Brazil. 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. Serum glucose (random) 
5. HBsAg ELISA 
6. HCV antibody assay 
7. HIV ELISA 
 
Urine hCG testing will be performed using urine pregnancy test kits that have been approved by 
Brazilian and/or US regulatory agencies. Urine dipstick testing will also be performed at the trial 
sites using an approved product.  
8.2.2 Special Assays or Procedures 
 
Anti-Na-GST-1 and anti-Na-APR-1 (M74) IgG antibody and B cell assays will be performed at 
the Clinical Immunology Laboratory of the Department of Microbiology, Immunology, and 
Tropical Medicine (MITM) of the George Washington University; the Dermatology, Allergy, and 
Clinical Immunology (DACI) Reference Laboratory of Johns Hopkins University; and the 
University of California at San Francisco. All cryopreservation of plasma, sera and peripheral 
blood mononuclear cells (PBMCs), as well as screening for levels of IgE against Na-APR-1 
(M74) and Na-GST-1 will take place at the clinical immunology laboratory of CPqRR in Belo 
Horizonte, Minas Gerais, Brazil.  Cryopreservation of sera, plasma and PBMCs will be in liquid 
nitrogen.  
 
8.2.2.1 
Anti-Na-APR-1 (M74) and Anti-Na-GST-1 Antibody Assays 
 
Antibodies to Na-APR-1 (M74) and Na-GST-1 will be measured in serum or plasma of study 
subjects using two different methods for comparison: a) a conventional qualified indirect 
enzyme-linked immunosorbent assay (ELISA) technique using a Standard Reference Serum; 
and, b) a custom made ImmunoCAP (Phadia, Inc.) for each antigen (Na-GST-1 and Na-APR-1 
(M74)). Antigen-specific IgG and IgG subclass antibody levels will be measured by ELISA and 
ImmunoCAP at baseline and at several time-points post-vaccination as outlined in Section 3 
and in Appendix B.  For the qualified conventional indirect ELISA, 96 microwell plates are 
coated with purified recombinant Na-APR-1 (M74) or Na-GST-1, 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 goat anti-human antibody will be added and incubated with plates, washed again, 
incubated with a chromogenic substrate, and antibody levels measured on an ELISA plate 
reader.  
 
Qualified ELISAs (one each for Na-GST-1 and Na-APR-1 (M74)) will also be used to detect IgE 
antibodies to each antigen, as screening tests to determine eligibility for study participation.  If 

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during screening for volunteer participation in the study, serum samples with anti-Na-GST-1 IgE 
results by ELISA are greater than the reactivity limit, they will then be tested by the ImmunoCAP 
method for confirmation. The ImmunoCAP method is considered to be the gold standard for 
measuring levels of antigen-specific IgE in serum. The ImmunoCAP tests will be performed at 
the DACI Reference Laboratory of Johns Hopkins University. 
 
Surface Plasmon Resonance (SPR) will be used to measure the affinity of antibody interactions 
with both recombinant antigens. SPR determines the immunoreactivity and affinity of antibody 
responses in numerous contexts as it measures rates of antibody-antigen association and 
dissociation based on the Law of Mass Action to assess antibody affinity.  
8.2.2.2 
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 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 
8.2.2.3 
Memory B Cell Measurement 
The ELISPOT method will be used to evaluate induction of memory B cells (MBCs) after each 
vaccination and maintenance of MBCs throughout the vaccination schedule to determine if 
MBCs act as surrogate markers (biomarkers) for later antibody production.  
8.2.2.4 
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-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.  
8.2.2.5 
Parasitology Assays 
 
Fecal exams will begin with an ether sedimentation assay to determine parasite infection status 
(positive or negative). Positive samples will undergo Kato Katz fecal thick smear exams to 
determine infection intensity (eggs per gram of feces).  

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8.2.3 Specimen Preparation, Handling, and Shipping 
8.2.3.1 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. 
8.2.3.2 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 clinical screening and safety laboratory evaluations will be transported from the 
Americaninhas site to the local (clinical) laboratory. 
 
Specimens for immune responses will be shipped from the Americaninhas site to CPqRR in 
Belo Horizonte, and then transported to the George Washington University in Washington, DC, 
in a blinded manner in batches as they become available.   
 

