Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel Co-administered With Different Toll-Like Receptor Agonists in Hookworm- Naïve Adults
Phase: PHASE2
Conditions: Hookworm Infections
Sponsor: Baylor College of Medicine

Protocol Cover Page 
Study Title: Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults 
NCT Number: NCT03172975 
Document Version & Date: v6.0 dated 03Oct2025 

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
i
Phase 2 Study to Assess the Safety, Efficacy and 
Immunogenicity of Na-GST-1/Alhydrogel® Co-administered 
with Different Toll-Like Receptor Agonists in Hookworm-
Naïve Adults
 DMID Protocol Number: 16-0116
DMID Funding Mechanism: NIAID Clinical Trial Implementation Cooperative 
Agreement (U01)
Pharmaceutical Support Provided by: Not Applicable
Other Identifying Numbers: IND Sponsor Protocol Number SVI-CH-02
IND Sponsor: Baylor College of Medicine
Principal Investigator: David Diemert, MD FRCP(C)
DMID Medical Officer:  Gregory Deye, MD
DMID Medical Monitor:  Mohamed Elsafy, MD
DMID Clinical Project Manager:  Walter Jones, RN, MPH 
Version Number: 6.0
03 October 2025
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
ii
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) 

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.
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
iii
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 federal regulations and ICH guidelines.
Site Investigator:
Signed:
Date:
Name: David Diemert, MD
Title: Principal Investigator
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
iv
TABLE OF CONTENTS
Statement of Compliance...............................................................................................................ii
Signature Page .............................................................................................................................iii
Table of Contents..........................................................................................................................iv
List of Abbreviations....................................................................................................................viii
Protocol Summary .........................................................................................................................x
1
Key Roles .............................................................................................................................1
2
Background Information and Scientific Rationale.................................................................2
2.1
Background Information............................................................................................2
2.1.1
Hookworm Infection and Justification for Vaccine Development ..................2
2.1.2
Study Site......................................................................................................3
2.1.3
Prior Clinical Experience with Hookworm Vaccines......................................3
2.1.4
The Na-GST-1/Alhydrogel® Hookworm Vaccine...........................................4
2.1.5
Immunogenicity Studies with Na-GST-1 .......................................................6
2.1.6
Clinical Experience with Na-GST-1/Alhydrogel® Hookworm Vaccine ...........7
2.1.7
Clinical Experience with AP 10-701 ............................................................10
2.1.8
Clinical Experience with CpG 10104...........................................................11
2.1.9
Clinical Experience with Controlled Human Hookworm Infection (CHHI) ...12
2.2
Rationale.................................................................................................................13
2.2.1
Rationale for the Study................................................................................13
2.2.2
Rationale for Na-GST-1 Doses and Dose Schedule to be Studied.............14
2.2.3
Rationale for Controlled Human Hookworm Infection Model (CHHI)..........14
2.2.4
Inclusion of Placebo....................................................................................15
2.2.5
Clinical Development Plan for the Human Hookworm Vaccine ..................15
2.3
Potential Risks and Benefits ...................................................................................16
2.3.1
Potential Risk ..............................................................................................16
2.3.2
Precautions Taken to Minimize Risks .........................................................18
2.3.3
Known Potential Benefits ............................................................................19
3
Objectives...........................................................................................................................20
3.1
Study Objectives.....................................................................................................20
3.2
Study Outcome Measures ......................................................................................21
3.2.1
Primary Outcome Measures .......................................................................21
3.2.2
Secondary Outcome Measures...................................................................21
3.2.3
Exploratory Outcome Measures..................................................................22
4
Study Design ......................................................................................................................23
4.1
Substudies (if applicable)........................................................................................24
5
Study Enrollment and Withdrawal.......................................................................................25
5.1
Subject Inclusion Criteria ........................................................................................25
5.2
Subject Exclusion Criteria.......................................................................................26
5.3
Treatment Assignment Procedures ........................................................................27
5.3.1
Randomization Procedures.........................................................................27
5.3.2
Masking Procedures ...................................................................................28
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
v
5.3.3
Reasons for Withdrawal and Discontinuation of Vaccinations....................28
5.3.4
Handling of Withdrawals .............................................................................30
5.3.5
Termination of Study...................................................................................31
6
Study InterventionS/Investigational ProductS.....................................................................32
6.1
Study Product Description ......................................................................................32
6.1.1
Acquisition...................................................................................................33
6.1.2
Formulation, Packaging, and Labeling........................................................33
6.1.3
Product Storage and Stability......................................................................34
6.2
Dosage, Preparation and Administration of Study Intervention/Investigational 
Product....................................................................................................................34
6.3
Modification of Study Intervention/Investigational Product for a Subject ................35
6.4
Accountability Procedures for the Study Intervention/Investigational Product(s)....35
6.5
Assessment of Subject Compliance with Study Intervention/Investigational 
Product....................................................................................................................36
6.6
Concomitant Medications/Treatments ....................................................................36
7
Study Schedule...................................................................................................................37
7.1
Screening................................................................................................................37
7.2
Enrollment/Baseline................................................................................................38
7.3
Follow-up ................................................................................................................38
7.4
Final Study Visit ......................................................................................................44
7.5
Early Termination Visit............................................................................................44
7.6
Unscheduled Visit ...................................................................................................45
7.7
Clearance of Infection.............................................................................................46
7.8
N. americanus Donor Sub-Study ............................................................................46
8
Study Procedures/Evaluations............................................................................................48
8.1
Clinical Evaluations.................................................................................................48
8.2
Laboratory Evaluations ...........................................................................................48
8.2.1
Clinical Laboratory Evaluations...................................................................48
8.2.2
Immunology Assays....................................................................................49
8.2.3
Parasitology Assays....................................................................................51
8.3
Specimen Preparation, Handling, and Shipping .....................................................51
8.3.1
Instructions for Specimen Preparation, Handling, and Storage ..................51
8.3.2
Specimen Shipment....................................................................................51
9
Assessment of Safety.........................................................................................................52
9.1
Specification of Safety Parameters.........................................................................52
9.2
Methods and Timing for Assessing, Recording, and Analyzing Safety 
Parameters .............................................................................................................53
9.2.1
Adverse Events...........................................................................................53
9.2.2
Vaccine Reactogenicity...............................................................................54
9.2.3
CHHI Reactogenicity...................................................................................56
9.2.4
Additional Adverse Event Severity Grading ................................................58
9.2.5
Serious Adverse Events..............................................................................59
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
vi
9.2.6
Procedures to be Followed in the Event of Abnormal Laboratory Test 
Values or Abnormal Clinical Findings .........................................................59
9.3
Reporting Procedures.............................................................................................59
9.3.1
Serious Adverse Events..............................................................................59
9.3.2
Regulatory Reporting for Studies Conducted Under DMID-Sponsored 
IND ..............................................................................................................60
9.3.3
Regulatory Reporting for Studies Not Conducted Under 
DMID-Sponsored IND .................................................................................60
9.3.4
Adverse Events of Special Interest (AESI)..................................................61
9.3.5
Medically-Attended Adverse Events (MAAEs)............................................61
9.3.6
Reporting of Pregnancy ..............................................................................62
9.4
Type and Duration of Follow-up of Subjects after Adverse Events.........................62
9.5
Halting Rules...........................................................................................................62
9.6
Unblinding...............................................................................................................63
9.7
Safety Oversight .....................................................................................................63
9.7.1
Independent Safety Monitor (ISM) ..............................................................63
9.7.2
Safety Monitoring Committee (SMC) ..........................................................64
10
Clinical Monitoring ..............................................................................................................66
10.1
Site Monitoring Plan................................................................................................66
11
Statistical Considerations ...................................................................................................67
11.1
Study Hypotheses...................................................................................................67
11.2
Sample Size Considerations...................................................................................67
11.3
Planned Interim Analyses .......................................................................................68
11.4
Final Analysis Plan..................................................................................................68
12
Source Documents and Access to Source Data/Documents .............................................75
13
Quality Control and Quality Assurance...............................................................................76
14
Ethics/Protection of Human Subjects .................................................................................77
14.1
Ethical Standard......................................................................................................77
14.2
Institutional Review Board ......................................................................................77
14.3
Informed Consent Process .....................................................................................77
14.3.1 Informed Consent/Assent Process (in Case of a Minor).............................78
14.4
Exclusion of Women, Minorities, and Children (Special Populations) ....................78
14.5
Subject Confidentiality ............................................................................................78
14.6
Study Discontinuation .............................................................................................79
14.7
Future Use of Stored Specimens............................................................................79
15
Data Handling and Record Keeping ...................................................................................80
15.1
Data Management Responsibilities ........................................................................80
15.2
Data Capture Methods............................................................................................80
15.3
Types of Data..........................................................................................................80
15.4
Timing/Reports........................................................................................................80
15.5
Study Records Retention........................................................................................80
15.6
Protocol Deviations.................................................................................................81
16
Publication Policy................................................................................................................82
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
vii
17
Literature References .........................................................................................................83
Supplements/Appendices............................................................................................................88
Appendix A: Schedule of EvenTS – VACCINATION PHASE......................................................89
Appendix A: Schedule of Events – CHHI Phase .........................................................................90
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
viii
LIST OF ABBREVIATIONS
AE
Adverse Event
AESI
Adverse Event of Special Interest
APR-1
Aspartic Protease-1
BCM
Baylor College of Medicine
CBC
Complete Blood Count
cGMP
Current Good Manufacturing Practices
CHHI
Controlled Human Hookworm Infection
CIOMS
Council for International Organizations of Medical Sciences
CpG
Cytosine-phosphate-Guanine Oligodeoxynucleotide
CRF
Case Report Form
DMID
Division of Microbiology and Infectious Diseases, NIAID, NIH
ELISA
Enzyme linked immunosorbent assay
FDA
Food and Drug Administration
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
HIV
Human Immunodeficiency Virus
IB
Investigator’s Brochure
ICF
Informed Consent Form
ICH
International Conference on Harmonisation
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
ODN
Oligodeoxynucleotide
PBMC
Peripheral Blood Mononuclear Cell
PI
Principal Investigator
SAE
Serious Adverse Event
SMC
Safety Monitoring Committee
SOP
Standard Operating Procedure
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
ix
SVI
Sabin Vaccine Institute (Albert B Sabin Vaccine Institute; Sabin) 
US
United States
WBC
White Blood Cell
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
x
PROTOCOL SUMMARY
Title:
Phase 2 Study to Assess the Safety, Efficacy and 
Immunogenicity of Na-GST-1/Alhydrogel® Co-administered with 
Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
Phase:
2
Population:
48 healthy male and non-pregnant female volunteers aged 18-
45 years, inclusive.
Number of Sites:
1 (The George Washington University, Washington, DC)
Study Duration:
22 months
Subject Participation 
Duration:
14 months
Description of 
Interventions:
a) 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.
b) CpG 10104 is an unmethylated cytosine-phosphate-guanine 
oligodeoxydinucleotide (CpG) that is a Toll-like Receptor-9 
agonist. The Na-GST-1/Alhydrogel® plus CpG 10104 
formulation will be prepared immediately prior to vaccination 
by adding an appropriate volume of CpG 10104 to Na-GST-
1/Alhydrogel® and withdrawing an appropriate volume to 
administer the desired amount of Na-GST-1 plus 500µg 
CpG 10104.  
c)
Gluco-Pyranosylphospho-Lipid A Aqueous Formulation 
(GLA-AF; referred to as AP 10-701 by the manufacturer) 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 AP 10-
701 to Na-GST-1/Alhydrogel® and withdrawing an 
appropriate volume to administer the desired amount of Na-
GST-1 plus 5µg AP 10-701.  
d) To maintain the study blind by ensuring that all subjects 
receive an injection at each vaccination point, sterile normal 
saline (0.9%) for injection will be administered to those 
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
xi
subjects randomized to be infectivity controls for the 
controlled human hookworm infection arm.
e) Infectious Necator americanus larvae (L3). N. americanus 
eggs will be obtained from the feces of a chronically infected 
human volunteer, who is negative for HIV, HBV, and HCV. 
Fecal material is processed following a qualified standard 
procedure, and after hatching from eggs, the N. americanus 
larvae are stored in the dark at ambient temperature (19-
25oC) until use. Controls for the manufacturing process are 
tests for viability (motility), species identification, and 
microbial bioburden of the larvae.
Objectives:
Primary:
1.
To compare the impact of vaccination with Na-GST-
1/Alhydrogel® delivered with or without AP 10-701 or CpG 
10104 on controlled human hookworm infection (CHHI) with 
N. americanus larvae in healthy, hookworm-naïve adults, as 
determined by the presence of eggs using a qualified 
flotation technique.
2.
To evaluate the safety and reactogenicity of Na-GST-1/ 
Alhydrogel® delivered with or without AP 10-701 or CpG 
10104 on Days 0, 56 and 112, in healthy, hookworm-naïve 
adults.
Secondary:
3.
To compare the impact of vaccination with Na-GST-
1/Alhydrogel® delivered with or without AP 10-701 or CpG 
10104 on fecal egg counts, as determined by the McMaster 
method, after controlled human hookworm infection (CHHI) 
with N. americanus larvae in healthy, hookworm-naïve 
adults.
4.
To assess the relationship between antibody responses to 
Na-GST-1 induced by vaccination with Na-GST-
1/Alhydrogel® delivered with or without AP 10-701 or CpG 
10104 and responses to CHHI in healthy, hookworm-naïve 
adults, as determined by the presence of eggs using a 
qualified flotation technique.
5.
To assess the duration of antibody responses to Na-GST-1 
induced by vaccination with Na-GST-1/Alhydrogel® delivered 
with or without AP 10-701 or CpG 10104. 
Exploratory:
1.
To assess the relationship between the functional capacity 
of vaccine-induced antibodies that neutralize the in vitro 
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
xii
activity of native Na-GST-1 enzyme and responses to CHHI 
in healthy, hookworm-naïve adults.
2.
To compare the impact of vaccination with Na-GST-
1/Alhydrogel® delivered with or without AP 10-701 or CpG 
10104 on levels of hookworm DNA as detected by real-time 
PCR in healthy, hookworm-naïve adults challenged with 
CHHI, as determined by the presence of eggs using a 
qualified flotation technique.
3.
To assess the impact of vaccination with Na-GST-
1/Alhydrogel® delivered with or without AP 10-701 or CpG 
10104 on the affinity of antibody-antigen interactions, and 
how affinity relates to responses to CHHI in healthy, 
hookworm-naïve adults, as determined by the presence of 
eggs using a qualified flotation technique.
4.
To assess the relationship between Na-GST-1 specific 
memory B cells induced by vaccination with Na-GST-
1/Alhydrogel® delivered with or without AP 10-701 or CpG 
10104 and responses to CHHI in healthy, hookworm-naïve 
adults, as determined by the presence of eggs using a 
qualified flotation technique. 
5.
To assess the relationship between innate immune 
responses to Na-GST-1/Alhydrogel® delivered with or 
without AP 10-701 or CpG 10104 and responses to CHHI in 
healthy, hookworm-naïve adults.
Description of Study 
Design:
Double blind, randomized, controlled, Phase 2 clinical trial in 
hookworm-unexposed adults living in the metropolitan area of 
Washington, DC. Subjects will receive three doses of the 
assigned vaccine formulation, or saline placebo, delivered 
intramuscularly on approximately Days 0, 56, and 112. 
Subjects will be challenged with 50 infectious N. americanus 
larvae 4 weeks after 3rd vaccination. Subjects will be evaluated 
weekly starting 5 weeks post-challenge to assess infection status. 
Albendazole will be administered 20 weeks post-challenge to 
cure infections. Subjects will be followed until 10 months after 
their final vaccination. 
Safety of vaccination 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. Safety of CHHI will be 
measured from the time of larval application (Day 140) through 
the first day of treatment with albendazole (Day 280).
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
xiii
Unsolicited non-serious adverse events (AEs) will be collected 
until approximately 1 month following each study vaccination 
and from study Day 140 (day of CHHI) through Day 297.  
New-onset chronic medical conditions, Serious Adverse Events 
(SAEs), and Adverse Events of Special Interest (AESIs) will be 
collected from the time of the first study vaccination through 
approximately 10 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 and CHHI.
Immunogenicity testing will include IgG antibody responses to 
Na-GST-1, by a qualified indirect enzyme-linked immunosorbent 
assay (ELISA), on serum obtained prior to each study 
vaccination and CHHI, and at time points after each vaccination 
and after CHHI (see Appendix A); the affinity of vaccine-induced 
antibodies against Na-GST-1 using Surface Plasmon 
Resonance; the functional activity of vaccine-induced antibodies 
via in vitro enzyme neutralization assay; antigen-specific 
memory B cell responses; and, the innate immune responses to 
each of the TLR receptor immunostimulants.  
Parasitological testing will include microscopic fecal egg 
detection by a qualified saline flotation technique, fecal egg 
counts by the McMaster method, fecal PCR for hookworm DNA, 
and peripheral eosinophil counts.
Recruitment and enrollment into the study will occur on an 
ongoing basis, with each group being recruited and vaccinated 
in sequence.
48 subjects will be enrolled into 4 groups of 12:
Subjects will be enrolled sequentially and upon enrollment will 
be randomized to one of the following IP assignments in a 
double-blind fashion:

Group 1 IP allocation (n=12 subjects): 
o
12 subjects will receive 100µg Na-GST-1/Alhydrogel® 
delivered by IM injection in the deltoid muscle.

Group 2 IP allocation (n=12): 
o
12 subjects will receive Na-GST-1/Alhydrogel® plus 
500µg CpG 10104 delivered by IM injection in the 
deltoid muscle.

Group 3 IP allocation (n=12): 
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
xiv
o
12 subjects will receive Na-GST-1/Alhydrogel® plus 
5µg AP 10-701 delivered by IM injection in the 
deltoid muscle.