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9 
ASSESSMENT OF SAFETY 
9.1 
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 12 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, feverishness (chills/shivering/sweating), fatigue 
(tiredness), malaise (general unwell feeling), myalgia (body aches/muscular pain 
exclusive of the injection site), arthralgia (joint pain exclusive of the injection site), 
headache, 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 the 
each 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. 
 
9.2 
Methods and Timing for Assessing, Recording, and Analyzing 
Safety Parameters 
9.2.1 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.  
 

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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 subject call on a study 
clinician to report an AE, it will be fully documented in their study chart, and discussed with the 
Principal Investigator. 
 
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. 
9.2.2 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 at injection site 
1 
Easily tolerated, does not interfere with activity 
 
2 
 
Repeated use of non-narcotic pain reliever for > 24 hours or 
interferes with daily activity 
 
3 
Any use of narcotic pain reliever or prevents daily activity 
Tenderness at injection site 
1 
2 
3 
Mild discomfort to touch 
Discomfort with movement 
Significant discomfort at rest 
Erythema at injection site 
1 
25 mm – 50 mm 
 
2 
51 mm – 100 mm 
 
3 
> 100 mm 
Induration/swelling at injection  
1 
25 mm – 50 mm and does not interfere with daily activity 
site 
2 
51 mm – 100 mm or interferes with daily activity 
 
3 
>100 mm or prevents daily activity 
Fever (oral) 
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 
 
Repeated use of non-narcotic pain reliever for >24 hours or 
interferes with daily activity 
 
3 
Any use of narcotic pain reliever or prevents daily activity 
Nausea 
1 
Easily tolerated, does not interfere with daily activity 
 
2 
Interferes with daily activity 
 
3 
Prevents daily activity 
Vomiting 
1 
1-2 episodes in 24 hours and does not interfere with activity 
 
2 
> 2 episodes in 24 hours or interferes with daily activity 
 
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 PO or topical treatment, or IV medication or steroids for 
<24 hours 
 
3 
Requiring IV medication or steroids for >24 hours 
Mucocutaneous Reaction /  
1 
Erythema; pruritus or localized macular rash 
Rash 
2 
Diffuse, maculopapular rash, dry desquamation 
 
3 
Vesiculation or moist desquamation or ulceration 
 
 
 
 
 

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Table 3: Assessment of Unsolicited Systemic Adverse Event Severity 
Systemic AE 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Anorexia 
Loss of appetite 
without decreased 
oral intake lasting 
greater than 48 
hours 
Loss of appetite 
associated with 
decreased oral intake 
without significant 
weight loss 
Loss of appetite associated with 
significant weight loss 
Diarrhea 
3 loose stools/24 
hours 
4-5 loose stools/24 
hours 
>6 loose stools or requires outpatient 
IV hydration 
Constipation 
Not Applicable 
Persistent 
constipation requiring 
regular use of dietary 
modifications, 
laxatives, or enemas 
Obstipation with manual evacuation 
indicated 
Fatigue 
No interference 
w/activity 
Some interference 
w/activity 
Significant, prevents daily activity 
 
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 
Vertigo 
Causes no or 
minimal interference 
with usual daily 
activities 
Causes greater than 
minimal interference 
with usual daily 
activities 
Inability to perform daily activities 
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 
rash 
Rash; flushing; 
urticaria; dyspnea 
Symptomatic bronchospasm, with or 
without urticaria; parenteral 
medications(s) indicated; allergy-
related edema/angioedema; 
hypotension 
 
 
 
 

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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 
101-115 
116-130 
>130 
Bradycardia –  
beats per minute 
50-54 
45-49 
<45 
Hypertension** 
(systolic, mm Hg) 
141-150 
151-155 
>155 
Hypertension** 
(diastolic, mm Hg) 
91-95 
96-100 
>100 
Hypotension** 
(systolic, mm Hg) 
85-89 
(and symptomatic) 
80-84 
(and symptomatic) 
<80 
Respiratory Rate –  
breaths per minute 
18-20  
21-25  
>25  
*Subject should be at rest for measurement of vital signs 
**With repeat testing at same visit 
Table 5: Assessment of Laboratory Adverse Event Severity 
HEMATOLOGY 
Grade 1 
(Mild) 
Grade 2 
(Moderate) 
Grade 3 
(Severe) 
Hemoglobin 
  Males 
  Females 
 
11.0 – 12.0 g/dL 
10.3 – 11.3 g/dL 
 
9.5 – 10.9 g/dL 
8.5 – 10.2 g/dL 
 
<9.5 g/dL 
<8.5 g/dL 
Platelets 
110,000 – 
130,000/mm3 
90,000 – 
109,999/mm3 
<90,000/mm3 
WBCs 
  (increase) 
10,800 – 
15,000/mm3 
15,001 – 
20,000/mm3 
<20,000/mm3 
WBCs 
  (decrease) 
2300 – 3200/mm3 
1400 – 2299/mm3 
<1400/mm3 
ANC 
  (decrease) 
750 – 1000/mm3 
500 – 749/mm3 
<500/mm3 
CLINICAL 
CHEMISTRIES 
Grade 1 
Grade 2 
Grade 3 
Serum creatinine 
  Males 
   
  Females 
 
1.5 – 1.7 mg/dL 
 
1.5 – 1.7 mg/dL 
 
1.8 – 2.0 mg/dL 
 
1.8 – 2.0 mg/dL 
 
>2.0 mg/dL or 
requires dialysis 
>2.0 mg/dL or 
requires dialysis 
ALT 
  Males 
  Females 
 
58 – 115 U/L 
64 – 128 U/L 
 
116 – 230 U/L 
129 – 255 U/L 
 
>230 U/L 
>255 U/L 
 
All local (injection-site) reactions occurring within the first 7 days after vaccination will be 
considered definitely related to vaccination. 
 
 
 
 

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9.2.3 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. 
9.2.4 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. 
9.3 
Reporting Procedures 
9.3.1 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 by 
email, telephone or fax within 1 working day of notification of the SAE occurrence to the 
Principal Investigator, via a scanned PDF version of an SAE form to: 
 
Dr. Maria Elena Bottazzi, PhD 
Deputy Director,  
Sabin Vaccine Institute Product Development Partnership 
1102 Bates St., Ste. 550, Houston, TX 77030 
832-824-0504, Alt. 713-798-1199, Fax 832-825-0549 
Email: bottazzi@bcm.edu 
 
In addition, the Principal Investigator will report all SAEs that are determined to be definitely or 
probably related to vaccination to the Institutional Review Boards (IRBs) that approved the study 

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(the IRB of the George Washington University and the local IRB in Brazil) according to the IRB’s 
reporting timelines and using the IRB-specified reporting forms, as the DMID 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.  
 
In addition, all SAEs must be submitted within 24 hours of site awareness on an SAE form to 
the DMID pharmacovigilance contractor, at the following address: 
  
DMID Pharmacovigilance Group 
Clinical Research Operations and Management Support (CROMS) 
6500 Rock Spring Dr. Suite 650 
Bethesda, MD 20814, USA 
 
SAE Hot Line: 1-800-537-9979 (US) or 1-301-897-1709 (outside US) 
SAE FAX Phone Number: 1-800-275-7619 (US) or 1-301-897-1710 (outside US) 
SAE Email Address: PVG@dmidcroms.com 
 
9.3.2 Regulatory Reporting for Studies Conducted Under DMID-Sponsored 
IND 
Not Applicable 
9.3.3 Regulatory Reporting for Studies Not Conducted Under 
DMID-Sponsored IND 
 
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 and ANVISA (the Brazilian national regulatory agency) 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 Sponsor, FDA and 
ANVISA according to the same procedure as for reporting SAEs, and according to the same 
timelines as described above.  
9.3.4 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:  

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• 
Neuroinflammatory disorders (optic neuritis, multiple sclerosis, demyelinating disease, 
transverse myelitis, Guillain-Barre 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  
9.3.5 Medically-Attended Adverse Events (MAAEs) 
Special attention will 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 qualified 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. 
9.3.6 Reporting of Pregnancy 
Pregnancies occurring in study subjects will be reported on the study-specific Pregnancy Report 
form.  No further study vaccinations will be administered to pregnant subjects, but with the 
subject’s permission all study mandated blood samples will be obtained and the subject will 
continue in follow-up for safety events.  Efforts will be made to follow all pregnancies reported 
during the course of the study to pregnancy outcome pending the subject’s permission. 
9.4 
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 product.  AEs limited to new-onset chronic medical conditions will 
be followed through approximately 9 months after the third study vaccination (Day 380). 
 