Group 4 IP allocation (n=12): 
o
12 subjects will receive sterile saline delivered by IM 
injection in the deltoid muscle
Estimated Time to 
Complete Enrollment:
6 months
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
xv
Schematic of Study Design: 
Controlled Human
Hookworm Infection
(50 L3)
N = 48
Group 1
100µg Na-GST-
1/Alhydrogel
®
(n=12)
Group 2
100µg Na-GST-
1/Alhydrogel
®
plus
500µg CpG 10104 
(n=12)
Group 3
Na-GST-
1/Alhydrogel
®
plus
5µg GLA-AF
(n=12)
Group 4
Sterile saline
placebo
(n=12)
Double blind, block 
randomization of all
study participants
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
1
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
Institutions:
Study Site: 
George Washington University Medical Faculty Associates
2150 Pennsylvania Avenue NW 
Washington, DC 20037
Contact Person: Caroline Thoreson, PA-C
Phone Number: 202-741-2443
E-mail: cthoreson@mfa.gwu.edu
Clinical Laboratory: 
Laboratory Corporation of America 
447 York Court, Burlington, NC 27215
Contact Person: Cheryle Green
Tel: 202-741-2371
Fax Number: 202-741-3055
E-mail: greenc@labcorp.com
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: Larissa Scholte
Tel: (202) 994-1409
E-mail: larissascholte@gwu.edu
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
2
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 
occur almost exclusively in school-aged children, it has been shown that high-intensity 
hookworm infections may also occur in adults (14-16).  
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Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
3
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 study will be conducted in healthy adult volunteers without history of hookworm infection at 
the George Washington University Medical Faculty Associates (GW MFA) and the George 
Washington University School of Medicine and Health Sciences, Department of Microbiology, 
Immunology, and Tropical Medicine (GW MITM), both in Washington, DC.
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 
several different dose concentrations of the vaccine was conducted in healthy, hookworm-naïve 
adults living in the United States, which showed the formulation to be safe, well tolerated and 
immunogenic (23).  
However, upon testing the vaccine in adults who had previously been infected with hookworm in 
Americaninhas, several volunteers in the lowest dose cohort to be vaccinated developed 
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Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
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generalized urticaria within 2 hours of immunization (24). Due to these immediate-type 
hypersensitivity reactions, vaccinations in this study were halted. Subsequent investigations 
revealed that the volunteers who developed urticaria upon their first dose of Na-ASP-2 had 
elevated levels of baseline (i.e., pre-vaccination) IgE to the vaccine antigen. Subsequently, a 
sero-epidemiological survey was conducted in an endemic region of Brazil; this study revealed 
that even in young children, a significant proportion of individuals have detectable levels of IgE 
to this protein, likely due to previous infection with N. americanus. In addition, similar findings 
were demonstrated for other larval proteins that were being considered as vaccine candidates. 
Due to these cumulative data, clinical development of the Na-ASP-2 and other larval-stage 
antigens as candidate vaccines was halted. Instead, the current strategy is to develop antigens 
expressed during the adult stage of the hookworm life cycle that play a role in digesting the host 
hemoglobin that is used by the worm as an energy source. These antigens do not induce 
specific IgE antibodies during natural infection, and hence have a low likelihood for inducing 
allergic reactions upon vaccination.  
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 (25). Following hemolysis, 
adult hookworms use an ordered cascade of hemoglobinases to cleave hemoglobin into smaller 
molecules (25-30). Aspartic protease-1 of N. americanus (Na-APR-1) is responsible for initiating 
the proteolytic cascade in hookworms, as described below. After hemoglobin digestion, the 
freed heme generates toxic oxygen radicals that can be bound and detoxified by molecules 
such as glutathione S-transferase-1 (GST-1) (31-33). GST-1 of N. americanus (Na-GST-1) is a 
critical enzyme that plays a role in parasite blood feeding; used as a vaccine, we hypothesize 
that the antigen will induce anti-enzyme neutralizing antibodies that will interfere with parasite 
blood-feeding and cause parasite death or reduce worm fecundity.  
Na-GST-1 is a 24-kDa protein with peroxidase enzymatic activity that catalyzes the conjugation 
of reduced glutathione to a variety of electrophiles (31-33). This hookworm protein belongs to 
the Nu class of nematode GSTs, which also includes GSTs from the blood-feeding parasite of 
ruminants, Haemonchus contortus, and the rodent nematode Heligomosoides polygyrus. This 
class is characterized by diminished peroxidase activity relative to other classes of GSTs, but 
elevated binding capacity for heme and related products (31, 33-36). X-ray crystallography of 
Na-GST-1 demonstrates that the protein can form homodimers in solution, which create 
atypically large binding cavities accessible to a diversity of ligands, including heme (Figure 1) 
(33). Na-GST-1 binds heme at high affinity in vitro (31, 36). Because both heme and hematin 
contain oxidative iron, these molecules are potent generators of toxic reactive oxygen species 
that could potentially damage parasite macromolecules. In vivo, hookworm GSTs may therefore 
bind and detoxify the heme and hematin byproducts generated during the blood degradation 
process.  
Figure 1: Three-dimensional surface plot of Na-GST-1. The path to the binding cavity is 
indicated by the red arrow (33).
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IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
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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 2); following challenge with A. 
caninum infective larvae, significantly lower host worm burdens and fecal egg counts were 
observed compared to control animals vaccinated only with adjuvant (31).  In hamsters, 
vaccination with recombinant Ac-GST-1 also resulted in substantially lower worm burdens (51-
54%) following heterologous challenge with N. americanus infective larvae compared to 
controls, as did vaccination with recombinant Na-GST-1 followed by homologous larval 
challenge (31, 32, 37).  Because of these encouraging preclinical results, Na-GST-1 was 
manufactured according to current good manufacturing practices (cGMP) and formulated on 
Alhydrogel® in preparation for clinical trials.
Figure 2: 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 (31).
Most importantly, extensive studies have been conducted to test for sensitization to the Na-
GST-1 protein in individuals living in a hookworm endemic area who have been repeatedly 
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IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
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exposed and infected with N. americanus. 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. Therefore, the likelihood of inducing immediate-
type hypersensitivity reactions by vaccinating individuals living in hookworm-endemic areas with 
Na-GST-1 is low and likely not more than that associated with any new vaccine antigen entering 
clinical trials. The situation with Na-GST-1 is therefore very different from that of Na-ASP-2 in 
that repeated infection with hookworm does not induce an IgE response to the antigen, most 
likely due to the fact that it is a protein found in the digestive tract of adult hookworms and is 
therefore relatively hidden from the human immune system. This lack of antigen-specific IgE in 
people living in an area of high transmission has served as a major justification for advancing 
development of Na-GST-1 as a candidate vaccine antigen.
Na-GST-1 has been successfully manufactured and tested in the laboratory and in animals with 
both Alhydrogel®, Alhydrogel® plus AP 10-701, and Alhydrogel® plus CpG 10104. Na-GST-1 has 
been shown to be pure, potent, and stable in both of these three formulations. 
The Na-GST-1 vaccine formulation to be tested in this clinical trial consists of the 24-kDa 
recombinant protein Na-GST-1, adsorbed to an adjuvant, Alhydrogel® (aluminum hydroxide 
suspension) with or without the addition of one of two Toll-Like Receptor (TLR) agonists: a) 
Gluco-Pyranosylphospho-Lipid A Aqueous Formulation (AP 10-701); or, b) CpG 10104.  AP 10-
701 or CpG 10104 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 3). In this study, mice were vaccinated 
with Na-GST-1/Alhydrogel® at antigen doses ranging from of 0.01 to 31.25 µg Na-GST-1 with or 
without CpG 10104 (5 µg) or CpG 10105 (5 µg). CpG 10105 is a CpG oligodeoxynucleotide 
sequence that is similar to CpG 10104 but that is not being proposed to be tested in the study 
described in this protocol. Mice were vaccinated twice intramuscularly at a 3-week interval, with 
blood collected for anti-Na-GST-1 IgG ELISA two weeks after the second immunization. This 
study demonstrated a large, highly significant increase in IgG specific for Na-GST-1 in the group 
administered Na-GST-1/Alhydrogel®/CpG 10104 compared to that administered only Na-GST-
1/Alhydrogel® as shown in Figure 3.    
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
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Figure 3: 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  
Na-GST-1/Alhydrogel® administered with or without AP 10-701
The Na-GST-1/Alhydrogel® hookworm vaccine has been tested with or without AP 10-701 in 
two completed and two ongoing Phase 1 clinical trials in humans in the United States (n=40), 
Brazil (n=162) and Gabon (n=32) either alone or co-administered with the Na-APR-1 
(M74)/Alhydrogel® hookworm vaccine. To date, no significant reactogenicity or safety issues 
have been observed in any of these trials. 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 AP 10-701 
(NCT01385189). Mild to moderate injection site pain and tenderness were observed in a 
minority of study subjects; other common adverse events included mild to moderate headache 
and nausea. No vaccine-related SAEs or Adverse Events of Special Interest occurred and the 
vaccine was well tolerated. The incidence of vaccine-related adverse events was not related to 
either dose of Na-GST-1 or co-administration status with AP 10-701. Antigen-specific IgG 
antibodies were induced in a dose-dependent fashion after the second and third vaccinations 
(Figure 4).
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
8
Figure 4: Mean anti-Na-GST-1 IgG antibody units induced by vaccination with Na-GST-
1/Alhydrogel® (with or without AP 10-701) in healthy American (SVI-11-01) vs. Brazilian 
(SVI-10-01). Butang HBV=hepatitis B vaccine (comparator); V=vaccination.
The trial of Na-GST-1 conducted 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 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 AP 10-701. 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 AP 10-701 (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 were followed for 12 
months after the final vaccination. In this study, Na-GST-1/Alhydrogel® (with or without AP 10-
701) was safe and well tolerated in both hookworm-unexposed and hookworm-exposed adults. 
No vaccine-related SAEs or Adverse Events of Special Interest occurred and the vaccine was 
well tolerated. The most commonly observed solicited adverse events were mild injection site 
pain and tenderness. The most common systemic solicited adverse event was mild headache. 
Neither the incidence nor the severity of these adverse events was related to the number of 
injections received. Similar to SVI-11-01, antigen-specific IgG antibodies were induced in a 
dose-dependent fashion (Figure 4).
Na-GST-1/Alhydrogel® co-administered with Na-APR-1 (M74)/Alhydrogel®
Na-GST-1/Alhydrogel® is currently being tested in two Phase 1 clinical trials in which it is being 
co-administered with the Na-APR-1 (M74)/Alhydrogel® hookworm vaccine. In the first of these 
studies, 32 healthy adults living in a hookworm-endemic area of Gabon received 3 vaccinations 
of up to 100 µg Na-GST-1/Alhydrogel® co-administered with Na-APR-1 (M74)/Alhydrogel® 
Study Day
Na-GST-1 IgG AU
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IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
9
(n=24), compared to a licensed hepatitis B vaccine (n=8). All subjects randomized to receive the 
hookworm vaccines received an injection of Na-GST-1 in one arm and an injection of Na-APR-1 
(M74) in the other arm, on each day of vaccination. Both Na-GST-1/Alhydrogel® and Na-APR-1 
(M74)/Alhydrogel® were mixed with 5 µg AP 10-701 prior to vaccination. Vaccinations in this trial 
(NCT02126462) were administered according to a 0, 1, 6-month schedule and were completed 
in September 2015. Interim results from this trial indicate that co-administration of Na-GST-
1/Alhydrogel/AP 10-701 when co-administered with Na-APR-1/Alhydrogel/AP 10-701 was well 
tolerated, with minimal reactogenicity. An interim analysis of anti-Na-GST-1 specific IgG 
antibody levels up to one month after the third set of vaccinations indicates that subjects in this 
hookworm-endemic area of Africa develop a humoral immune response to vaccination after the 
second vaccination that was boosted upon vaccination at 6 months after the first set of 
vaccinations (Figure 5).
Figure 5: Geometric mean anti-Na-GST-1 IgG antibody units in healthy Gabonese 
adults vaccinated with Na-GST-1/Alhydrogel® plus AP 10-701 and co-administered 
with Na-APR-1 (M74)/Alhydrogel® at 0, 1, and 6 months.
Vertical bars are 95% confidence intervals
In the second currently ongoing Phase 1 trial, 60 healthy adults are being vaccinated with the 
Na-APR-1 (M74)/Alhydrogel hookworm vaccine either with or without co-administration of Na-
GST-1/Alhydrogel in a hookworm endemic area of Brazil (NCT02476773). This study is being 
conducted with funding from a U01 grant from the National Institute of Allergy and Infectious 
Diseases (NIAID Grant #1U01AI116414-01). The aim of this trial is to determine if there is any 
immunological interference when the two hookworm vaccine antigens are co-administered to 
subjects chronically exposed to hookworm infection. Vaccinations in this trial were initiated in 
January 2016 and are currently ongoing. No Serious Adverse Events or Adverse Events of 
Special Interest have been reported in this study. Interim results from this clinical trial indicate 
that co-administration of the two vaccines is well tolerated; mild to moderate injection site pain 
and tenderness were the most common vaccine-related adverse events. 
Na-GST-1/Alhydrogel® administered with or without CpG 10104
The Na-GST-1/Alhydrogel® hookworm vaccine is currently being tested with or without CpG 
10104 in one ongoing Phase 1 clinical trial in humans in the United States (n=24). To date, no 
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IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
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significant reactogenicity or safety issues have been observed in this trial. In study SVI-GST-03 
in healthy, hookworm-naïve American adults conducted in Washington, DC, 24 volunteers 
received three vaccinations with up to 100 µg Na-GST-1/Alhydrogel® administered with or 
without 500 µg CpG 10104 (NCT02143518). Vaccinations were completed in October 2015 and 
extended follow-up is ongoing. In this study, the most common reported adverse events have 
been mild to moderate injection site pain and tenderness; other common adverse events have 
included mild to moderate headache, nausea, myalgia and arthralgia. Immunogenicity 
conducted on serum samples collected up to and including 1 month following the final 
vaccination demonstrates that the addition of CpG 10104 to Na-GST-1/Alhydrogel® significantly 
improved antigen-specific IgG antibody responses over those induced by Na-GST-
1/Alhydrogel® alone (Figure 6).
Figure 6: Anti-Na-GST-1 IgG antibody responses as measured by indirect ELISA in 
hookworm-naïve adults vaccinated with Na-GST-1/Alhydrogel® with or without co-
administration of CpG 10104. AU = arbitrary antibody units; V=vaccination.
2.1.7 Clinical Experience with AP 10-701
In the clinical trial described in this protocol, Na-GST-1/Alhydrogel® will be tested in combination 
with the Toll-like Receptor 4 (TLR4) agonist, Gluco-Pyranosylphospho-Lipid A in Aqueous 
Formulation (AP 10-701, Infectious Diseases Research Institute [IDRI], Seattle, WA). AP 10-701 
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 (38). 
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 (39-41), leishmaniasis (42), and hepatitis B (43).  In 
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IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
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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 AP 10-701 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 (44, 45).
As mentioned previously, up to 5 µg of AP 10-701 has been administered to over 100 human 
volunteers in combination with both Na-GST-1/Alhydrogel® and Na-APR-1 (M74)/Alhydrogel®. In 
addition, an oil-in-water emulsion of GLA (GLA-SE) has been used in combination with the 
Fluzone® trivalent killed influenza vaccine in a Phase 1 trial. In this study, doses up to 2.5 µg of 
GLA-SE were safe and well-tolerated and significantly enhanced influenza-specific antibody 
responses (46).
2.1.8 Clinical Experience with CpG 10104
Unmethylated cytosine-guanine dinucleotides (CpGs) are found in bacterial DNA in the 
expected frequency predicted by random usage, whereas their occurrence is suppressed 4-fold 
in vertebrate DNA. In vertebrate DNA CpG motifs are also usually methylated. Bacterial CpG-
DNA motifs are recognized by the human innate immune system via Toll-like Receptor-9 (TLR-
9), a pathogen-associated molecular pattern (PAMP) receptor that is expressed, in particular, by 
antigen-presenting dendritic cells. Interactions between CpG-DNA and TLR9 rapidly activate 
antigen-presenting dendritic cells to upregulate co-stimulatory molecules and to produce Th1-
polarizing cytokines such as interleukin-12 and interferon gamma (47). CpG 10104 is a short 
synthetic oligodeoxynucleotide of the following sequence: 5’-TCG TCG TTT CGT CGT TTT 
GTC GTT-3’. 
CpG 10104 has been tested in humans in combination with Na-GST-1/Alhydrogel in one Phase 
1 trial to date, as described above in Section 2.1.6. In addition, ProMune® (CpG 7909), a 
human TLR9 agonist similar to CpG 10104, has been evaluated in clinical trials in combination 
with the BioThrax® anthrax vaccine(48), Engerix-B® hepatitis B virus vaccine(49), AMA1-
C1/Alhydrogel® and BSAM-2/Alhydrogel® candidate malaria vaccines(50, 51), a number of 
therapeutic cancer vaccines, cancer chemotherapies, and other therapeutic interventions.(52) 
No serious adverse events related to ProMune® have been reported. Phase 3 evaluation of the 
HEPLISAV® Hepatitis B vaccine formulated with CpG 1018 ISS (a different CpG sequence than 
will be tested in the study described in this protocol) has also been completed, with results 
indicating improved immunogenicity as compared to currently available hepatitis B vaccines.(53) 
The US FDA has requested additional safety information with trials of sufficient size to evaluate 
rare autoimmune events following administration of HEPLISAV®.
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IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
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2.1.9 Clinical Experience with Controlled Human Hookworm Infection 
(CHHI)
A total of nine experimental human hookworm infection studies have been reported in the 
literature and two more (EudraCT 2008-005008-24 and NCT01940757) are currently underway 
in the United Kingdom and Washington, DC, respectively (54-61). The completed studies were 
conducted to investigate the natural history of hookworm infection in humans but also to test the 
therapeutic efficacy of experimental infection on various inflammatory and allergic diseases 
such as celiac disease, inflammatory bowel disease and allergic rhinoconjunctivitis. The 
important points regarding these previous hookworm challenge studies are that: a) experimental 
infections were overall well-tolerated, with minimal significant adverse events; b) hookworm 
infection is curable with well-tolerated licensed medications; and, c) the time to patency (as 
determined by detection of egg in feces) is predictable. These studies, conducted under 
approvals from ethical review committees in the US, Australia and the UK, have enrolled a total 
of over 100 adults who have been challenged with between 10-100 infectious N. americanus 
larvae.  
In the previous studies, the time until hookworm eggs were detectable by microscopic 
examination of feces was consistently between 4 and 8 weeks after challenge.  All experimental 
infections were completely cured by administration of a benzimidazole anthelminthic drug (either 
albendazole or mebendazole) orally. The most common adverse effects of challenge infection in 
these studies included transient skin rash at the site of larval application and gastrointestinal 
discomfort beginning approximately 4 to 6 weeks post-challenge that was characterized by 
flatulence, abdominal pain, nausea, vomiting, and diarrhea. 
2.1.9.1
Experience with CHHI at George Washington University
A Phase 1 clinical trial of CHHI is currently underway at George Washington University (GWU) 
in Washington, DC, the same study site as will be used for the study described in this clinical 
protocol. This feasibility study (NCT01940757) is being conducted by Dr. David Diemert serving 
as study Principal Investigator. In it, 20 volunteers between the ages of 18 and 45 years who 
have no history of infection or exposure to hookworm were enrolled into 2 cohorts in a 
staggered fashion. In the first cohort volunteers received 25 N. americanus larvae whereas in 
the second cohort they received 50 larvae. All subjects (except 2 who were enrolled into a donor 
sub-study, see below) were cured of their infections by administering albendazole between 12 
and 18 weeks post-infection.
In this study, adverse events have been mostly related to papulovesicular rash and pruritus at 
the site of larval application on the skin, and mild-to-moderate gastrointestinal symptoms most 
likely related to development of adult hookworms in the gastrointestinal tract. The skin reactions 
occurred immediately after application of larvae, have been graded by the study subjects as 
mild or moderate in most cases, and these have in some cases lasted for up to one month, 
sometimes with residual hyperpigmentation that gradually fades over time. Gastrointestinal 
symptoms consisting principally of mild to moderate abdominal bloating, flatulence and episodic 
nausea begin to occur approximately 4-6 weeks after infection. Skin reactions were more 
common in the 50 L3 cohort compared to the 25 L3 cohort, although there were no significant 
differences between the cohorts in incidence or severity of gastrointestinal complaints. 
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
13
In the first cohort (25 L3), 3 of 10 subjects developed hookworm infections with eggs detectable 
in fecal samples by microscopy and PCR, whereas in the second (50 L3) cohort, 9 of 10 did. 
Mean egg counts, as estimated by the McMaster Method using all collected fecal samples, 
varied from 0 to 33 eggs per gram (epg) of feces in the 25 L3 cohort and from 0 to 133 epg in 
the 50 L3 cohort. Given the high rate of infection in the 50 L3 cohort, this will be the dose used 
in the Phase 2 clinical trial described in this protocol.   
The larvae that were used in this initial study at GWU were obtained from the University of 
Nottingham. Two of the study subjects in the 50 L3 group have enrolled into a sub-study in 
which they remain untreated and are followed monthly to provide fecal samples that can be 
used to harvest hookworm eggs (donors). The same production method used at the University 
of Nottingham has been instituted at GWU to produce N. americanus larvae from these 
hookworm eggs that can then be used to infect future human subjects. Therefore, the larvae 
that will be used to experimentally infect study volunteers in the proposed Phase 2 trial at GWU 
will be supplied by the Department of MITM at GWU. The Baylor College of Medicine (BCM) 
currently holds an IND to the US FDA for the N. americanus hookworm larvae (IND#015752) to 
permit their use in the U01 clinical trial described herein, in which healthy hookworm-naïve 
volunteers are vaccinated with the Na-GST-1/Alhydrogel® hookworm vaccine and then 
challenged with an experimental hookworm infection.  
2.2
Rationale
2.2.1 Rationale for the Study
A product that combines Na-GST-1 shows promise as an effective hookworm vaccine because 
vaccination of laboratory animals with recombinant Na-GST-1 results in significant protection 
from challenge infections (29, 37). Therefore, vaccination of humans with recombinant Na-GST-
1 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 candidate antigen in healthy adult volunteers from hookworm endemic 
areas (rural Brazil and Gabon) and non-endemic areas (urban Brazil and USA). As of May 1, 
2017, regulatory sponsorship of these vaccine candidates has been transferred to the Baylor 
College of Medicine (Houston, TX). These trials have tested three different formulations of Na-
GST-1: adsorbed on Alhydrogel®, plus the addition of two different TLR agonist 
immunostimulants to the Alhydrogel® formulation, AP 10-701 and CpG 10104.  These Phase 1 
trials have demonstrated that all three formulations are well tolerated, have not resulted in 
significant adverse events, and induce antigen-specific IgG antibodies.  
Since there are no known immune correlates of protection against natural hookworm infection, it 
is unclear how the immunogenicity observed in the Phase 1 trials of Na-GST-1 will translate into 
efficacy. Following the traditional pediatric vaccine clinical development pathway, the next steps 
would be to conduct age de-escalation Phase 1 trials into children, followed by large pediatric 
Phase 2 and 3 field trials. Given the transmission dynamics of hookworm even in highly 
endemic areas, it is estimated that it would take at least two years and require over 1000 
children to be enrolled into a Phase 2 proof-of-concept trial of a single vaccine candidate in 
which the main endpoint would be mean fecal egg count. Given the length of time, the effort, 
and the cost involved, it would not be feasible to conduct multiple such Phase 2 trials of the 
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
14
different vaccine formulations that have progressed past the Phase 1 stage. Therefore, it is 
critical that a new paradigm is developed in which an estimate of efficacy can be made early in 
development so that different candidate formulations can be compared and the most promising 
selected for advancement into pediatric testing. 
In addition to use of the CHHI model in this trial to test proof-of-efficacy of various formulations 
of the Na-GST-1 vaccine candidate, we will utilize several state-of-the-art immune-profiling 
technologies (see Section 8.2) to characterize and compare the immune responses to Na-GST-
1 induced by the different adjuvant preparations and immunostimulants (e.g., AP 10-701 and 
CpG 10104) and how these responses relate to the impact on CHHI. In particular, we will 
compare conventional indirect enzyme-linked immunosorbent assay (ELISA) techniques with 
cutting-edge antibody (Surface Plasmon Resonance or SPR), innate immunity and memory B 
cell (MDB) techniques that offer a more comprehensive profiling of the humoral immune 
response when administering vaccine antigens using a “systems vaccinology” approach. This 
will enable us to identify immune variables that are associated with, and can discriminate 
between, the different immunization groups. The introduction of these novel assays and 
systems analysis methodology 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 Na-GST-1 Doses and Dose Schedule to be Studied 
For the study proposed in this clinical protocol, each subject will be vaccinated three times, on 
Days 0, 56, and 112, by intramuscular injection in the deltoid muscle. 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, and it is the schedule that 
has been tested in all but one of the Phase 1 trials of Na-GST-1, to date. 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. The doses of Na-GST-1, AP 10-701 and CpG 10104 are the maximum doses that have 
been tested in the human clinical trials conducted to date, as described above. This is justified 
since these doses were all well tolerated and there appears to be a dose effect for Na-GST-1 in 
terms of immunogenicity.
2.2.3 Rationale for Controlled Human Hookworm Infection Model 
(CHHI)
There have been several attempts to create permissive animal models for hookworm infection, 
but without much success (62). The two major laboratory animal models for studying hookworm 
infection are A. caninum infections in dogs, which closely resembles human hookworm 
infection, and A. ceylanicum and N. americanus in the golden hamster (Mesocricetus auratus) 
(63, 64). While both are better than other animal models, neither canines nor hamsters 
adequately reproduce the endpoints of human hookworm disease--specifically, including host 
eggs counts and adult worm recovery. Hence, an experimental hookworm infection model is 
being developed to provide early proof-of-concept that a hookworm vaccine targeting the 
blood-feeding pathway of adult hookworms is feasible and efficacious. This model consists of 
vaccinating healthy, hookworm-naïve adults with candidate hookworm vaccine formulations, 
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
15
followed by challenging them with third stage infective N. americanus larvae (L3) to assess the 
effect of vaccination on infection. Such a model will provide an indication of potential efficacy of 
the vaccine antigens under development, prior to age de-escalation studies into children as 
described above.  
A number of human challenge models have been established to evaluate investigational clinical 