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9.5 
Halting Rules 
 
If a dose of study vaccine is considered significantly reactogenic (see below and Section 9.2.2), 
dose escalation and/or additional vaccinations will be suspended until reviewed by the SMC, 
Independent Safety Monitor, DMID Medical Monitor, and IND sponsor (SVI). Any 
recommendation of the Independent Safety Monitor, DMID Medical Monitor, and 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 IRBs.  
 
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, 
• 
One 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, 
• 
Two 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 140 visit, after 
which 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 Safety Monitor, and the SMC. If deemed necessary for urgent safety reasons, the 
Independent Safety 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 
Safety 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. 
 
The decision to completely unblind the study prior to Day 140 or permanently stop the study 
prior to Day 380 will take the form of a formal recommendation by the SMC to the study 
Sponsor. The Principal Investigator must then notify the IRBs of this decision. 

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9.6 
Safety Oversight  
9.6.1 Independent Safety Monitor (ISM) 
 
An independent Safety Monitor will be appointed for oversight of subject safety in this trial. The 
ISM will be a local, qualified physician who will be available to advise the investigators on trial-
related medical questions or problems. The ISM will work with the DMID Medical Monitor to 
ensure adequacy of adverse event monitoring and reporting. Should the ISM not be available, 
he/she will recommend an alternative to serve as a substitute ISM. 
 
The ISM primary responsibility will be to monitor subject safety. The Principal Investigator is 
responsible for ensuring that the ISM is aware of any new safety information that becomes 
available during the course of the trial.   
9.6.2 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 3 and Group 5 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 3 and Group 5 of the study. If no criteria for halting the 
study are met (see Section 9.5 above) vaccinations will proceed with approval from the SMC.   
 
Written approval (via fax or email) to proceed to vaccinating Group 3 and Group 5 must be 
obtained from the SMC prior to vaccine administration. Both the SMC and ISM 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 that it is reported to the IRBs. 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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10 
CLINICAL MONITORING 
10.1 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 
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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11 
STATISTICAL CONSIDERATIONS 
11.1 Study Hypotheses 
This study is not designed to statistically test a specific hypothesis. Rather, it is intended to 
assess the safety, reactogenicity, and immunogenicity of two novel vaccines being co-
administered to humans for the first time. The chosen sample size principally facilitates the 
assessment of safety, as discussed in Section 11.2, Sample Size Considerations.  Given that, 
this study will explore, primarily by using descriptive statistics, to assess the following 
hypotheses: 
1. Co-administration of the Na-APR-1 (M74)/Alhydrogel® and Na-GST-1/Alhydrogel® 
vaccines will not increase the reactogenicity or safety risk above that of either 
administered alone. 
2. Co-administration of the Na-APR-1 (M74)/Alhydrogel® and Na-GST-1/Alhydrogel® 
vaccines will not decrease the functional activity of induced IgG antibodies to either 
antigen compared to when either is administered alone. 
3. Co-administration of the Na-APR-1 (M74)/Alhydrogel® and Na-GST-1/Alhydrogel® 
vaccines will not decrease the IgG antibody response to either antigen compared to 
when either is administered alone. 
4. Co-administration of the Na-APR-1 (M74)/Alhydrogel® and Na-GST-1/Alhydrogel® 
vaccines will not decrease the avidity of induced IgG antibodies to either antigen 
compared to when either is administered alone. 
5. Co-administration of the Na-APR-1 (M74)/Alhydrogel® and Na-GST-1/Alhydrogel® 
vaccines will not decrease the induction of specific MBCs to either antigen compared to 
when either is administered alone. 
 
11.2 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.  

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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. 
11.3 Planned Interim Analyses  
11.3.1 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) vs. Na-APR-1 (M74) co-administered with Na-GST-1), by Na-
APR-1 (M74) dose, and by GLA-AF dose.  

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Laboratory results (hematological and clinical chemistry) will be examined for trends over time 
and any clinically significant values for individuals will be reported. 
11.3.2 Immunogenicity Review 
 
The proportion of subjects with detectable anti-Na-GST-1 and anti-Na-APR-1 (M74) responses, 
and the duration of responses, will be summarized as descriptive measures. Geometric mean 
antibody responses will be compared between Groups 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, 119, and 126 for interim analysis, with Days 
140, 200, 290, and 380 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® alone and those 
co-administered Na-GST-1/Alhydrogel®.  
 
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. 
11.4 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 between the investigational vaccines, when they are 
administered separately or when they are co-administered. 
 
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%.  
 
Note: For the analyses described below, historical safety data from a Phase 1 clinical trial 
conducted at the same study site and in the same subject population (study SVI-10-01, see 
Section 2.1.6 above for a description of this trial) will be used to obtain a comparison of Na-
GST-1/Alhydrogel. As well, stored serum and cell specimens from those study participants will 
be used in the immunological assays to obtain data on subjects administered Na-GST-
1/Alhydrogel without co-administered Na-APR-1 (M74). These specimens from SVI-10-01 will 
be assayed at the same time as those newly collected from the study described in this protocol. 
 

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Primary Objective: To evaluate the safety and reactogenicity of three different dose 
concentrations of Na-APR-1 (M74) adjuvanted with Alhydrogel® or Alhydrogel® plus GLA-AF 
and administered either with or without Na-GST-1, in healthy Brazilian adults. 
 
AEs will be coded according to Medical Dictionary of Regulatory Activities (MedDRAÔ) 
preferred terms. The frequency, severity, and relationship of AEs per each vaccine formulation 
and dose cohort of Na-APR-1 (M74) and co-administration status with Na-GST-1 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 dose cohort and 
formulation of Na-APR-1 (M74) and formulation (i.e., no GLA-AF or 5 μg GLA-AF). 
• 
AEs will be summarized by severity and relationship to vaccine formulation by 
individuals and dose of Na-GST-1, Na-APR-1 (M74), and GLA-AF. 
• 
The frequency of vaccine-related SAEs will be tallied as well as summarized by body 
system, by vaccine formulation and dose cohort. 
 
Primary Outcome Measures: 
The following summary parameters will be evaluated for each dose and formulation of Na-
APR-1 (M74) alone or 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. 
Analysis Plan: 
 
The proportion of participants with at least one injection site AE will be compared by Na-
APR-1 (M74) vaccine formulation and dose cohort and co-administration status with Na-
GST-1. 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. 
 
Laboratory results (hematological and clinical chemistry) will be examined for trends over 
time and any clinically significant values for individuals will be reported. 

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Secondary Objective: To assess the impact of co-administering Na-APR-1 (M74) and Na-GST-1 
on antibody production by measuring the antibody levels to each antigen on study Day 126.  
 
Secondary Outcome Measures: 
 
The following parameters will be evaluated for each dose and formulation of Na-APR-1 
(M74) alone or co-administered with Na-GST-1: 
1. The IgG level by an indirect enzyme-linked immunosorbent assay (ELISA) on 
approximately Day 126. 
2. The IgG level by ImmunoCAP on approximately Day 126. 
 
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), 
GLA-AF (0 or 5 μg), and Na-GST-1 (0, 100μg). 
e. For antigen-specific log IgG levels, a three-way ANOVA will be conducted, with Na-
GST-1, Na-APR (M74) and GLA-AF doses as factors.  
f. 
Pair-wise comparisons across all combinations of Na-APR-1 (M74) doses, GLA-AF 
doses (i.e., 0 or 5 µg), and Na-GST-1 co-administration status will be assessed using 
Tukey’s HSD.   
 
 
Tertiary Objective 1: To assess the impact of co-administration of Na-GST-1 on the duration of 
the antibody responses to Na-APR-1 (M74), and vice versa.  
 
Tertiary Outcome Measure 1: The IgG antibody response, by an indirect enzyme-linked 
immunosorbent assay (ELISA) at approximately 7, 14, and 28 days after each vaccination, 
and approximately 3, 6, and 9 months after the third dose. 
Tertiary Outcome Measure 2: The IgG antibody response, by ImmunoCAP at approximately 
7, 14, and 28 days after each vaccination, and approximately 3, 6, and 9 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. 