products under development. Among these, thousands of volunteers around the world have 
received experimental dengue, malaria (65), influenza (66), enterotoxigenic E. coli (67), and 
Shigella infections (68), to name a few. Model development includes identification of the optimal 
dose and route of administration of an appropriate challenge strain or challenge organism, 
which results in safe but measurable infection, mirroring natural infection as closely as possible. 
Through the development and use of these models, preliminary assessment of vaccine or drug 
efficacy against targeted bacteria, viruses, and parasites can be obtained earlier in clinical 
development, for more rapid evaluation of go/no-go criteria, and better deployment of resources 
toward the most promising vaccine or drug candidates. Recent vaccine candidates evaluated 
via human challenge models include the RTS,S Plasmodium falciparum malaria vaccine and the  
influenza vaccine (66, 69).
The controlled human hookworm infection (CHHI) model is being developed as a tool to assess 
the potential efficacy of novel hookworm vaccine candidates early in their clinical development.  
The prior studies described above demonstrate that experimental infection with N. americanus 
can be induced in a safe and controlled manner, with predictable and manageable adverse 
effects. N. americanus is an easily curable infection using readily available licensed medications 
that result in rapid resolution of symptoms should they occur and be intolerable to study 
participants.  
2.2.4 Inclusion of Placebo
Placebo recipients are included in order to maintain blinding, provide safety reference data, 
provide controls for the immunogenicity assays, and to confirm the infectivity of the N. 
americanus Larval Inoculum (i.e., infectivity controls) during the CHHI stage of the trial. 
2.2.5 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, Na-GST-1 will 
be tested in healthy, hookworm-naïve adults living in area where hookworm is not endemic, to 
provide proof-of-efficacy before large-scale, long-duration and expensive pediatric Phase 2 and 
3 field trials are undertaken in endemic areas. 
The decision to proceed to Phase 2 and 3 testing in children will be taken after the Sponsor has 
reviewed the final clinical study report of the study described in this protocol.  Phase 1 testing of 
Na-GST-1/Alhydrogel is currently planned to start in the third quarter of 2016 in children aged 5-
10 years living in a hookworm endemic area of Gabon. After safety and immunogenicity of this 
product is shown in children, and if the results of the Phase 2 trial described in this protocol 
indicate efficacy of at least one of the Na-GST-1 formulations, further and more extensive 
testing will be pursued, ultimately culminating in Phase 2 and 3 trials in this target population in 
which protection against natural infection will be the primary endpoint. 
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
16
In the first pediatric Phase 2 trial, 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 Risk
Risks to subjects are those associated with venipuncture, intramuscular injection of the Na-
GST-1 vaccine formulations, possible reactions to the vaccine formulations, possible reactions 
to CHHI, possible reactions to albendazole, and breach of confidentiality. 
2.3.1.1
Risks of Venipuncture
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.  
2.3.1.2
Risks of Vaccination
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.  Thus, 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. 
Transient, clinically insignificant decreases in white blood cell counts and platelet counts have 
been observed in the two weeks following receipt of other CpG ODN adjuvant preparations.  
CpG 10104 is an immune activator, and has the potential to overcome the normal tolerance of 
the immune system for self antigens.  In a number of murine models, administration of CpG 
ODN has been associated with development of certain autoimmune phenomenon.  However, 
the relevance of this finding to humans is unclear because while TLR9 receptors are widely 
distributed among immune cells in mice, they appear to be restricted to B cells and 
plasmacytoid dendritic cells in humans.  No subject in any study to date with CpG 7909 
oligonucleotides, a closely related CpG sequence to CpG 10104, has developed signs or 
symptoms of autoimmune disease.
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
17
In animal studies, CpG ODN given by themselves only rarely induce the production of anti-
double-stranded DNA antibodies. However, if the CpG 7909 ODN is administered as a complex 
together with a foreign protein or other antigen that can elicit T cell help, then it is likely that 
antibodies against the CpG 7909 ODN will be produced, and these may cross-react with 
double-stranded DNA. Vaccination of humans with a CpG 7909 ODN together with an antigen 
capable of binding to the ODN could be expected to result in the production of IgG anti-ODN 
antibodies, which could cross react with self DNA.
This phenomenon has been observed in some clinical studies with CpG ODNs involving use of 
CpG 7909 as an antineoplastic agent, in which study subjects have developed detectable 
antibody to double-stranded DNA. These antibodies have generally declined to baseline after 
cessation of CpG 7909, and have not been associated with other autoimmune phenomenon, 
and their possible clinical significance is unknown. Subjects in the study described in this 
protocol will be closely monitored for laboratory evidence (e.g., anti-double stranded DNA 
antibodies or rheumatoid factor) or clinical signs or symptoms of autoimmunity  
Female subjects will be cautioned of the unknown risk of the Na-GST-1/Alhydrogel® vaccine 
administered with or without AP 10-701 or CpG 10104, and of hookworm infection (and of the 
albendazole used to treat hookworm infection) to the fetus.  A female of childbearing potential, 
unless surgically sterile (i.e., bilateral tubal ligation, bilateral oophorectomy, or complete 
hysterectomy), at least 2 years postmenopausal, practices abstinence, or has a vasectomized 
partner, must practice effective contraception (including oral, intra/transdermal, intravaginal, or 
implanted hormonal contraceptives; intrauterine device (IUD); double barrier methods [condom 
plus foam or spermicide, diaphragm plus foam or spermicide], or the Essure procedure) until 
documented clearance of hookworm infection following treatment with albendazole. Female 
subjects will be counseled by a study team member, or referred to the health provider of their 
choice, for evaluation and institution of an appropriate contraceptive method.
Female subjects will be cautioned of the theoretical risk of albendazole during pregnancy.  Per 
the FDA’s categorization of drug risks to a fetus, albendazole is Category C, indicating that 
animal reproduction studies have shown an adverse effect on the fetus.  However, 
teratogenicity in humans has not been observed, and a study of over 800 women treated with 
albendazole during the second and third trimesters demonstrated no adverse effects (70). Use 
in the first trimester, however, is still not recommended, but potential benefits may warrant use 
of the drug in pregnant women despite potential risks. Albendazole has been shown to cause 
embryotoxicity and skeletal malformations in pregnant rats and rabbits. Female subjects will be 
counseled on the potential risks of albendazole use during pregnancy at each weekly visit.
2.3.1.3
Risks of Experimental Infection with N. americanus Larval Inoculum
Expected adverse effects of being inoculated with N. americanus infective larvae are local skin 
reactions at the site of application of the larvae and gastrointestinal symptoms upon maturation 
of the parasites into adult hookworms in the small intestine of the host.  Reported skin reactions 
include localized maculopular or papulovesicular rash that may be significant and prolonged (up 
to a month) when higher doses (≥50) of larvae are applied. Occasional respiratory symptoms 
may be experienced during the migration of larvae through the lungs, including dry cough, 
shortness of breath, and wheeze. Reported gastrointestinal symptoms include abdominal 
bloating and pain, flatulence, diarrhea, nausea, and vomiting.
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
18
As with any investigational product, there is a theoretical possibility of risks of CHHI about which 
we have no present knowledge.  Subjects will be informed of any such risks should further data 
become available during the course of this or other clinical studies. Risks associated with 
chronic hookworm infection include iron deficiency, iron-deficiency anemia (associated with 
fatigue and malaise) and chronic gastrointestinal symptoms including flatulence, abdominal 
bloating, abdominal pain, diarrhea, nausea, and vomiting. However, these usually only occur 
after several months or years of infection, which will not occur during this study.
Volunteers in this study who are experimentally infected with the N. americanus Larval Inoculum 
may develop patent infections and shed hookworm eggs in their feces.  It is extremely unlikely 
that a bystander individual – for example, someone living in the same household as the study 
subject – could become infected with N. americanus due to exposure to these hookworm eggs 
that are shed, for several reasons. First, to become infectious to humans, hookworm eggs must 
hatch to release larvae into the environment. Hatching and development into infective larvae 
require a moist and warm environment (optimally between 23-33˚C) and a certain period of 
time, generally estimated to be at least a week. Second, N. americanus larvae can infect 
humans only by coming into contact with skin; they are not infectious by oral ingestion.  
Therefore, in order for a study subject to transmit their hookworm infection to another person, 
they would have to defecate such that their feces did not get disposed in a toilet and were 
incubated at a warm temperature in adequate moisture for over a week. The incubated feces 
would then have to come into contact with another person’s skin. Since almost all residents of 
the Washington, DC, area have access to flush toilets, the prospect of them depositing their 
feces outside of a toilet and into a warm, moist environment such that the hookworm eggs could 
incubate and hatch is quite far-fetched. However, to prevent this extremely unlikely event from 
occurring, all study subjects will be counseled to practice good hygiene and to always defecate 
in a flush toilet and to dispose of all fecally contaminated matter immediately into a toilet.
2.3.1.4
Risks of Albendazole
Albendazole is a licensed anthelminthic medication that is used ubiquitously throughout the 
world to treat intestinal nematode infections. Expected adverse effects associated with 
administration of three daily doses of albendazole include abdominal pain, nausea, vomiting, 
and diarrhea. 
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, AP 10-701, and CpG 10104 have been produced 
according to current Good Manufacturing Practices (GMP). The N. americanus larvae that will 
be used in this study have been produced according to the procedure outlined in the IND for this 
product. The vaccine products and N. americanus larvae 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.  
Subjects will be monitored throughout their infection for the development of anemia, by means 
of regular complete blood counts. Any subject who develops significant anemia during the study 
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
19
that meets one of the halting rules (see Section 9.5) will be treated with albendazole for the 
infection, according to the procedure outlined in Section 7.7 of the protocol. 
Maintenance of Confidentiality
Subjects 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.
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
20
3
OBJECTIVES
3.1
Study Objectives
Primary:
1.
To compare the impact of vaccination with Na-GST-1/Alhydrogel® delivered with or without 
AP 10-701 or CpG 10104 on controlled human hookworm infection (CHHI) with N. 
americanus larvae in healthy, hookworm-naïve adults, as determined by the presence of 
eggs using a qualified flotation technique.
2.
To evaluate the safety and reactogenicity of Na-GST-1/ Alhydrogel® delivered with or 
without AP 10-701 or CpG 10104 on Days 0, 56 and 112, in healthy, hookworm-naïve 
adults.
Secondary:
1.
To compare the impact of vaccination with Na-GST-1/Alhydrogel® delivered with or without 
AP 10-701 or CpG 10104 on fecal egg counts, as determined by the McMaster method, 
after controlled human hookworm infection (CHHI) with N. americanus larvae in healthy, 
hookworm-naïve adults.
2.
To assess the relationship between antibody responses to Na-GST-1 induced by 
vaccination with Na-GST-1/Alhydrogel® delivered with or without AP 10-701 or CpG 10104 
and responses to CHHI in healthy, hookworm-naïve adults, as determined by the presence 
of eggs using a qualified flotation technique.
3.
To assess the duration of antibody responses to Na-GST-1 induced by vaccination with Na-
GST-1/Alhydrogel® delivered with or without AP 10-701 or CpG 10104. 
Exploratory:
1.
To assess the relationship between the functional capacity of vaccine-induced antibodies 
that neutralize the in vitro activity of native Na-GST-1 enzyme and responses to CHHI in 
healthy, hookworm-naïve adults.
2.
To compare the impact of vaccination with Na-GST-1/Alhydrogel® delivered with or without 
AP 10-701 or CpG 10104 on levels of hookworm DNA as detected by real-time PCR in 
healthy, hookworm-naïve adults challenged with CHHI, as determined by the presence of 
eggs using a qualified flotation technique.
3.
To assess the impact of vaccination with Na-GST-1/Alhydrogel® delivered with or without AP 
10-701 or CpG 10104 on the affinity of antibody-antigen interactions, and how affinity relates 
to responses to CHHI in healthy, hookworm-naïve adults, as determined by the presence of 
eggs using a qualified flotation technique.
4.
To assess the relationship between Na-GST-1 specific memory B cells induced by 
vaccination with Na-GST-1/Alhydrogel® delivered with or without AP 10-701 or CpG 10104 
and responses to CHHI in healthy, hookworm-naïve adults, as determined by the presence 
of eggs using a qualified flotation technique. 
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
21
5.
To assess the relationship between innate immune responses to Na-GST-1/Alhydrogel® 
delivered with or without AP 10-701 or CpG 10104 and responses to CHHI in healthy, 
hookworm-naïve adults.
3.2
Study Outcome Measures
3.2.1 Primary Outcome Measures
Efficacy
The following parameters will be evaluated for each formulation of Na-GST-1, in comparison to 
the saline placebo control:
1.
Proportion of subjects with detectable hookworm eggs, at any time point, in fecal 
samples, as determined by microscopy using the qualified saline flotation technique.
Safety
The following parameters will be evaluated for each formulation of Na-GST-1, in comparison to 
the saline placebo control:
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 solicited adverse events, graded by severity, on the day of CHHI through 
study Day 280.
3.
Frequency of study vaccine-related Serious Adverse Events from the time of the first 
study vaccination through approximately 10 months after the last study vaccination.
4.
Frequency of clinical safety laboratory adverse events. 
5.
Frequency of unsolicited adverse events, graded by severity, from the time of each study 
vaccination through approximately 1 month after each study vaccination; and from the 
time of CHHI through treatment with albendazole (Day 297). 
6.
Frequency of new-onset chronic medical conditions through approximately 10 months 
after the third study vaccination.
7.
Frequency of Adverse Events of Special Interest through approximately 10 months after 
the third study vaccination.
3.2.2 Secondary Outcome Measures
The following parameters will be evaluated for each formulation of Na-GST-1, in comparison to 
the saline placebo control:
Efficacy
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
22
1.
Fecal egg counts as determined by microscopy using the McMaster method, during 
Weeks 5 through 20 post-CHHI.
Immunogenicity
2.
The anti-Na-GST-1 IgG antibody response, by a qualified indirect enzyme-linked 
immunosorbent assay (ELISA) at approximately 14 days after each vaccination, and 
approximately 1, 2, 4, 6, 7, 8 and 10 months after the third dose.
3.2.3 Exploratory Outcome Measures
1.
The functional capacity of vaccine-induced antibodies to neutralize the in vitro activity of 
the native Na-GST-1 enzyme. 
2.
Levels of N. americanus DNA in fecal samples, as measured by real-time PCR, during 
Weeks 5 through 20 post-CHHI.
3.
The affinity of the antibody interactions with recombinant Na-GST-1 antigen at 
approximately 14 days after each vaccination, and approximately 1, 2, 4, 6, 7, 8 and 10 
months after the third dose.
4.
The production of memory B cells specific for Na-GST-1 on days of vaccination; 
approximately 7 and 14 days following each vaccination; on day of CHHI; and then 7 
and 14 days, and 5, 7, 13, and 20 weeks post-CHHI. 
5.
Innate immune responses on days of vaccination approximately 7 and 14 days following 
each vaccination; on day of CHHI; and then 7 and 14 days, and 5, 7, 13, and 20 weeks 
post-CHHI. 
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
23
4
STUDY DESIGN
The study will be a randomized, double blind, placebo-controlled Phase 2 clinical trial in healthy 
hookworm-naïve adult volunteers. This study is designed to evaluate the efficacy, safety, 
reactogenicity, and immunogenicity of Na-GST-1/Alhydrogel® administered with or without the 
point-of-injection addition of the immunostimulants AP 10-701 or CpG 10104. To assess the 
impact of vaccination with these different vaccine formulations on infection, subjects will be 
challenged with 50 infectious N. americanus larvae by CHHI 4 weeks after the 3rd vaccination. 
Safety parameters will be monitored throughout the study.
Volunteers will be invited to participate in the study by means of verbal, email, and listserve 
announcements and poster, newspaper, and online advertisements (including ResearchMatch). 
After providing written informed consent, volunteers will undergo eligibility screening, including a 
complete medical history; physical examination; hematology testing; liver and renal function 
testing; anti-double stranded DNA (anti-dsDNA) and rheumatoid factor (Rf) testing; Human 
Immunodeficiency Virus (HIV), Hepatitis B, and C testing; fecal occult blood testing; fecal 
examination for ova and parasites; and, urinalysis (for protein and glucose).  Urine pregnancy 
testing will be performed on all female volunteers. All clinically significant abnormalities will be 
reviewed with volunteers and they will be referred for follow-up care if appropriate. After 
screening, those volunteers determined to be eligible, based on the inclusion and exclusion 
criteria described in Section 5 in this protocol, will be invited to participate in the study.  No 
exemptions from the inclusion or exclusion criteria will be granted on inclusion/exclusion. 
In the study, 48 volunteers will be enrolled and randomized into 4 groups. Groups will not be 
enrolled separately; rather, eligible volunteers will be randomly assigned to a group upon 
enrollment in a double-blind fashion (i.e., neither the subject nor the investigators will know to 
which group the subject has been assigned). In the first group, volunteers will receive 100µg 
Na-GST-1/Alhydrogel®. In the second group, volunteers will receive either 100µg Na-GST-
1/Alhydrogel® co-administered with 500µg CpG 10104. In the third group, volunteers will receive 
either 100µg Na-GST-1/Alhydrogel® co-administered with 5µg AP 10-701. In the fourth group, 
volunteers will receive sterile saline.  
As with other aluminum hydroxide-adsorbed vaccines, hypersensitivity reactions would be 
expected to occur within the first 24 hours after vaccination, 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 be assessed on Days 3, 7, 14 
and 28 following each vaccination either in the study clinic or by telephone. See Appendix A for 
a detailed description of the scheduled clinical and laboratory evaluations.
Subjects will be challenged by CHHI with 50 infectious N. americanus larvae 4 weeks after 3rd 
vaccination. After CHHI, subjects will be observed for immediate reactions following the 
application of larvae for at least 1 hour. Fecal samples will be collected at least every other 
week starting 5 weeks post-CHHI until 2 weeks following treatment with albendazole at 20 
weeks post-challenge to cure infections. 
Subjects will be followed until 10 months after their final vaccination, for a total of 14 months of 
study participation. See Appendix A for a detailed description of the scheduled clinical and 
laboratory evaluations during the Vaccination and CHHI phases of the trial.IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
24
The Safety Monitoring Committee (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 if any of the halting criteria listed in Section 9.5 are met or, in the clinical judgment of 
the SMC, Independent Safety Monitor, and/or DMID Medical Monitor, continuation would pose 
an unacceptable safety risk to the subjects.
4.1
Substudies (if applicable)
Not Applicable.
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
25
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 48 subjects will be enrolled. The study population will be enrolled from the 
metropolitan Washington, DC area.
Volunteers agreeing to participate will first provide written informed consent. Signing the 
informed consent form will provide consent for both screening procedures and study procedures 
following enrollment. 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. Volunteers will not be 
considered enrolled in the study until they have received their first dose of vaccine.
5.1
Subject Inclusion Criteria
1.
Males and non-pregnant females between 18 and 45 years, inclusive.
2.
Good general health as determined by means of the screening procedures1.
 Existing medical diagnoses or conditions (except those in the Subject Exclusion 
Criteria below) must be deemed as stable chronic medical conditions.  A stable 
chronic medical condition is defined as no change in prescription medication, dose, 
or frequency of medication in the last 90 days and health outcomes of the specific 
disease are considered to be within acceptable limits in the last 180 days. Any 
change due to change of health care provider, or that is done for financial reasons, 
as long as in the same class of medication, will not be considered a violation of this 
inclusion criterion. Any change in prescription medication due to improvement of a 
disease outcome, as determined by the Principal Investigator or a co-investigator, 
will not be considered a violation of this inclusion criterion. Individuals may be taking 
chronic or as needed medications if, in the opinion of the Principal Investigator or co-
investigator, they pose no additional risk to safety or assessment of reactogenicity 
and immunogenicity. Topical, nasal, and inhaled medications (with the exception of 
corticosteroids as outlined in the Subjects Exclusion Criteria [Section 5.2]), vitamins, 
and contraceptives are permitted.
3.
Available for the duration of individual subject study participation (14 months).
4.
Willingness to participate in the study as evidenced by signing the informed consent 
document.
5.
Able to understand and comply with planned study procedures.
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
26
5.2
Subject Exclusion Criteria
1.
Pregnancy as determined by a positive urine human choriogonadotropin (hCG) test 
(if female).
2.
Subject unwilling to use effective contraception for a minimum of 30 days prior to 
vaccination and up until documentation of clearance of hookworm infection post-
CHHI (if female and not surgically sterile, abstinent from intercourse with a male 
partner, in a monogamous relationship with a vasectomized partner, at least 2 years 
post-menopausal, or determined otherwise by medical evaluation to be sterile).
3.
Currently lactating and breast-feeding or plans to breastfeed at any given time from 
the first study vaccination until clearance of hookworm infection post-CHHI (if 
female).
4.
Evidence of clinically significant neurologic, cardiac, pulmonary, hepatic, 
rheumatologic, autoimmune, gastrointestinal, diabetes, or renal disease by history, 
physical examination, and/or laboratory studies.2
2Includes the conditions and diagnoses defined as AESIs in Section 9.3.4.
5.
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).
6.
Known or suspected immunodeficiency or immunosuppression as a result of an 
underlying illness or treatment.3
3Causes for immunosuppression may include, but are not limited to, poorly-controlled 
diabetes mellitus, chronic liver disease, renal insufficiency, active neoplastic disease 
or a history of hematologic malignancy, connective tissue disease, organ transplant.
7.
Laboratory evidence of liver disease (alanine aminotransferase [ALT] greater than 
1.25-times the upper reference limit).
8.
Laboratory evidence of renal disease (serum creatinine greater than 1.25-times the 
upper reference limit, or urine dipstick testing positive for glucose or more than trace 
protein).
9.
Laboratory evidence of hematologic disease (hemoglobin <11.1 g/dl [females] or 
<12.5 g/dl [males]; absolute leukocyte count <3400/mm3 or >10.8 x 103/mm3; 
absolute eosinophil count >500/mm3; or platelet count <140,000/mm3).
10. 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.
11. Planned participation in another investigational vaccine or drug trial within 30 days of 
starting this study or until the last study visit.4
4This may include other licensed or unlicensed vaccines, drugs, biologics, devices, 
blood products, or medications.
12. Volunteer has had medical, occupational, or family problems as a result of alcohol or 
illicit drug use during the past 24 months.
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
27
13. Positive fecal occult blood test at screening.
14. Infection with a pathogenic intestinal helminth as determined by stool examination for 
ova and parasites at screening. 
15. History of iron deficiency anemia or laboratory evidence of iron deficiency (serum 
ferritin concentration below the lower reference limit). 
16. History of hypoalbuminemia.
17. History of a severe allergic reaction or anaphylaxis.
18. Severe asthma as defined by the need for daily use of corticosteroid and/or long-
acting beta agonist inhalers, or emergency clinic visit or hospitalization within 6 
months of the volunteer’s expected first vaccination in the study.
19. Positive test for hepatitis B surface antigen (HBsAg).
20. Positive confirmatory test for HIV infection.
21. Positive confirmatory test for hepatitis C virus (HCV) infection.
22. Using or intends to continue using oral or parenteral corticosteroids, high-dose 
inhaled corticosteroids (>800 μg/day of beclomethasone dipropionate or equivalent) 
or other immunosuppressive or cytotoxic drugs within 30 days of the volunteer’s 
expected first vaccination in this study or planned use during the study.
23. Receipt of a live vaccine within 4 weeks or a killed vaccine within 2 weeks prior to the 
volunteer’s expected first vaccination in the study.
24. Receipt of immunoglobin or other blood products (with exception of Rho D 
immunoglobulin) within 90 days of the planned first study vaccination. 
25. Known allergy to albendazole, amphotericin B or gentamicin.
26. History of previous infection with hookworm or continuous residence for more than 6 
months in a community where hookworm is endemic.
27. Current or past scars, tattoos, or other disruptions of skin integrity at the intended 
site of larval application.
28. Previous receipt of the Na-GST-1/Alhydrogel® hookworm vaccine.
29. History of a surgical splenectomy.
30. Pre-existing autoimmune or antibody-mediated diseases including but not limited to: 
systemic lupus erythematosis, rheumatoid arthritis, multiple sclerosis, Sjogren’s 
syndrome, autoimmune thrombocytopenia; or laboratory evidence of possible 
autoimmune disease determined by a positive anti-dsDNA titer, positive rheumatoid 
factor, and/or proteinuria (greater than trace protein on urine dipstick testing).
31. Two or more first-degree family members with autoimmune or antibody-mediated 
disease including but not limited to those listed in exclusion criterion #30.
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). After undergoing a clinical interview and directed 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. 
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
28
Within each group, randomization will be done through use of a randomization code, furnished 
to the study vaccine manager by the study statistician. 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 in the GW MFA Investigational Drug Service 
pharmacy or the office of the study vaccine manager. 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.
Vaccine/placebo assignment will be done by block randomization. That is, for each block of 4 
subjects who are sequentially enrolled into the trial, 1 will be assigned to receive Na-GST-
1/Alhydrogel®, 1 to Na-GST-1/Alhydrogel® plus CpG 10104, 1 to Na-GST-1/Alhydrogel® plus AP 
10-701, and 1 to sterile saline placebo.
5.3.2 Masking Procedures
Due to the double-blind nature of this Phase 2 clinical trial, neither study subjects nor study 
team personnel will know to which group an individual subject has been assigned. Investigators 
and subjects will be blinded to the vaccine/placebo allocation until all subjects have completed 
their final study visit (i.e., Visit #39 at Day 380), the primary (efficacy and safety) and secondary 
IgG immunogenicity outcomes (i.e., anti-Na-GST-1 IgG antibody results by ELISA) have been 
monitored and entered into the database, and the database has been locked for analysis. 
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 
doses of the Na-GST-1 formulations are of different opacity than that of sterile saline (slightly 
turbid vs. clear, respectively), 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.
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.
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
29