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c. Geometric mean antibody responses will be compared between vaccine formulation 
and dose groups at each time point. Comparisons between groups will be made by a 
one-way 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, 6 and 9 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), GLA-AF (0 or 5μg), and Na-GST-1 (0, 100μg) 
h. For antigen-specific log IgG levels, three-way ANOVAs will be conducted, with Na-
APR-1 (M74) dose, GLA-AF dose, and Na-GST-1 co-administration status as 
factors.  
i. 
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 and 
formulation of Na-APR-1 (M74) and Na-GST-1 co-administration status will be 
explored. This will be accomplished using Proc Mixed in SAS 9.3 so the analysis will 
take account of the correlation between measurements on the same participant. 
 
 
Tertiary Objective 2: To assess the impact of co-administration of Na-GST-1 on the distribution 
of IgG subclass responses to Na-APR-1 (M74) and vice versa.  
 
Tertiary Outcome Measure 3: The IgG subclass distribution (IgG1, IgG3, and IgG4) by plate 
based ImmunoCAP on approximately Day 126. 
 
Tertiary Outcome Measure 4: The IgG subclass distribution by plate based ImmunoCAP 
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) 
IgG subclass responses will be summarized at each time point as a descriptive 
measure.   
b. IgG 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 formulation 
and dose groups. Comparisons between groups will be made by a one-way analysis 

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of variance (ANOVA) with pair-wise comparisons between groups made by 
contrasts. 
d. For antigen-specific IgG subclass (IgG1-IgG4) levels, a three-way ANOVA will be 
conducted, with Na-APR-1 (M74) and GLA-AF doses, and Na-GST-1 co-
administration status, as factors.  
e. 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 and formulation of Na-APR-1 (M74) and Na-GST-1 
co-administration status will be explored. This will be accomplished using Proc Mixed 
in SAS 9.3 so the analysis will take account of the correlation between 
measurements on the same participant. 
 
 
Tertiary Objective 3: To assess the impact of co-administration of Na-GST-1 and Na-APR-1 
(M74) on the functional capacity of vaccine-induced antibodies to neutralize the in vitro activity 
of the native enzymes (Na-GST-1 and wild type Na-APR-1).  
 
 
Tertiary Outcome Measure 5: The functional capacity of vaccine-induced antibodies to 
neutralize the in vitro activity of the native enzymes.  
 
Analysis Plan:  
a. Percent inhibition of native enzyme activity will be displayed graphically for the 
various study groups at each time point. 
b. Three-way ANOVAs with Na-APR-1 (M74), Na-GST-1, and GLA-AF as factors and 
percent inhibition as the outcome will be estimated. 
c. A longitudinal model will be built to describe percent inhibition of native enzyme 
activity over time. Using a longitudinal panel model, differences in percent inhibition 
by dose and formulation of Na-APR-1 (M74) and co-administration with Na-GST-1 
will be explored. This will be accomplished using Proc Mixed in SAS 9.3, so the 
analysis will take account of the correlation between measurements on the same 
participant. 
 
 
Tertiary Objective 4: To assess the impact of co-administration of Na-GST-1 and Na-APR-1 
(M74) on the affinity of the antibody interactions with both recombinant antigens. 
 
Tertiary Outcome Measure 6: The affinity of the antibody interactions with both recombinant 
antigens at approximately 7, 14, and 28 days after each vaccination, and 3, 6, and 9 months 
after the third dose. 
 
Analysis Plan: 
 
a. Antibody affinity will be displayed graphically for the various study groups at each 
time point. 
b. Three-way ANOVAs with Na-APR-1 (M74), Na-GST-1, and GLA-AF as factors and 
overall affinity as the outcome will be estimated. 

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c. A longitudinal model will be built to describe increasing antibody affinity (affinity 
maturation) over time. Using a longitudinal panel model, differences in affinity 
maturation by dose and formulation of Na-APR-1 (M74) and co-administration with 
Na-GST-1 will be explored. This will be accomplished using Proc Mixed in SAS 9.3, 
so the analysis will take account of the correlation between measurements on the 
same participant. 
 
Tertiary Objective 5: To assess the impact of co-administration of Na-GST-1 and Na-APR-1 
(M74) on the production of memory B cells specific for each antigen.  
 
Tertiary Outcome Measure 7: The production of memory B cells specific for each antigen on 
days of vaccination, approximately 28 days following each vaccination, and 6 and 9 months 
after the third dose.  
 
Analysis Plan: 
 
a. The amount of Na-GST-1 and Na-APR-1 (M74) specific memory B cells will be 
expressed as a percentage of total lymphocytes present in the blood.  
b. The percentage of specific memory B cells will be displayed graphically by study 
group at each time point. 
c. Two separate longitudinal panel analyses will test the null hypothesis that average 
percentages for each of memory B cells are the same in Na-APR-1 (M74) groups 
with and without co-administration of Na-GST-1 over time. This will be accomplished 
using Proc Mixed in SAS 9.3 so the analysis will take account of the correlation 
between measurements on the same participant.   
d. Mann-Whitney tests will assess the null hypothesis that cytokine levels are the same 
in the groups at the primary time point of Day 126, two weeks after final vaccination.  
 
 
Exploratory Objective: To assess the cellular immune responses to the Na-GST-1 and Na-APR-
1 (M74) antigens following immunization. 
 
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 test the null hypothesis that cytokine and 
chemokine levels are the same when Na-APR-1 (M74) is administered with and 
without or Na-GST-1 and examine for trends over time.  
 
 

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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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12 
SOURCE DOCUMENTS AND ACCESS TO SOURCE 
DATA/DOCUMENTS 
Complete source documentation (clinical evaluations and test results) will be collected for every 
study subject 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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13 
QUALITY CONTROL AND QUALITY ASSURANCE 
Following a written Sponsor-accepted site quality management plan, 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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14 
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). 
14.1 Ethical Standard 
 
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); 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]); and, Brazilian Resolution Nº 466/12 
(that replaced Resolution Nº 196/96) on Research Involving Human Subjects.  
14.2 Institutional Review Boards 
 
The investigators will be responsible for obtaining full IRB approvals for the study from the local 
Brazilian IRB (at the Federal University of Minas Gerais) and George Washington University 
IRBs. 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 
14.3 Informed Consent 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 US 21 CFR 50.25 and Brazilian 
Resolution Nº 466/12 on Research Involving Human Subjects. For SVI-DBL-01 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 volunteer. Individual sessions may be 
on the same day as the community meeting, or scheduled for a later date. During the individual 
session a member of the study team will read the consent form together with the volunteer, 
regardless of whether or not the volunteer is illiterate. The study team member will explain why 
the volunteer is being invited to participate in the study and will clarify all of the volunteer’s 

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questions. Throughout the informed consent process volunteers will be encouraged to ask 
questions, and if they wish, to discuss the study with family or community members prior to 
making their decision.  
 
Volunteers agreeing to participate will first provide written informed consent and take a 
true/false comprehension questionnaire. The questionnaire is 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. Volunteers 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 volunteer, the 
volunteer’s questions were answered, and the volunteer answered all comprehension questions 
correctly. The original signed informed consent form for each volunteer will be maintained as 
part of that volunteer’s study records. A copy of the informed consent form will be provided to 
every volunteer.   
14.3.1 Informed Consent/Assent Process (in Case of a Minor) 
Not Applicable 
14.4 Exclusion of Women, Minorities, and Children (Special 
Populations) 
 
Healthy adults age 18 to 45 meeting all protocol defined inclusion and exclusion criteria will be 
invited to participate. Females of childbearing potential, unless surgically sterile (i.e., bilateral 
tubal ligation, bilateral oophorectomy, or complete hysterectomy), at least 2 years 
postmenopausal, or practicing abstinence, must use an effective method of avoiding pregnancy 
(including oral, transdermal, or implanted contraceptives, intrauterine device, female condom 
with spermicide, diaphragm with spermicide, cervical cap, or use of a condom with spermicide 
by the sexual partner) until at least one month following the last immunization. Female 
participants will be counseled by a study clinician or nurse, or referred to the local health center 
for evaluation and institution of an appropriate contraceptive method.  
 
This clinical trial will enroll healthy, non-pregnant volunteers from the general population of the a 
study site in the Brazilian state of Minas Gerais located in and around the town of 
Americaninhas, without regard to gender or racial/ethnic group.  That is, no specific racial/ethnic 
groups will be targeted or excluded from participation in these trials.  In addition, both (non-
pregnant) women and men will be equally encouraged to participate, and it is expected that 
roughly equal numbers of each gender will consent to participate in this Phase 1 study, based 
on the experience of the study team that will conduct the proposed clinical trial and which has 
enrolled subjects into similar vaccine trials at the same site.   
 