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, or the CHHI, may not be administered to a subject if any 
of the following criteria are met:

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 or prior to planned CHHI. Any clinical safety 
laboratory parameter may be re-evaluated only once at the central (clinical) laboratory 
prior to the second or third study vaccination, or CHHI. If the clinical safety laboratory 
value decreases to Grade 2 or less, the subject may receive the second or third study 
vaccination, or CHHI. The second or third study vaccination, or the CHHI, 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 or CHHI and 
the documentation of approval should be filed in the subject’s chart.

Severe or sustained reaction or disability related to a preceding study vaccination.

New onset of illness or condition that meets exclusion criteria.

Subject no longer meets eligibility criteria.
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
30

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.
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 vaccine will be encouraged to remain in 
the study for follow-up safety assessments and collection of venous blood samples for 
immunogenicity testing and fecal samples for parasitological 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.
Subjects who have received N. americanus challenge by CHMI who withdraw or are withdrawn 
before Day 280 post-CHHI and who have not been treated with albendazole for hookworm 
infection must receive early presumptive treatment for hookworm or else agree to continue be 
followed until Day 297 (Visit #37) post-CHHI. Subjects who elect to withdraw from the study will 
not be subjected to additional research portions of the study (research blood collections, fecal 
collections, etc.) although they will be strongly advised to continue with the safety portions (i.e., 
fecal testing for hookworm eggs, safety laboratory tests) of the study. 
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 will be encouraged to remain in 
this study for safety follow-up assessments (at a minimum, by telephone if not in person) 
continuing through approximately study Day 140 for subjects who do not receive CHHI, or study 
Day 189 (approximately 6 weeks post-CHHI) for subjects who receive CHHI. In the latter case, 
volunteers will be encouraged to remain in the study until Day 189 to ensure that treatment with 
albendazole is given after the point where the hookworms would have become adult worms, 
since the medication is not effective against the immature forms of the worm.
If voluntary withdrawal occurs, the subject will be asked for permission to continue scheduled 
safety evaluations and to complete an end-of-study evaluation. If the withdrawal is due to an AE 
or an SAE, appropriate medical care and supervision of the subject should continue until the AE 
or SAE resolves or the subject's condition becomes stable. 
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
31
If it is felt that inclusion of the study subject’s data for analysis is compromised, the study 
subject will be terminated from the study, and the relevant study data may not be included in 
analysis. However, all data generated before withdrawal that is not compromised will be 
included. This does not preclude the ethical responsibility of the investigators to ensure the 
safety of the subject and offer curative therapy for hookworm if they have received CHHI. 
If possible, subjects who leave the study area will be traced and visited by clinical investigators 
to collect safety follow-up data. In the case of subjects who fail to appear for a follow-up 
assessment, extensive efforts (i.e., at least three documented phone calls and certified mail) will 
be made to locate or recall them, or at least to determine their health status and provide 
treatment with albendazole if CHHI has been performed. These efforts will be documented in 
the subjects' records. Any subject who does not adhere to the protocol requirements must be 
classified (e.g., non-compliant, AE, lost-to-follow-up, or other) and the classification will be 
recorded on the appropriate Case Report Form. 
Pregnancies occurring in study subjects will be reported on the study Pregnancy Report Form. 
No further study vaccinations or CHHI will be administered to pregnant subjects.  
Subjects who withdraw, or are withdrawn or terminated from the study, or are lost to follow-up 
after being randomized will not be replaced. Subjects who have signed the informed consent 
form and who meet all inclusion/exclusion criteria after the screening visit and procedures will 
be randomized to vaccine formulation/placebo assignment until the study sample size for 
randomized subjects has been met.
5.3.5 Termination of Study 
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 IRB of the George 
Washington University. 
The study Sponsor has the right to terminate this study at any time. Reasons for terminating the 
study may include, but are not limited to, the following: 

Incidence or severity of AEs indicating a potential health hazard 

Data recording is inaccurate or incomplete 

The Investigator has not adhered to the protocol or applicable regulatory guidelines in 
conducting the study
In addition, the study may be discontinued at the discretion of the U.S. FDA or the study site.
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
32
6
STUDY INTERVENTIONS/INVESTIGATIONAL 
PRODUCTS
Investigators will receive the current version of the Investigator’s Brochures for Na-GST-
1/Alhydrogel® (with or without AP 10-701), Na-GST-1/Alhydrogel® (with or without CpG 10104), 
and Necator americanus Larval Inoculum, which comprehensively describe all available 
preclinical and human experience with the experimental vaccine formulations and CHHI. 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
Na-GST-1/Alhydrogel®
Na-GST-1 contains recombinant N. americanus Glutathione-S-Transferase-2 (Na-GST-1), 
which is expressed in modified Pichia pastoris. Na-GST-1 from the human hookworm N. 
americanus is a 24 kDa protein with peroxidase enzymatic activity that catalyzes the 
conjugation of reduced glutathione to a variety of electrophiles, including toxic compounds 
generated as products of blood degradation by the adult hookworm. When recombinant Na-
GST-1 is used as a vaccine, it is hypothesized that it will induce neutralizing antibodies that will 
interfere with parasite blood-feeding and cause parasite death or reduce worm fecundity. The 
three formulations that will be used in this study consist of:
(a) Na-GST-1 adsorbed to Alhydrogel®;
(b) Na-GST-1 adsorbed to Alhydrogel® and administered with the novel immunostimulant 
AP 10-701, or Gluco-Pyranosylphospho-Lipid A Aqueous Formulation (GLA-AF), a 
synthetic lipid A molecule that is a Toll-Like Receptor (TLR)-4 agonist;
(c) Na-GST-1 adsorbed to Alhydrogel® and administered with the novel immunostimulant 
CPG 10104, a synthetic oligodeoxynucleotide molecule that is a Toll-Like Receptor 
(TLR)-9 agonist.
AP 10-701
AP 10-701, previously referred to as Gluco-Pyranosylphospho-Lipid A Aqueous Formulation 
(GLA-AF), is a TLR-4 agonist. Point-of-injection formulations with this immunostimulant will be 
prepared immediately prior to vaccination by adding an appropriate volume of AP 10-701 to Na-
GST-1/Alhydrogel® and withdrawing an appropriate volume to administer the desired amount of 
Na-GST-1/Alhydrogel® plus 5mcg AP 10-701.
CPG 10104
CPG 10104 is a TLR-9 agonist. Point-of-injection formulations with this immunostimulant will be 
prepared immediately prior to vaccination by adding an appropriate volume of CPG 10104 to 
Na-GST-1/Alhydrogel® and withdrawing an appropriate volume to administer the desired amount 
of Na-GST-1/Alhydrogel® plus 500mcg CPG 10104.
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
33
Normal Saline, 0.9% Sodium Chloride
Normal saline will be used as the placebo. It consists of a sterile 0.9% physiologic solution of 
sodium chloride that has no pharmacologic activity.
6.1.1 Acquisition
Na-GST-1/Alhydrogel® for this study will be supplied to the study site by the Baylor College of 
Medicine. Vials of CpG 10104 and AP 10-701 will be purchased from the Infectious Diseases 
Research Institute (IDRI).  
Normal saline (0.9% sodium chloride injection, USP) and albendazole will be purchased 
commercially by the GWU Investigational Drug Service.
The Necator americanus Larval Inoculum that will be used for CHHI in this study will be supplied 
by the Department of Microbiology, Immunology, and Tropical Medicine of GWU under a current 
Investigational New Drug application to the US FDA (IND#015752).  Briefly, N. americanus eggs 
will be obtained from the feces of chronically infected human volunteers who are subjects in a 
GWU IRB-approved clinical trial of CHHI (NCT#01940757). These individuals are regularly 
tested to ensure that they are negative for HIV, HBV, and HCV. Fecal material is processed 
following a qualified standard procedure, and after hatching from eggs, the N. americanus 
larvae are stored in the dark at ambient temperature (19-25oC) until use. Controls for the 
manufacturing process are tests for viability (motility), species identification, and microbial 
bioburden of the larvae. Donors are tested for HIV, HBV, and HCV prior to release of each new 
batch of larvae and release will not be accepted if any of these tests are positive.  
Na-GST-1/Alhydrogel®, CpG 10104, AP 10-701, and the sterile saline placebo will be 
transported to the Investigational Drug Service at GWU MFA 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-GST-1/Alhydrogel®
Na-GST-1/Alhydrogel® is supplied as a sterile milky-white suspension (when shaken slightly).  
Each 2.0 ml vial contains 1.35 ml of a 0.1 mg/ml suspension of Na-GST-1 adsorbed to 0.8 
mg/ml of Alhydrogel® in a buffer consisting of 10% glucose and 10 mM imidazole, pH 7.4.  
Glucose acts as an excipient and imidazole as the buffer based on evidence that these 
components specifically 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 Walter Reed Army Institute of Research.
6.1.2.2
CpG 10104
CpG 10104 will be supplied as a clear to slightly hazy, colorless aqueous solution at a 
concentration of 2mg/mL, in multi-dose vials containing 0.8 ml each. Appropriate volumes of 
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
34
CpG 10104 will be withdrawn and added to a vial containing Na-GST-1/Alhydrogel® (as 
described in the study Pharmacy Manual). The mixture must be administered not more than 4 
hours after mixing the CpG 10104 with Na-GST-1/Alhydrogel®. CpG 10104 has been 
manufactured according to cGMP by the Infectious Diseases Research Institute (IDRI).  
6.1.2.3
Glucopyranosyl-Lipid A Aqueous Formulation (AP 10-701)
AP 10-701 will be supplied as a 0.5 mL clear to slightly hazy, colorless aqueous solution in 
multi-dose vials containing 25 µg/mL of GLA without preservative. Appropriate volumes of AP 
10-701 will be withdrawn from the multi-dose vials using a syringe and added to a vial 
containing Na-GST-1/Alhydrogel® (described in the Investigator’s Brochure and study Pharmacy 
Manual). The mixture must be administered not more than 24 hours after mixing the AP 10-701 
with Na-GST-1/Alhydrogel®. AP 10-701 has been manufactured according to cGMP by the 
Infectious Diseases Research Institute (IDRI).  
6.1.2.4
Placebo (Normal Saline, 0.9% Sodium Chloride, USP)
Normal saline (0.9% sodium chloride injection, USP) is a sterile, nonpyrogenic, isotonic solution.  
Each mL contains sodium chloride 9 mg and contains no preservatives, bacteriostatic, 
antimicrobial agent, or added buffer. The solution may contain hydrochloric acid and/or sodium 
hydroxide for pH adjustment (pH 5.3, range 4.5-7.0).
6.1.2.5
N. americanus Larval Inoculum
N. americanus larval inoculum will be supplied as a clear, colorless suspension in individual 
cryotube vials containing 50 infectious N. americanus larvae each in sterile water. 
6.1.3 Product Storage and Stability
The vaccine, CpG, and AP 10-701 will be stored at the site in a refrigerator at 2oC to 8oC until 
just prior to administration and will not be frozen. Normal saline placebo will be stored at the site 
at room temperature (20oC to 25oC). All study product vials will be stored in the upright position.
N. americanus Larval Inoculum will be maintained at 19oC to 25oC until just prior to 
administration.
6.2
Dosage, Preparation and Administration of Study 
Intervention/Investigational Product
Doses of Na-GST-1/Alhydrogel® administered without AP 10-701 or CpG 10104 will be 
prepared the site Investigational Pharmacist by withdrawing appropriate volumes into syringes 
of appropriate size. Study product doses will be prepared using aseptic technique in a biosafety 
cabinet. The Na-GST-1/Alhydrogel® plus AP 10-701 formulation will be prepared by adding 
appropriate volume of AP 10-701 solution to vials of Na-GST-1/Alhydrogel® within 24 hours of 
vaccination. The Na-GST-1/Alhydrogel® plus CpG formulation will be prepared by adding 
appropriate volume of CpG solution to vials of Na-GST-1/Alhydrogel® within 4 hours of 
vaccination.  Doses of the sterile saline control will be prepared by withdrawing the appropriate 
volume into syringes of appropriate size. 
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
35
Doses of vaccine/placebo will be administered by qualified study personnel in the deltoid muscle 
of the appropriate arm after disinfecting the skin with an alcohol swab and allowing it to dry.  
Individual doses of N. americanus larvae will be prepared at MITM and transported to the study 
site at ambient temperature, immediately prior to application on a study subject’s skin. Doses of 
the N. americanus larval inoculum will be prepared by pipetting the entire contents of the dose 
vial containing 50 motile larvae onto a sterile absorbent pad under a fume hood. The absorbent 
pad will then be directly applied to the subject’s forearm and immobilized there using a 
transparent sterile adhesive dressing for at least one hour before removing and placing the pad 
back into the dose vial for counting of residual larvae at MITM.
6.3
Modification of Study Intervention/Investigational Product for a 
Subject
There will be no dose modifications. If a subject’s second and/or third dose of study vaccination 
or CHHI 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, or the CHHI, 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 or CHHI 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 vaccine, AP 10-701, CpG 10104 and sterile saline placebo supplies must be received by 
a designated person at the GWU MFA Investigational Drug Service pharmacy, 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®, the sterile saline placebo, CpG10104, and 
AP 10-701) received, the quantities administered to study subjects, and the amounts remaining 
at the conclusion of the study.  
After administration of vaccine or AP 10-701 or CpG 10104 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®, CpG 
10104, and AP 10-701 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.  
Supplies of N. americanus larval inoculum will be prepared and stored at the Department of 
MITM at GWU. Individual doses of larvae will be prepared on the planned day of inoculation and 
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
36
transported to the study clinic for administration to study subjects.  At the study clinic, the larval 
doses must be received by a designated person, handled and stored safely and properly and 
kept in a secure location prior to administration. MITM personnel will be responsible for 
maintaining accurate records of the supplies of the N. americanus larval inoculum, the quantities 
administered to study subjects, and the amounts remaining at the conclusion of the study
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 vaccine, from study Day 140 to 297, 
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.
Medications that might interfere with the evaluation of the investigational vaccine formulations 
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 investigational vaccine, with the exception of the inactivated influenza vaccine 
that should not be administered within 2 weeks of receipt of study vaccine (i.e., 2 weeks prior to 
planned study vaccination and 2 weeks after study vaccination). 
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
37
7
STUDY SCHEDULE
The following sections provide a detailed listing of the procedures and studies to be performed 
in this protocol at designated time points. The total volume of blood (approximately 1091 mL) to 
be collected from each volunteer over 14 months is less than the volume collected when 
donating three units of blood and should not compromise the health of trial subjects.
See Appendix A for the schedule of study procedures.
7.1
Screening 
Volunteers aged 18-45 years, inclusive, will be invited to undergo screening for the study.  
During this initial screening visit, a member of the study team will review the consent form with 
the volunteer. The study team member will explain the study to the volunteer and will clarify all 
of the volunteer’s questions. Volunteers will be encouraged to ask questions. The volunteer may 
either sign the consent form immediately or later after further consideration. 
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.
Elicit a complete medical history, including medication history, and for female subjects, a 
menstrual and contraceptive history and/or history of surgical sterility.
3.
Administer a complete physical examination.
4.
Obtain blood for hematology, biochemistry, anti-dsDNA, rheumatoid factor, serum ferritin, 
and tests for HIV and viral hepatitis (B and C).
5.
Obtain urine for urine dipstick testing for glucose and protein, as well as urine pregnancy 
testing in females.
6.
Counsel females to avoid becoming pregnant during the study.
7.
Obtain feces for fecal occult blood testing and baseline parasitology. 

Volunteers unable to provide a fecal sample during the screening visit may provide a 
sample prior to the planned Day 0 study visit. A fecal sample must be obtained prior to 
determination of eligibility and larval administration. 

Explain procedures for fecal sample collection, storage, and transport to the clinic.
8.
Explain the procedure for at-home fecal sample collection, storage, and transport to the 
clinic on or before Day 0.
Screening steps 2-8 must be performed within 90 days of 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) may be repeated to ensure 
continued eligibility for the study (blood screening tests can be repeated a maximum of one time 
for this reason).  Abnormal screening test results may be repeated to confirm the result, at the 
discretion of the investigator.
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
38
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 (Visit #1: 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, rheumatoid factor, anti-dsDNA, anti-Na-GST-1 
antibody and cellular immunology assays.
5.
For females, obtain a urine sample for pregnancy testing.  Test must be negative to 
proceed; a positive test will exclude the volunteer from the trial.
6.
Record vital signs (blood pressure, oral temperature, and heart rate). Vital signs recorded 
pre-vaccination on Day 0 will be considered as baseline.
7.
Subjects will be enrolled and randomly assigned to a study group by the vaccine manager 
prior to the first study vaccination.
8.
Administer the vaccine in the deltoid muscle. Record the site of injection (i.e., right or left 
arm) on the appropriate data collection form.
9.
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, describe proper use of digital thermometers, injection-site 
reaction measurement tools, and subject symptom diaries. 
10. Record vital signs at the end of the 1-hour post-immunization wait period.
7.3
Follow-up 
Study Day 3 (Visit #2) – Telephone Call
1.
Review interim medical history emphasizing any acute signs or symptoms (including entries 
on the post vaccination symptom memory aid).
2.
Collect concomitant medication information.
Study Day 7 (Visit #3)
1.
Perform basic history and physical exam (including injection site), emphasizing examination 
of any acute complaints.
2.
Record vital signs (oral temperature, pulse, and blood pressure).
3.
Review and collect subject post vaccination symptom memory aid.
4.
Obtain blood for cell-mediated immunity (CMI) assays.
Study Day 14 (Visit #4)
1.
Perform basic history and physical exam (including injection site), emphasizing examination 
of any acute complaints.
2.
Record vital signs (oral temperature, pulse, and blood pressure).
3.
Review and collect subject post vaccination symptom memory aid (if not collected on Visit 
#3/Study Day 7).
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
39
4.
Obtain blood for hematology, biochemistry, rheumatoid factor, anti-dsDNA, anti-Na-GST-1 
antibody and CMI assays.
Study Day 28 (Visit #5) – Telephone Call
1.
Review interim medical history emphasizing any acute signs or symptoms (including entries 
on the post vaccination symptom memory aid).
2.
Collect concomitant medication information.
Study Day 56 (Visit #6: 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, rheumatoid factor, anti-dsDNA, anti-Na-GST-1 
antibody and CMI assays.
3.
For females, obtain a urine sample for pregnancy testing.  Test must be negative to 
proceed; 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.  
During the 1-hour post-immunization wait period, study staff will discuss signs and 
symptoms of potential AEs, describe proper use of digital thermometers, injection-site 
reaction measurement tools, and subject symptom diaries.
7.
Record vital signs at the end of the 1-hour post-immunization wait period.
Study Day 59 (Visit #7) – Telephone Call
1.
Review interim medical history emphasizing any acute signs or symptoms (including entries 
on the post vaccination symptom memory aid).
2.
Collect concomitant medication information.
Study Day 63 (Visit #8)
1.
Perform basic history and physical exam (including injection site), emphasizing examination 
of any acute complaints.
2.
Record vital signs.
3.
Review and collect subject post vaccination symptom memory aid.
4.
Obtain blood for CMI immunology assays.
Study Day 70 (Visit #9)
1.
Perform basic history and physical exam (including injection site), emphasizing examination 
of any acute complaints.
2.
Record vital signs.
3.
Review and collect subject post vaccination symptom memory aid (if not collected on Day 63 
visit).
4.
Obtain blood for hematology, biochemistry, rheumatoid factor, anti-dsDNA, anti-Na-GST-1 
antibody and CMI assays.  
Study Day 84 (Visit #10) – Telephone Call
1.
Review interim medical history emphasizing any acute signs or symptoms (including entries 
on the post vaccination symptom memory aid).
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
40
2.
Collect concomitant medication information.
Study Day 112 (Visit #11: 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, rheumatoid factor, anti-dsDNA, anti-Na-GST-1 
antibody and CMI assays.
3.
For females, obtain a urine sample for pregnancy testing.  Test must be negative to 
proceed; 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.  
During the 1-hour post-immunization wait period, study staff will discuss signs and 
symptoms of potential AEs, describe proper use of digital thermometers, injection-site 
reaction measurement tools, and subject symptom diaries.
7.
Record vital signs at the end of the 1-hour post-immunization wait period.
Study Day 115 (Visit #12) – Telephone Call
1.
Review interim medical history emphasizing any acute signs or symptoms (including entries 
on the post vaccination symptom memory aid).
2.
Collect concomitant medication information.
Study Day 119 (Visit #13)
1.
Perform basic history and physical exam (including injection site), emphasizing examination 
of any acute complaints.
2.
Record vital signs.
3.
Review and collect subject post vaccination symptom memory aid.
4.
Obtain blood for CMI assays.
Study Day 126 (Visit #14)
1.
Perform basic history and physical exam (including injection site), emphasizing examination 
of any acute complaints.
2.
Record vital signs.
3.
Review and collect subject post vaccination symptom memory aid (if not collected during 
Day 119 visit).
4.
Obtain blood for hematology, biochemistry, rheumatoid factor, anti-dsDNA, anti-Na-GST-1 
antibody and cellular immunology assays.  
5.
Obtain fecal sample for parasitological testing.
Study Day 140 (Visit #15: Application of N. americanus Larval Inoculum)
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, antibody and cellular immunology assays.
5.
Obtain a urine sample for pregnancy testing (females only).  Pregnancy test must be 
negative to proceed; a positive test will exclude the volunteer from the receiving the CHHI.
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
41
6.
Record vital signs (blood pressure, oral temperature, and heart rate).
7.
Administer the N. americanus Larval Inoculum.
8.
Observe for at least 1 hour after application of the N. americanus Larval Inoculum to 
evaluate for immediate adverse reactions. During the 1-hour post-application wait period, 
study staff will:

Discuss signs and symptoms of potential Adverse Events (AEs);

Discuss proper use of the digital thermometer, skin reaction measurement tool, and 
subject post-CHHI symptom memory aid, and;