There will not be any research conducted on children under the age of 18 years as part of this 
trial.  Children between the ages of 18-21 years, inclusive, will be eligible to participate in this 
Phase 1 trial, given that the legal age of consent in Brazil is 18 years. 

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14.5 Subject Confidentiality 
 
All study-related information will be stored securely at the study site. All subject information 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 US National 
Institutes of Health, and the FDA or Brazilian regulatory authorities. 
14.6 Study Discontinuation 
If the trial is discontinued, subjects who sign the informed consent form, and are randomized 
and vaccinated will continue to be followed for safety assessments. No further study 
vaccinations will be administered. 
14.7 Future Use of Stored Specimens 
 
Some of the biological samples collected from study participants may be stored at the local site 
and some at CPqRR in Belo Horizonte or at the MITM at GWU. Stored samples may be shared 
with other investigators at other institutions for the purposes of conducting the tests outlined in 
this study 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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15 
DATA HANDLING AND RECORD KEEPING 
15.1 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. 
15.2 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 is derived from 
source documents should be consistent with source documents and the discrepancies should 
be explained. 
15.3 Types of Data 
Data for this study will include clinical, safety, and outcome measures (e.g., clinical laboratory 
values, reactogenicity, and immunogenicity data). 
15.4 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 2 have been compiled, for 
the purposes of deciding whether it is safe to precede with enrollment into Group 3 of the study. 
The second interim SMC safety report will be completed after the safety data from the Day 14 
visits of Group 4 have been compiled, for the purposes of deciding whether it is safe to precede 
with enrollment into Group 5 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 140 visits is available and the study 
investigators unblinded. This report will serve as the basis for deciding whether to continue with 

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future Phase 1 testing of the vaccines in children. A final report containing all safety and 
immunology data will be prepared after trial completion. 
15.5 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 Brazilian regulatory authorities. 
15.6 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 
actively participating IRBs 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, an IRB problem report will be 
generated and submitted to the IRBs according to their guidelines and reporting timelines.  

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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 publically 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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17 
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Phase 1 Study of Na-APR-1 (M74) Co-Administered with Na-GST-1 
Version 5 
 
22 July 2015 
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77 
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Phase 1 Study of Na-APR-1 (M74) Co-Administered with Na-GST-1 
Version 5 
 
22 July 2015 
 
 
 
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78 
 
SUPPLEMENTS/APPENDICES

Phase 1 Study of Na-APR-1 (M74) Co-Administered with Na-GST-1 
Version 5 
 
22 July 2015 
 
 
 
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79 
APPENDIX A: SCHEDULE OF EVENTS 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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 Asessment 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine Dipstick 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine Pregnancy Test 
(Females) 
 
X 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HCV 
10 mL 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HBsAg 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HIV 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Glucose (Random) 
5 mL 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
ALT 
X 
X 
 
 
 
X 
 
X 
 
 
 
X 
 
X 
 
 
 
X 
 
 
 
 
Creatinine 
X 
X 
 
 
 
X 
 
X 
 
 
 
X 
 
X 
 
 
 
X 
 
 
 
 
CBC2  
2 mL 
X 
X 
 
 
 
X 
 
X 
 
 
 
X 
 
X 
 
 
 
X 
 
 
 
 
Serum and Plasma 
Antibody Assays 
10 mL X 
X 
 
 
X 
X 
X 
X 
 
 
X 
X 
X 
X 
 
 
X 
X 
X 
X 
X 
X 
Cellular Assays 
30 mL 
 
X 
 
 
X 
X 
X 
X 
 
 
X 
X 
X 
X 
 
 
X 
X 
X 
X 
X 
X 
Daily Blood  
Volume (mL) 
 
27 47 
 
 
30 
47 
40 
47 
 
 
30 
47 
40 
47 
 
 
30 
47 
40 
40 
40 
40 
Total Blood  
Volume (mL) 
 
27 74 
 
 
114 161 201 248 
 
 
288 335 375 422 
 
 
462 509 549 589 629 669 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Stool Examination 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
VACCINATIONS 
 
 
1 
 
 
 
 
 
2 
 
 
 
 
 
3