Review procedures for fecal sample collection, storage, and transport to the clinic.
9.
At the end of the observation period, remove absorbent pad used to apply the Larval 
Inoculum and place in a screw-top tube for transport to GWU MITM.
Study Day 143 (Visit #16) – Telephone Call
1.
Review interim medical history emphasizing any acute signs or symptoms (including entries 
on the post vaccination symptom memory aid).
2.
Collect concomitant medication information.
Study Day 147 (Visit #17)
1.
Perform basic history and physical exam (including larval application site), emphasizing 
examination of any acute complaints. 
2.
Record vital signs.
3.
Obtain blood for antibody and cellular assays.
4.
Review subject post-CHHI symptom memory aid.
Study Day 154 (Visit #18)
1.
Perform basic history and physical exam (including larval application site), emphasizing 
examination of any acute complaints.
2.
Record vital signs.
3.
Review and collect post-CHHI symptom memory aid.
4.
Give subject two weekly post-CHHI symptom memory aids. 
5.
Obtain blood for hematology, biochemistry, antibody and cellular assays.
Study Day 175 (Visit #19)
1.
Perform basic history and physical exam (including larval application site), emphasizing 
examination of any acute complaints.
2.
Record vital signs.
3.
Collect prior weeks’ weekly symptom memory aids and give a weekly GI symptom memory 
aid. 
4.
Obtain blood for hematology, antibody and cellular assays.
5.
Obtain fecal sample for parasitological testing. 
Study Day 182 (Visit #20)
1.
Perform basic history and physical exam, emphasizing examination of any acute complaints.
2.
Record vital signs.
3.
Collect prior week’s weekly GI symptom memory aid and give new weekly GI symptom 
memory aid. 
4.
Obtain fecal sample for parasitological testing. 
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
42
Study Day 189 (Visit #21)
1.
Perform basic history and physical exam, emphasizing examination of any acute complaints.
2.
Record vital signs.
3.
Obtain fecal sample for parasitological testing. 
4.
Collect prior week’s weekly GI symptom memory aid and give new weekly GI symptom 
memory aid. 
5.
Obtain blood for hematology, antibody and cellular assays.
Study Day 196 (Visit #22) – Telephone Call
1.
Review interim medical history emphasizing any acute signs or symptoms (including entries 
on the GI symptom memory aid).
2.
Collect concomitant medication information.
Study Day 203 (Visit #23)
1.
Perform basic history and physical exam, emphasizing examination of any acute complaints.
2.
Record vital signs.
3.
Obtain fecal sample for parasitological testing.
4.
Collect prior week’s weekly GI symptom memory aid and give new weekly GI symptom 
memory aid.
5.
Obtain blood for hematology.
Study Day 210 (Visit #24) - Telephone Call
1.
Review interim medical history emphasizing any acute signs or symptoms (including entries 
on the GI symptom memory aid).
2.
Collect concomitant medication information.
Study Day 217 (Visit #25)
1.
Perform basic history and physical exam, emphasizing examination of any acute complaints.
2.
Record vital signs.
3.
Obtain fecal sample for parasitological testing.
4.
Collect prior weeks’ GI symptom memory aids and give two new weekly GI symptom 
memory aids. 
5.
Obtain blood for hematology.
Study Day 224 (Visit #26) - Telephone Call
1.
Review interim medical history emphasizing any acute signs or symptoms (including entries 
on the GI symptom memory aid).
2.
Collect concomitant medication information.
Study Day 231 (Visit #27)
1.
Perform basic history and physical exam, emphasizing examination of any acute complaints.
2.
Record vital signs.
3.
Obtain fecal sample for parasitological testing.
4.
Collect prior weeks’ GI symptom memory aids and give two new weekly GI symptom 
memory aids. 
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
43
5.
Obtain blood for hematology, antibody and cellular assays.
Study Day 238 (Visit #28) - Telephone Call
1.
Review interim medical history emphasizing any acute signs or symptoms (including entries 
on the GI symptom memory aid).
2.
Collect concomitant medication information.
Study Day 245 (Visit #29)
1.
Perform basic history and physical exam, emphasizing examination of any acute complaints.
2.
Record vital signs.
3.
Obtain fecal sample for parasitological testing.
4.
Collect prior weeks’ GI symptom memory aids and give two new weekly GI symptom 
memory aids. 
5.
Obtain blood for hematology.
Study Day 252 (Visit #30) – Telephone Call
1.
Review interim medical history emphasizing any acute signs or symptoms (including entries 
on the GI symptom memory aid).
2.
Collect concomitant medication information.
Study Day 259 (Visit #31)
1.
Perform basic history and physical exam, emphasizing examination of any acute complaints.
2.
Record vital signs.
3.
Obtain fecal sample for parasitological testing.
4.
Collect prior weeks’ GI symptom memory aid and give two new weekly GI symptom memory 
aids. 
5.
Obtain blood for hematology.
Study Day 266 (Visit #32) – Telephone Call
1.
Review interim medical history emphasizing any acute signs or symptoms (including entries 
on the GI symptom memory aid).
2.
Collect concomitant medication information.
Study Day 273 (Visit #33)
1.
Perform basic history and physical exam, emphasizing examination of any acute complaints.
2.
Record vital signs.
3.
Obtain fecal sample for parasitological testing.
4.
Collect prior weeks’ GI symptom memory aid and give new weekly GI symptom memory aid. 
Study Day 280 (Visit #34)
1.
Perform basic history and physical exam, emphasizing examination of any acute complaints.
2.
Record vital signs.
3.
Obtain fecal sample for parasitological testing.
4.
Collect prior week’s weekly GI symptom memory aid.
5.
Obtain blood for hematology, antibody and cellular assays.
6.
For females, obtain a urine sample for pregnancy testing.  Test must be negative to proceed 
with albendazole administration.
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
44
7.
Dispense 3-dose treatment of albendazole 400 mg once a day by mouth. 
8.
Observe administration of 1st dose of albendazole, and provide 2 remaining doses to subject 
for at-home use. 
Study Day 290 (Visit #35)
1.
Obtain fecal sample #1 collected approximately 1 week post-treatment with albendazole, 
for test of cure.
Study Day 294 (Visit #36)
1.
Obtain fecal sample #2 collected approximately 1.5 weeks post-treatment with 
albendazole, for test of cure. 
Study Day 297 (Visit #37)
1.
Obtain fecal sample #3 collected approximately 2 weeks post-treatment with albendazole, 
for test of cure.
2.
Obtain blood for antibody and cellular assays.
3.
Only for subjects with laboratory evidence of eosinophilia post-CHHI: obtain blood for 
hematology testing (complete blood count with white blood cell differential).
4.
For females, obtain a urine sample for pregnancy testing (follow-up post albendazole 
treatment).  
Study Day 320 (Visit #38)
1.
Perform basic history and physical exam (including injection site), emphasizing examination 
of any acute complaints.
2.
Record vital signs.
3.
Obtain blood for rheumatoid factor, anti-dsDNA, anti-Na-GST-1 antibody assays.  
7.4
Final Study Visit
Study Day 380 (Visit #39)
1.
Perform basic history and physical exam, emphasizing examination of any acute complaints.
2.
Obtain blood for anti-Na-GST-1 antibody assays.
7.5
Early Termination Visit
Every attempt will be made to retain subjects once they have been enrolled.  However, if for 
safety reasons, withdrawal of consent, or loss to follow-up, a subject is deemed by the 
investigators and/or SMC to be not eligible to receive the study product as per protocol, he/she 
will discontinue vaccinations.  Volunteers will be encouraged to continue attending scheduled 
study visits to be followed for safety. Study subjects who discontinue the vaccinations or study 
participation early will not be replaced. Withdrawal from the study for any reason will not impact 
the subject’s medical care.
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
45
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 or during the CHHI phase of the study).

Information regarding adverse events/Serious Adverse Events will be assessed and 
recorded on the appropriate data collection form.

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 15 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 15 and if not recently obtained).
Subjects who have undergone CHHI and who insist upon terminating the study prematurely and 
prior to completion of Day 197 will be recommended to take albendazole for hookworm infection 
(400 mg orally for three consecutive days). Follow-up care will be offered regardless of study 
participation, if so desired by the subject.
7.6
Unscheduled Visit 
Unscheduled visits may occur at any time during the study.  Procedures conducted at these 
visits may include any of the procedures detailed in Appendix A as appropriate at the discretion 
of the investigator depending on the nature of the unscheduled visit.  Any of the following 
activities may be performed:

Review concomitant medications (if prior to 28 days after a study vaccination or between 
study Days 140 to 297).

Review solicited adverse events (if prior to 28 days after a study vaccination or between 
study Days 140 to 280).

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).
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
46

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 site principal investigator or 
sub-investigator, 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).
7.7
Clearance of Infection
In order to confirm that treatment with albendazole results in cure of experimental hookworm 
infection, participants will be asked to provide fecal samples 1, 1½ and 2 weeks following 
treatment (Days 290, 294 and 297), which will be examined for the presence of eggs by 
microscopy. Three negative samples are required as confirmation of successful treatment. As 
treatment with even a single dose of albendazole is currently highly effective (20, 71), and as 
this study is being conducted in a hookworm non-endemic area, cure following a 3-dose course 
of treatment is expected for all subjects (72). Nevertheless, if eggs are detected in post-
treatment fecal samples, the participant will be given a second course of albendazole, with the 
same procedure for confirmation of successful treatment.
In addition, treatment with albendazole will be offered to any study participant who wishes to 
withdraw from the study (whether due to the occurrence of adverse events or for personal 
reasons) prior to study Day 280. In such an instance, the participant will be strongly encouraged 
to provide post-treatment fecal samples to ensure cure of the infection.  Fecal samples at 1 
week, 2 weeks and 60 days following albendazole treatment will be requested for subjects who 
terminate the study early.
If a female becomes pregnant during the course of the study, provisions for treatment of 
hookworm infection will be offered (i.e., albendazole treatment after the first trimester of 
pregnancy). 
7.8
N. americanus Donor Sub-Study
Transition to and enrollment into the Donor Sub-Study of protocol SVI-CH-01 will be scheduled 
at or before Study Day 280. In this sub-study participants will agree to continue being infected 
with N. americanus and to provide routinely scheduled fecal samples of not more than three per 
month.  Participants will not receive the 3-dose treatment of albendazole 400 mg scheduled at 
Study Day 280, but will be permitted to remain infected initially for up to 6 years. During the 
donor sub-study, participants will be tested for infection with HIV and viral hepatitis (B and C) 
when they provide a fecal sample that may be used to prepare larvae for application to human 
study subjects, if this testing has not been done in the previous 30 days. If any participant in the 
donor sub-study develops new infection with HIV, hepatitis B or hepatitis C, they will be 
withdrawn from the sub-study and provided treatment for the N. americanus infection as 
described in the protocol for study SVI-CH-01.
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
47
Study participants who enroll into the Donor Sub-Study of protocol SVI-CH-01 will remain as 
participants in protocol SVI-CH-02/DMID 16-0116 until Visit #39 as described above. 
Participants will complete all study procedures described in Sections 7.3 and 7.4 and will be 
considered to have completed protocol SVI-CH-02/DMID 16-0116 after Visit #39 (study day 
380) is completed. 
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
48
8
STUDY PROCEDURES/EVALUATIONS
8.1
Clinical Evaluations
Subjects will be monitored for local and systemic adverse events during specific protocol-
defined periods. As with other aluminum hydroxide-adsorbed vaccines, hypersensitivity 
reactions would be expected to occur within the first 24 hours after vaccination, 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 at the study clinic on Days 7 and 14 following each vaccination. A telephone 
call with the subject will take place on Days 3 and 28 following vaccinations 1 and 2 to monitor 
for adverse events. Day 28 following vaccination 3 falls on the day of CHHI and the subject will 
be assessed at the study clinic. 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. 
Following CHHI, local larval application site reactions are expected to occur within the first 
month after application of the larvae. Subjects will therefore be observed for immediate 
reactions following each larval application for at least 1 hour. Subjects will have a telephone call 
on day 3 following CHHI to monitor for adverse events. A clinical assessment at the study clinic 
will be performed on Days 7 and 14 following CHHI. Gastrointestinal AEs are expected to occur 
after approximately week 4 after CHHI; subjects will therefore be evaluated weekly from weeks 
5 through 20 after CHHI, until treatment with albendazole. 
All AEs will be graded for severity and relationship to study vaccine or larval application, 
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 clinician. 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, and gastrointestinal, hematologic or respiratory disorders that may potentially be 
related to CHHI. 
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 tests listed below will be performed at the site’s clinical 
laboratory.  One or more of the laboratory parameters may be repeated at any time during the 
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
49
study as determined by the PI, if indicated by an AE.  A clinically significant abnormal value 
should be repeated within 10 days if possible and followed up as clinically relevant.
1. Complete blood count plus white blood cell (WBC) differential* 
2. Serum creatinine
3. Alanine aminotransferase (ALT)
4. Rheumatoid factor (RF)
5. Anti-double stranded DNA antibody (anti-dsDNA)
6. HBsAg ELISA
7. HCV serology
8. HIV serology
9. Serum ferritin
*The following CBC parameters will be assessed for safety throughout the trial: WBC count, 
absolute neutrophil count, absolute eosinophil count, hemoglobin concentration and platelet 
count.
Urine pregnancy testing will be performed at the clinical trial site using test kits that have been 
approved by the FDA. Urine dipstick testing will also be performed at the trial site using an 
approved product.
Parasitological and immunology assays (see below) will be performed in the laboratories of the 
Department of MITM at the GWU, and at Baylor College of Medicine (fecal PCR for hookworm 
DNA).   
8.2.2 Immunology Assays
Anti-Na-GST-1 IgG antibody, other humoral, innate immunity and cellular assays will be 
performed at the Department of MITM of the George Washington University School of Medicine 
and Health Sciences. All cryopreservation of plasma, sera and peripheral blood mononuclear 
cells (PBMCs) will be in liquid nitrogen. 
8.2.2.1
Anti-Na-GST-1 Antibody Assays
Antibodies to Na-GST-1 will be measured in serum or plasma of study subjects using an indirect 
ELISA assay. Antigen-specific IgG and IgG1-4 antibody levels will be measured at baseline and 
at several time-points post-vaccination. For the indirect ELISA method, microwell plates will be 
adsorbed with purified recombinant Na-GST-1, blocked, and incubated with test sample sera or 
plasma, and then incubated overnight at approximately 4 C. Plates will then be washed and a 
horseradish peroxidase conjugated anti-human antibody (e.g., anti-human IgG) will be added 
and incubated with plates. Plates will be washed again, incubated with a chromogenic 
substrate, and then the Optical Density (OD) of the samples read using an automated ELISA 
plate reader. Levels of antibodies against Na-GST-1 in sera or plasma will be converted to 
Arbitrary Units of antibody by homologous interpolation of their OD readings from a standard 
reference curve derived from serial dilutions of a Standard Reference Serum unique to each 
antibody isotype. 
Surface Plasmon Resonance (SPR) will be used to measure the affinity of antibody interactions 
with recombinant Na-GST-1 antigen. SPR determines the immunoreactivity and affinity of 
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
50
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
An in vitro assay has been developed to measure the functional capacity of antibodies induced 
by vaccination with recombinant Na-GST-1 to inhibit or neutralize the catalytic activity of the 
native form of this protein. Serum or plasma samples collected from study subjects will be 
tested for their neutralizing capacity at several post-vaccination time points. These assays will 
be performed at the Clinical Immunology Laboratory of the Department of MITM of the George 
Washington University.
8.2.2.3
Memory B Cell Measurement
The ELISPOT method will be used to evaluate the induction of memory B cells (MBCs) after 
each vaccination and the 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 and Innate Immunity Assays
PBMCs will be isolated by Ficoll gradient and the circulating dendritic cell (DC) population 
isolated by affinity columns according to standard protocol. The DC population will be cultured in 
vitro and the cells will either remain “un-stimulated” or be “stimulated” with a TLR7/8 agonist (1 
μg/ml, 4-amino-2-ethoxymethyl-α, α-dimethyl-1H-imidazoquinoline-1-ethanol). After 24 hours of 
stimulation, the supernatants will be collected and assayed for cytokine production (e.g., 
Interleukin (IL)-12, IFN-gamma, TNF-alpha, IL-4, IL-5 and IL-18) by commercially available 
ELISA kits. The pellet collected from the culture will be assayed for the cytokines by real-time 
(RT) PCR. In addition, purified DCs will be directly assayed for positive co-stimulatory molecules 
such as CD40, CD80, CD86 CD83, HLA-DR, PD-L1, and ICOSL by flow cytometry using 
commercially available labeled antibodies. The Natural Killer (NK) cell response generated in 
response to vaccination with Na-GST-1 administered with a TLR4 or TLR9 agonist will be 
compared to the response when Na-GST-1 is administered without these TLR agonists. At 
various points post-vaccination and post-CHHI, purified PBMCs will be stained with labeled anti-
CD56 and the cells will be assayed for functionality by measuring cytokine production by 
intracellular staining. As functionality of NK cells can also be measured by their cytolytic activity, 
the cells will be analyzed for granzyme B and activation markers (e.g., NKG2D and NKP46) by 
flow cytometry. The cytotoxic activity of NK cells may also be assayed by using 51Cr-labeled 
K562 targets. 
In addition, circulating DC and purified T cell populations will be separated by affinity columns. 
The DC will be cultured (1x105 cells/well) overnight and pulsed with the recombinant Na-GST 
antigen. The following day, purified T cells from the same individual will be added to the culture 
and after 48 hours of incubation the T cells will be evaluated for proliferation (Ki67 labeling), 
cytokine production (IL-2, IL-4, IL-5, IFN , TNF ), co-stimulatory molecules (CD40, CD40L, 
CD80, CD86, and ICOS), and inhibitory receptors (PD-1, PDL-1, LG-3, 2B4) by polychromatic 
flow cytometry.  
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
51
8.2.3 Parasitology Assays
8.2.3.1
Fecal Egg Detection by Microscopy
Fecal exams will begin with the super-saturated saline flotation technique to determine 
hookworm infection status (i.e., positive or negative). Fecal samples will be analyzed by the 
McMaster technique to determine the intensity of hookworm infection. To perform the McMaster 
examination, a fecal suspension is applied to a slide with counting chambers. Egg counts are 
performed under light microscopy.   
8.2.3.2
Adult Worm Counts
During the 3-day albendazole treatment period at the end of the study, subjects will be asked to 
provide all fecal material passed, which will be examined for expelled worms. Worms will be 
washed in phosphate-buffered saline and stored in ethanol. The worms will be clarified in a 
phenol solution and the mouthparts examined under a microscope.
8.2.3.3
Polymerase Chain Reaction (PCR) Test for N. americanus
A quantitative PCR assay will be used to detect and quantify N. americanus DNA in fecal 
samples collected from study participants. DNA will be isolated from a small sample of feces 
(~50 mg), and Necator DNA amplified by real-time quantitative PCR using primers and probe 
specific for the internal transcribed spacer 2 (ITS2) of N. americanus (73).
8.3
Specimen Preparation, Handling, and Shipping
8.3.1 Instructions for Specimen Preparation, Handling, and Storage
Instructions for specimen preparation, handling, and storage are included in the protocol-
specific Manual of Procedures (MOP), as appropriate.
Blood specimens for clinical screening and safety labs will be tested locally by the LabCorp® 
clinical laboratory.
Urine dipstick tests and urine pregnancy tests will be performed in the research clinic laboratory 
at the GW MFA.
Fecal specimens obtained for parasitology testing will be processed as specified in the MOP 
and transported to the Department of MITM at GWU for processing and storage.
Blood specimens for isolation of serum, plasma, and PBMCs will be collected as specified in the 
study MOP and transported to the Department of MITM at GWU for processing and storage.
8.3.2 Specimen Shipment
Specimen shipment (primarily fecal samples to Baylor College of Medicine, Houston, TX) 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.
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
52
9
ASSESSMENT OF SAFETY
9.1
Specification of Safety Parameters
Safety will be assessed by the frequency and severity of: 
1.
Study product (vaccine or CHHI)-related serious adverse events occurring from the time 
of the first study vaccination through approximately 10 months after the last study 
vaccination.
2.
Study vaccine-related solicited Adverse Events – reactogenicity events occurring on the 
day of each study vaccination through 14 days after each study vaccination:
a) Injection site reactions including erythema (redness), induration (hardness)/swelling, 
pain, and tenderness.
b) Systemic reactions including fever, myalgia (body aches/muscular pain exclusive of 
the injection site), arthralgia (joint pain exclusive of the injection site), headache, 
nausea, and vomiting.
3.
CHHI-related solicited adverse events – reactogenicity events occurring on the day of 
CHHI through study Day 280 (time of treatment with first dose of albendazole):
a) Application site reactions including pruritus, rash, induration (hardness)/swelling, 
pain, and tenderness.
b) Gastrointestinal reactions including abdominal pain, abdominal bloating, flatulence, 
nausea, vomiting, and diarrhea.
c)
Respiratory reactions (possibly due to migration of larvae through the lungs) 
including cough and sore throat.
4.
Clinical safety laboratory adverse events occurring throughout the study. Parameters to 
be evaluated include WBC, ANC, absolute eosinophil count, hemoglobin concentration, 
and platelet count; ALT; creatinine; rheumatoid factor; and, anti-ds DNA antibodies.
5.
Unsolicited Adverse Events – non-serious adverse events occurring from the time of 
each study vaccination through approximately 28 days after each vaccination and from 
study Day 140 (day of CHHI) through Day 297.
6.
New-onset chronic medical conditions occurring from the time of the first study 
vaccination through approximately 10 months after the last study vaccination.
7.
Adverse Events of Special Interest occurring from the time of the first study vaccination 
through approximately 10 months after the last study vaccination.
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
53
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 
product (i.e., vaccine, placebo or CHHI), and whether or not considered study product related. 
This includes an exacerbation of pre-existing conditions and intercurrent illnesses. 
All AEs will be graded for severity and relationship to study vaccine or to CHHI. A licensed 
clinician (i.e. physician, nurse practitioner, physician assistant) listed on the Form FDA 1572 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 1-5:
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 or CHHI 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 study product or the nature of 
the event is such that the study product 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 study product 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 product 
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 CHHI) or reproduced on re-
vaccination.
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
54
2.
A reaction of similar nature having previously been observed with this study product.
3.
The event having often been reported in the literature for similar types of vaccines or for 
CHHI.
9.2.2 Vaccine Reactogenicity
All subjects will be observed for at least 60 minutes after each study vaccination to detect and 
treat any immediate AEs. Subjects will also complete study memory aids after each vaccination 
beginning with the day of vaccination and through 14 days after vaccination. Injection site 
reactions including bruising, redness, induration (hardness)/swelling, pain, and tenderness will 
be recorded on the study aids. Systemic reactions including fever, myalgia (body 
aches/muscular pains exclusive of the injection site), arthralgia (joint pains exclusive of the 
injection site), headache, nausea, and vomiting will also be recorded.
Subjects will also be asked to record any medications taken and any emergency room or 
healthcare provider visits (other than routine check-ups). The subject memory aid will be 
reviewed with the subject at subsequent clinic visits. 
Vaccine 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:
Table 1: Assessment of Solicited or Expected Adverse Event Severity – Vaccination 
Phase
Adverse Event
Grade
Severity
Pain at injection site
1
Aware of pain but it does not interfere with daily activity and no 
pain medication is taken
2
Aware of pain; there is interference with daily activity or it requires 
use of pain medication
3
Aware of pain and it prevents daily activity
Tenderness at injection site
1
Area immediately surrounding injection site hurts only when 
touched or with arm motion, and it does not interfere with daily 
activity 
2
Area immediately surrounding injection site hurts when touched or 
with arm motion, and it interferes with daily activity
3
Area immediately surrounding injection site hurts when touched or 
with arm motion, and it prevents daily activity
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
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
55
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
* Oral temperature assessed on Day 0 (Visit 1) prior to the first study vaccination will be considered as 
baseline. Temperatures obtained during the clinic visits will be recorded.
† Subjects must not eat or drink anything hot or cold, or smoke within 10 minutes prior to taking oral 
temperature.
** Not at injection site.
Table 2: Assessment of Unsolicited Systemic Adverse Event Severity – Vaccination Phase
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 
Causes greater than 
minimal interference 
Inability to perform daily activities
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
56
with usual daily 
activities
with usual 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
Urticaria
Requiring no 
medications
Requiring PO or 
topical treatment, or 
IV medication or 
steroids for <24 
hours
Requiring IV medication or steroids 
for >24 hours
9.2.3 CHHI Reactogenicity
All subjects will be observed for at least 60 minutes after application of the N. americanus larvae 
during CHHI to detect and treat any immediate AEs. Subjects will also complete study memory 
aids after CHHI beginning with the day of larval application and through Day 280 when they will 
be treated with albendazole to cure their infections. For the first 4 weeks after application of the 
N. americanus Larval Inoculum, the memory aids will be used to record pruritus, pain, 
tenderness, rash and swelling at the site of larval application, as well as cough and sore throat 
(to capture symptoms that may possibly be related to larval migration through the lungs). 
Starting on Day 175 (approximately 5 weeks after CHHI) of the study until the participant 
receives their first dose of albendazole, the memory aids will be used to record nausea, 
vomiting, diarrhea, abdominal pain, abdominal bloating and flatulence.  Female participants of 
childbearing potential will additionally capture on the memory aids the form of contraception 
they are using. The memory aids will include a printed reminder of the potential risks associated 
with use of albendazole during pregnancy. 
Subjects will also be asked to record any medications taken and any emergency room or 
healthcare provider visits (other than routine check-ups) through Day 297. The subject memory 
aid will be reviewed with the subject at subsequent clinic visits. 
CHHI reactogenicity events are AEs that are known to occur with hookworm infection. The 
following Toxicity Grading Scales will be used to grade solicited local (larval application site) and 
systemic (subjective and quantitative) reactions:
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
57
Table 3: Assessment of Solicited or Expected Adverse Event Severity – CHHI Phase
Adverse Event
Grade
Severity
Pain at site of larval application
1
Aware of pain but it does not interfere with daily activity 
and no pain medication is taken
2
Aware of pain; there is interference with daily activity or 
it requires use of pain medication
3
Aware of pain and it prevents daily activity
1
Area immediately surrounding application site hurts only 
when touched or with arm motion, and it does not 
interfere with daily activity 
Tenderness at site of larval 
application
2
Area immediately surrounding application site hurts 
when touched or with arm motion, and it interferes with 
daily activity
3
Area immediately surrounding application site hurts 
when touched or with arm motion, and it prevents daily 
activity
1
25 mm – 50 mm and does not interfere with daily activity
Rash at site of larval application
2
51 mm – 100 mm or interferes with daily activity
3
> 100 mm or prevents daily activity
1
25 mm – 50 mm and does not interfere with daily activity
Induration/swelling at site of 
larval application
2
51 mm – 100 mm or interferes with daily activity
3
> 100 mm or prevents daily activity
Pruritus at site of larval 
application
1
Easily tolerated, does not interfere with daily activity
2
Interferes with daily activity
3
Prevents daily activity
Abdominal pain
1
Easily tolerated, does not interfere with daily activity
2
Interferes with daily activity
3
Prevents daily activity
Abdominal bloating
1
Easily tolerated, does not interfere with activity
2
Interferes with daily activity
3
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
Diarrhea
1
2-3 loose stools/24 hours
2
4-5 stools/24 hours
3
> 6 loose stools or requires outpatient IV hydration
Flatulence
1
Easily tolerated, does not interfere with daily activity
2
Interferes with daily activity
3
Prevents daily activity
Cough
1
Easily tolerated, does not interfere with daily activity
2
Interferes with daily activity
3
Prevents daily activity
Sore throat
1
Easily tolerated, does not interfere with daily activity
2
Interferes with daily activity
3
Prevents daily activity
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
58
The severity of unsolicited adverse events post-CHHI will be graded as per Table 2 above.
9.2.4 Additional Adverse Event Severity Grading
Pulse and blood pressure will be graded as follows (Table 4):
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 (bpm)
101-115
116-130
>130
Bradycardia – 
beats per minute
50 - 54 or 45-50 bpm if 
baseline <60 bpm
45 - 49 or 40-44 if 
baseline <60bpm
< 45 or  <40bpm if 
baseline <60bpm
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
Absolute 
eosinophil count 
(increase)
700 – 1500/mm3
1501 – 5000/mm3
>5000/mm3
CLINICAL
CHEMISTRIES
Grade 1
Grade 2
Grade 3
Serum 
creatinine
1.5 – 1.7 mg/dL
1.8 – 2.0 mg/dL
>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
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
59
9.2.5 Serious Adverse Events
An SAE is an AE, whether considered related to a study product 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.
9.2.6 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
AEs will be followed until resolution even if this extends beyond the study-reporting period.  
Resolution of an AE is defined as the return to pretreatment status or stabilization of the 
condition with the expectation that it will remain chronic. 
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 24 hours 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
Texas Children’s Hospital Center for Vaccine Development
Baylor College of Medicine and Texas Children’s Hospital
1102 Bates St., Ste. 550, Houston, TX 77030
832-824-0504, Alt. 713-798-1199, Fax 832-825-0549
Email: bottazzi@bcm.edu
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
60
In addition, the Principal Investigator will report all SAEs that are determined to be definitely or 
probably related to study product to the Institutional Review Board (IRB) that approved the study 
(the IRB of the George Washington University) 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
The site will notify the study ISM when an SAE is provided to the Sponsor and the DMID 
Pharmacovigilance Group. The DMID Medical Monitor and DMID Clinical Project Manager will 
be notified of the SAE by the DMID Pharmacovigilance Group. The DMID Medical Monitor will 
review and assess the SAE for potential impact on study subject safety and protocol conduct.
At any time after completion of the study, if the Principal Investigator or appropriate co-
investigator becomes aware of an SAE that is suspected to be related to study product, the 
Principal Investigator or appropriate co-investigator will report the event to the Sponsor and the 
DMID Pharmacovigilance Group.
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 (Baylor College of 
Medicine) will report events that are both serious and unexpected that are possibly, probably, or 
definitely related to the study product, to the FDA within the required timelines: fatal and life-
threatening events within 7 calendar days (by phone, fax, or internet) and all other SAEs in 
writing within 15 calendar days. All SAEs not listed as possibly, probably, or definitely related 
will be reported to the FDA at least annually in a summary format.
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
61
All Adverse Events of Special Interest (AESIs; see below) will be reported to Sponsor, and FDA 
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 Na-GST-1 in combination with either AP 10-701 or 
CpG 10104). The occurrence of the following AESI’s will be closely monitored: 

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 
In addition, disorders that may potentially be related to experimental hookworm infection will be 
defined as AESIs that will be closely monitored following CHHI, including: 

Gastrointestinal disorders (Crohn’s Disease, Ulcerative Colitis, Irritable Bowel 
Syndrome) 

Gastrointestinal bleeding

Anemia (greater than Grade 1 low absolute hemoglobin concentration as defined in 
Table 5)

Pneumonitis
The new onset of AESI’s during the study will be reported to the Sponsor, the DMID Medical 
Monitor, and the FDA in the same fashion as described above in Section 9.3.1 for SAEs.
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 
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
62
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 or CHHI 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. If a 
woman becomes pregnant after CHHI but before study Day 280, management of her infection 
will be followed as per Section 7.7 of this protocol (i.e., deferral of treatment with albendazole 
until after the first trimester due to the uncertain risk of this medication during the first trimester). 
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 vaccine, and from time of CHHI up to study Day 297 (to include 
the albendazole treatment period).  AEs limited to AESI’s and new-onset chronic medical 
conditions will be followed through approximately 6 months after the larval application (Day 380, 
approximately 10 months after the final vaccination).
SAEs will be followed from the time of the first study vaccination (Day 0 [Visit 1]) through 
resolution even if this extends beyond the study-reporting period (approximately 10 months after 
the third study vaccination [Day 380, Visit 39]).  Resolution of an AE/SAE is defined as the 
return to pretreatment status or stabilization of the condition with the expectation that it will 
remain chronic.
Follow-up procedures, evaluations, and outcomes will be recorded on the appropriate data 
collection form.
9.5
Halting Rules
If the study vaccine or CHHI is considered significantly reactogenic or results in significant AEs 
(see below and Section 9.2), additional vaccinations or CHHIs will be suspended until reviewed 
by the SMC, Independent Safety Monitor, DMID Medical Monitor, and IND sponsor (BCM). Any 
recommendation of the Independent Safety Monitor, DMID Medical Monitor, and SMC to 
resume or suspend further injections or CHHIs (either for an individual subject or the full study) 
will be communicated in writing to the sponsor and Principal Investigator. All communications 
from the SMC will subsequently be forwarded by the investigators to the IRB. 
The following criteria will be used to define significant reactogenicity:
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
63

One or more participants experience a protocol defined SAE that is determined to be 
possibly, probably, or definitely related to a study vaccine or CHHI, 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 CHHI, 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 CHHI, OR,

Two or more participants experience a protocol defined Grade 2 hypersensitivity reaction 
that is probably or definitely related to a study vaccine or CHHI, OR,

Three or more participants experience the same objective physical finding or laboratory 
abnormality of Grade 3 or higher (with the exception of isolated Grade 3 erythema or 
swelling at the site of vaccination or Grade 3 erythema and/or rash at the site of 
application of the larvae), that is determined to be probably or definitely related to a 
study vaccine or CHHI, OR,

Three or more participants experience the same Grade 2 or higher safety laboratory 
abnormality (excluding eosinophilia [or increased white blood cell count secondary to 
eosinophilia] after CHHI) or Grade 3 clinical AE that is possibly, probably or definitely 
related to a study vaccine or CHHI.
9.6
Unblinding
The study will be double-blinded until all study subjects have completed their final study visit.  
Prior to final study unblinding, 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 the final study Day 380 visit or to 
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 IRB of this 
decision.
9.7
Safety Oversight 
9.7.1 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 
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
64
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's 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.  
9.7.2 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.
The SMC will review safety data at the following time points:

Ad hoc when a study halting rule is met (see Section 9.5) or for immediate concerns 
regarding observations during this trial, or as needed.

1-2 weeks before the first CHHI application to review safety data collected to date. 

After the first 20 subjects have completed their study Day 175 (5 weeks after CHHI) 
visits. 

Final review meeting: 6 to 8 months after clinical database lock to review the cumulative 
unblinded safety data for the study.
Data will be provided in a standard summary format. The SMC may be asked to provide 
recommendations in response to questions posed by the study Principal Investigator, the 
Sponsor, or DMID.
The SMC will operate under the rules of a DMID-approved charter that will be written at the 
organizational meeting of the SMC. At this time, each data element that the SMC needs to 
assess will be clearly defined. Procedures for SMC reviews and meetings will be defined in the 
charter. The SMC will review applicable data to include, but not limited to, study progress and 
subject, clinical, safety, and reactogenicity data.  Reports may include enrollment and 
demographic information, medical history, concomitant medications, physical assessments, 
clinical laboratory values, subject compliance with study procedures/interventions, and solicited 
and unsolicited AE/SAEs.  Additional data may be requested by the SMC, and interim statistical 
reports may be generated as deemed necessary and appropriate by the Sponsor. The SMC will 
receive data in aggregate and presented by group.  The SMC may also be provided with 
expected and observed rates of the expected AEs in an unblinded fashion, and may request 
vaccine assignment be unblinded for an individual subject if required for safety assessment.  
The SMC will review grouped and unblinded data in the closed session only.  As an outcome of 
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
65
each review/meeting, the SMC will make a recommendation as to the advisability of proceeding 
with study vaccinations or CHHI (as applicable), and to continue, modify, or terminate this trial.
The study Sponsor, Principal Investigator or the SMC chair may convene the SMC on an ad hoc 
basis according to protocol criteria or if there are immediate concerns regarding observations 
during the course of this trial. The ISM and DMID Medical Monitor will be responsible for 
reviewing SAEs in real time.  The SMC will review SAEs on a regular basis and ad hoc during 
this trial.
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 IRB. Additionally, any new information that may adversely 
affect the safety of the subjects or the conduct of the study will be submitted to the SMC as it 
becomes available.
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
66
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.  
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
67
11
STATISTICAL CONSIDERATIONS
11.1 Study Hypotheses
This study is not designed to test statistically significant small differences in efficacy between 
different adjuvant formulations of the Na-GST-1 hookworm vaccine as assessed in the CHHI 
model. Rather, it is intended to provide an indication of an impact on infection as assessed by 
CHHI, and to detect large differences in efficacy with acceptable power.  Furthermore, the study 
intends to assess the safety, reactogenicity, and immunogenicity of three novel adjuvant 
formulations of Na-GST-1: Na-GST-1 formulated on Alhydrogel, Na-GST-1/Alhydrogel® 
administered with AP 10-701, and Na-GST-1/Alhydrogel® administered with CpG 10104. 
The chosen sample size principally facilitates the assessment of efficacy (as estimated by 
infection status determined by the saturated saline flotation technique for hookworm egg 
detection in fecal samples following CHHI) as well as safety, as discussed in Section 11.2, 
Sample Size Considerations.  Given that, this study will attempt to assess the following 
hypotheses:
1.
Administration of the Na-GST-1/Alhydrogel® vaccine with either AP 10-701 or CpG 
10104 will result in a reduced proportion of volunteers infected with hookworm following 
CHHI, as determined by the saline flotation technique, compared to placebo controls.
2.
Co-administration of Na-GST-1/Alhydrogel® vaccine with either AP 10-701 or CpG 
10104 will increase the IgG antibody response to Na-GST-1 compared to when Na-
GST-1/Alhydrogel® is administered alone.
3.
Co-administration of Na-GST-1/Alhydrogel® vaccine with either AP 10-701 or CpG 
10104 will increase the affinity of induced IgG antibodies to Na-GST-1 compared to 
when Na-GST-1/Alhydrogel® is administered alone.
4.
Co-administration of the Na-GST-1/Alhydrogel® vaccine with either AP 10-701 or CpG 
10104 will increase the induction of specific MBCs to Na-GST-1 compared to when Na-
GST-1/Alhydrogel® is administered alone.
5.
Co-administration of the Na-GST-1/Alhydrogel® vaccine with either AP 10-701 or CpG 
10104 increases the neutralizing capacity of induced IgG antibodies to Na-GST-1 
compared to when Na-GST-1/Alhydrogel® is administered alone.
6.
Co-administration of Na-GST-1/Alhydrogel® vaccine with either AP 10-701 or CpG 
10104 will increase the avidity of induced IgG antibodies to Na-GST-1 compared to 
when Na-GST-1/Alhydrogel® is administered alone.
11.2 Sample Size Considerations
Sample size considerations are based on the proportion of subjects with detectable hookworm 
eggs at any time, as measured by the saturated saline flotation technique. The primary analysis 
of the proportions will consist of comparing the proportion in each of the three Na-GST-1 
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IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
68
intervention groups to the proportion in the placebo control group (group 4) using a one-sided 
Fisher exact test, for a total of three comparisons. The Holm-Bonferroni procedure will be used 
to control the familywise error rate at 0.05. Based on the results from the initial CHHI trial that 
was conducted at GWU (NCT01940757), we expect an infected proportion of 90% in group 4 
(i.e., infectivity controls), and a proportion less the 20% in at least one of the intervention 
groups. At alpha=0.05/3, this yields a power of 87.7%. The power is 73.2% for an infection rate 
of 30% in intervention and 96.3% for an infection rate of 10%.
11.3 Planned Interim Analyses 
None planned.
11.4 Final Analysis Plan
The purpose of this trial is to estimate vaccine efficacy as assessed by CHHI, adverse event 
rates and patterns of immune responses as well as to compare these rates and patterns 
between the investigational Na-GST-1 vaccine formulations.
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 the different Na-GST-1 vaccine 
formulations, as compared to saline placebo. Statistical tests will use a significance level of 5% 
(either two-sided or one-sided, depending on the analysis being conducted; this will be specified 
a priori).
Primary Objective #1: To compare the impact of vaccination with Na-GST-1/Alhydrogel® 
delivered with or without AP 10-701 or CpG 10104 on controlled human hookworm infection 
(CHHI) with N. americanus larvae in healthy, hookworm-naïve adults, as determined by the 
presence of eggs using a qualified flotation technique.
Primary Outcome #1 Measure:
The following parameters will be evaluated for each formulation of Na-GST-1:
1.
Proportion of subjects with detectable hookworm eggs, at any time point, in fecal 
samples, as determined by microscopy using the qualified saline float technique.
Analysis Plan: 
a.
The proportion of participants with detectable hookworm eggs in fecal samples by 
microscopy, at any time point post-CHHI, will be summarized as a descriptive measure 
by study group.  
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
69
b.
The proportion of participants with detectable hookworm eggs in fecal samples by 
microscopy will be summarized as a descriptive measure by study group at each time 
point.  
c.
The proportion of subjects with detectable hookworm eggs in each of the intervention 
groups (Groups 1, 2 and 3) will be compared to the proportion of subjects in the placebo 
control group (Group 4) using a one sided Fisher exact test.  The p-values from the three 
tests will be adjusted using the Holm-Bonferroni procedure to control the familywise error 
rate at 0.05.  Power calculations are based on this outcome.
Primary Objective #2: To evaluate the safety and reactogenicity of Na-GST-1/ Alhydrogel® 
delivered with or without AP 10-701 or CpG 10104 on Days 0, 56 and 112, in healthy, 
hookworm-naïve adults.
AEs will be coded according to Medical Dictionary of Regulatory Activities (MedDRA ) 
preferred terms. The frequency, severity, and relationship of AEs per each vaccine formulation 
of Na-GST-1 will be presented in tabular form using the MedDRA coded term and organized 
by MedDRA System, Organ, and Class (SOC) designations. 
1.
The frequency of solicited local injection site and systemic AEs, and unsolicited AEs will 
be summarized by SOC and preferred term.
2.
Line listings of clinical and laboratory AEs classified as solicited injection site, solicited 
systemic, or unsolicited and will be displayed in tables stratified by formulation group 
(i.e., Na-GST-1/Alhydrogel, Na-GST-1/Alhydrogel plus AP 10-701, Na-GST-1/Alhydrogel 
plus CpG 10104, or saline placebo).
3.
AEs will be summarized by severity and relationship to vaccine by individuals and 
formulation of Na-GST-1.
4.
The frequency of vaccine-related SAEs and AESIs will be tallied as well as summarized 
by body system, by vaccine formulation.
Primary Outcome #2 Measures:
The following summary parameters will be evaluated for each formulation of Na-GST-1, in 
comparison to saline placebo control:
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 solicited adverse events, graded by severity, on the day of CHHI through 
study Day 280.
3.
Frequency of study vaccine-related Serious Adverse Events from the time of the first 
study vaccination through approximately 10 months after the last study vaccination.
4.
Frequency of clinical safety laboratory adverse events. 
5.
Frequency of unsolicited adverse events, graded by severity, from the time of each study 
vaccination through approximately 1 month after each study vaccination; and from the 
time of CHHI through treatment with albendazole (Day 297). 
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
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6.
Frequency of new-onset chronic medical conditions through approximately 10 months 
after the third study vaccination.
7.
Frequency of Adverse Events of Special Interest through approximately 10 months after 
the third study vaccination.
Analysis Plan:
a.
The proportion of subjects with at least one injection site AE will be compared by Na-
GST-1 vaccine formulation. We will test the null hypotheses that the type and 
number of adverse events is the same across all groups by Fisher’s exact test.
b.
We will test the null hypothesis that the frequency of adverse events grouped by 
SOC term is the same in all groups using exact Poisson tests of the ratio of the rates. 
There will be no adjustment for multiple comparisons.
c.
Laboratory results (hematological and clinical chemistry) will be examined for trends 
over time and any clinically significant values for individuals will be reported.
Secondary Objective #1: To compare the impact of vaccination with Na-GST-1/Alhydrogel® 
delivered with or without AP 10-701 or CpG 10104 on fecal egg counts, as determined by the 
McMaster method, after CHHI with N. americanus larvae in healthy, hookworm-naïve adults.
Secondary Outcome #1 Measure:
The following parameters will be evaluated for each formulation of Na-GST-1, in comparison 
to the saline placebo control:
1.
Fecal egg counts as determined by microscopy using the McMaster method, during 
Weeks 5 through 20 post-CHHI.
Analysis Plan:
a.
Fecal egg counts as determined by microscopy of the subjects in each of the 
intervention groups (Groups 1, 2 and 3) will be compared to the eggs counts of the 
subjects in Group 4 using a Wilcoxon-Mann-Whitney rank-sum test. The p-values 
from the three tests will be adjusted using the Holm-Bonferroni procedure to control 
the familywise error rate at 0.05.  
Secondary Objective #2: To assess the relationship between antibody responses to Na-GST-1 
induced by vaccination with Na-GST-1/Alhydrogel® delivered with or without AP 10-701 or CpG 
10104 and responses to CHHI in healthy, hookworm-naïve adults, as determined by the 
presence of eggs using a qualified flotation technique.
Secondary Objective #3: To assess the duration of antibody responses to Na-GST-1 induced by 
vaccination with Na-GST-1/Alhydrogel® delivered with or without AP 10-701 or CpG 10104.
Secondary Outcome #2 and #3 Measures:
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
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The following parameters will be evaluated for each formulation of Na-GST-1, in comparison 
to the saline placebo control:
1.
The anti-Na-GST-1 IgG antibody response, by a qualified indirect enzyme-linked 
immunosorbent assay (ELISA) at approximately 14 days after each vaccination, and 
approximately 1, 2, 4, 6, 7, 8 and 10 months after the third dose.
Analysis Plan: 
a.
The proportion of participants with detectable anti-Na-GST-1 IgG responses will be 
summarized as a descriptive measure.  
b.
IgG levels will be displayed graphically by study group using notched box plots at 
each time point.
c.
Geometric mean antibody responses will be compared between vaccine formulation 
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.
Geometric mean antibody responses for each antigen will be compared using a 
regression model with main effects for the doses of Na-GST-1 (0 or 100μg), AP 10-
701 (0 or 5 μg), and CpG 10104 (0 or 500μg).
e.
For antigen-specific log IgG levels, a three-way ANOVA will be conducted, with Na-
GST-1, CpG 10104 and AP 10-701 doses as factors. 
f.
Pair-wise comparisons of IgG levels on study Day 126 (2 weeks after the third 
vaccination) across all combinations of Na-GST-1 doses (i.e., 0 [placebo] or 100 µg), 
AP 10-701 doses (i.e., 0 or 5 µg), and CpG 10104 doses (0 or 500μg) will be 
assessed using Tukey’s HSD.  
g.
Percent change in IgG antibody levels from days of vaccination to days 7,14 and 28 
post-vaccination will be compared between vaccine formulation and dose groups. 
Values will be reported with 95% confidence intervals.
h.
Percent change in IgG antibody levels from vaccination day #1 to #2, from 
vaccination day #2 to #3, and from vaccination day #3 to day of CHHI will be 
compared between vaccine formulation and dose groups to assess the sustainability 
of the response from trough to trough and post-CHHI. Values will be reported with 
95% confidence intervals.
i.
Percent change in IgG antibody levels from the peak following the third vaccination, 
to 1, 2, 4, 6, 7, 8 and 10 after the third vaccination. Values will be reported with 95% 
confidence intervals.
j.
Percent change in IgG antibody levels from the day of CHHI to 1, 3, 5, 6, 7 and 9 
months post-CHHI. Values will be reported with 95% confidence intervals.
k.
A longitudinal model will be built to describe the IgG levels over time. Using a 
longitudinal panel model, differences in antibody isotype levels by formulation of 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 
subject.
l.
Logistic regression will be explored for the binary response of hookworm infection 
post-CHHI as a function of antibody response, and a Poisson regression of the event 
counts (i.e., infection) as a function of IgG antibody response. In these models, 
vaccination group will be a covariate, rather than just using the IgG responses.
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
72
Exploratory Objective #1: To assess the relationship between the functional capacity of vaccine-
induced antibodies that neutralize the in vitro activity of native Na-GST-1 enzyme and 
responses to CHHI in healthy, hookworm-naïve adults.
Exploratory Outcome #1 Measure:
The following parameters will be evaluated for each formulation of Na-GST-1, in comparison 
to the saline placebo control:
1.
The functional capacity of vaccine-induced antibodies to neutralize the in vitro activity 
of the native Na-GST-1 enzyme. 
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-GST-1, CpG 10104, and AP 10-701 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 formulation of 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 subject.
Exploratory Objective #2: To compare the impact of vaccination with Na-GST-1/Alhydrogel® 
delivered with or without AP 10-701 or CpG 10104 on levels of hookworm DNA as detected by 
real-time PCR in healthy, hookworm-naïve adults challenged with CHHI.
Exploratory Outcome #2 Measure:
The following parameters will be evaluated for each formulation of Na-GST-1, in comparison 
to the saline placebo control:
1.
Levels of N. americanus DNA in fecal samples, as measured by real-time PCR, 
during Weeks 5 through 20 post-CHHI.
Analysis Plan: 
a.
The proportion of participants with detectable hookworm DNA in fecal samples by 
PCR, at any time point post-CHHI, will be summarized as a descriptive measure by 
study group.  
b.
The proportion of participants with detectable hookworm DNA in fecal samples by 
PCR will be summarized as a descriptive measure by study group at each time point. 
c.
Estimated fecal egg counts as determined by PCR of the subjects in each of the 
vaccination groups (Groups 1, 2 and 3) will be compared to the eggs counts of the 
subjects in Group 4 using a Wilcoxon-Mann-Whitney rank-sum test. Current PCR 
hookworm spiking studies and previous egg/DNA correlations are used to determine 
estimated egg counts (74). The p-values from the three tests will be adjusted using 
the Holm-Bonferroni procedure to control the familywise error rate at 0.05.  
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
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Exploratory Objective #3 To assess the impact of vaccination with Na-GST-1/Alhydrogel® 
delivered with or without AP 10-701 or CpG 10104 on the affinity of antibody-antigen 
interactions, and how affinity relates to responses to CHHI in healthy, hookworm-naïve adults, 
as determined by the presence of eggs using a qualified flotation technique.
Exploratory Outcome #3 Measures:
The following parameters will be evaluated for each formulation of Na-GST-1, in comparison 
to the saline placebo control:
1.
The affinity of the antibody interactions with recombinant Na-GST-1 antigen at 
approximately 14 days after each vaccination, and approximately 1, 2, 4, 6, 7, 8 and 
10 months after the third dose.
Analysis Plan: 
a.
Antibody affinity will be displayed graphically for the various study groups at each 
time point post-vaccination and post-CHHI.
b.
Three-way ANOVAs with Na-GST-1, CpG 10104 and AP 10-701 as factors and 
overall affinity as the outcome will be estimated.
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 formulation of 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 subject.
d.
Logistic regression will be explored for the binary response of hookworm infection 
post-CHHI as a function of antibody affinity, and a Poisson regression of the event 
counts (i.e., infection) as a function of IgG antibody affinity. In these models, 
vaccination group will be a covariate, rather than just using the IgG affinity.
Exploratory Objective #4: To assess the relationship between Na-GST-1 specific memory B 
cells induced by vaccination with Na-GST-1/Alhydrogel® delivered with or without AP 10-701 or 
CpG 10104 and responses to CHHI in healthy, hookworm-naïve adults, as determined by the 
presence of eggs using a qualified flotation technique.
Exploratory Outcome #4 Measures:
The following parameters will be evaluated for each formulation of Na-GST-1, in comparison 
to the saline placebo control:
1.
The production of memory B cells specific for Na-GST-1 on days of vaccination; 
approximately 7 and 14 days following each vaccination; on day of CHHI; and then 7 
and 14 days, and 5, 7, 13, and 20 weeks post-CHHI. 
Analysis Plan: 
a.
The amount of Na-GST-1 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 the different Na-GST-1 
formulation groups over time. This will be accomplished using Proc Mixed in SAS 9.3 
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
74
so the analysis will take account of the correlation between measurements on the 
same subject.  
d.
Wilcoxon-Mann-Whitney tests will assess the null hypothesis that memory B cell 
levels are the same in the groups at the primary time point of Day 126, two weeks 
after final vaccination. 
e.
Logistic regression will be explored for the binary response of hookworm infection 
post-CHHI as a function of memory B cell production, and a Poisson regression of 
the event counts (i.e., infection) as a function of memory B cell counts. In these 
models, vaccination group will be a covariate, rather than just using the B cell counts.
Exploratory Objective #5 To assess the relationship between innate immune responses to Na-
GST-1/Alhydrogel® delivered with or without AP 10-701 or CpG 10104 and responses to CHHI 
in healthy, hookworm-naïve adults.
Exploratory Outcome #5 Measure:
The following parameters will be evaluated for each formulation of Na-GST-1, in comparison 
to the saline placebo control:
1.
Innate immune responses on days of vaccination approximately 7 and 14 days 
following each vaccination; on day of CHHI; and then 7 and 14 days, and 5, 7, 13, 
and 20 weeks post-CHHI. 
Analysis Plan: 
a.
Changes in innate immune responses will be described using descriptive statistics.
A longitudinal panel analysis will test the null hypothesis that innate immune responses are the 
same among the different formulations of Na-GST-1 and examine for trends over time.
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
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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.
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
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13
QUALITY CONTROL AND QUALITY ASSURANCE
Following a written site clinical quality management plan that is accepted by the Sponsor and by 
DMID, 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 and to DMID.
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.
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
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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 Council for 
Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use 
(ICH) (1996) Guideline for good clinical practice E6 (R1); and the US Code of Federal 
Regulations (Protection of Human Subjects [21 CFR 50], Institutional Review Boards [21 CFR 
56], and Obligations of Clinical Investigators [21 CFR 312]). 
14.2 Institutional Review Board
The investigators will be responsible for obtaining full IRB approvals for the study from the 
George Washington University IRB. Before the start of the study, the appropriate documents 
(including the protocol, Investigator’s Brochures, and informed consent form) will be submitted 
to the IRB. The IRB 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 prior to any protocol-specified procedures being conducted.  Informed consent will 
be obtained in accordance with US 21 CFR 50.25.
Informed consent will be documented by the use of a written consent form approved by the 
GWU IRB. All relevant information will be provided in both oral and written form in a way that is 
understandable to the subject. Ample time and opportunity will be given for the subject to inquire 
about details of the study.  
The Principal Investigator (or the Investigator’s designee) will explain the nature of the study 
and will inform the subject that participation is voluntary and that they can withdraw at any time.  
The volunteer will be informed about the study’s purpose, goals, expected benefits and risks, 
and potential risks that are currently unforeseeable. They will be provided with a description of 
the procedures and an estimated duration of time that will be required to participate in the study, 
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
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and they will be informed of alternatives to participation in the study. The volunteers will receive 
an explanation as to what options are available if injury occurs as a result of participation in the 
study and whom to contact in the event of a study-related injury. They will also be informed 
whom they should contact for answers to any questions relating to the study.  The volunteer will 
be informed that his/her signature on the informed consent form indicates that he/she has 
decided to participate in the study having discussed the information presented. 
The original signed informed consent form for each volunteer will be maintained as part of the 
volunteer’s study records by the Principal Investigator.  A copy of the signed 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 clearance of their hookworm infection 
with albendazole. 
This clinical trial will enroll healthy, non-pregnant volunteers from the general population of 
Washington, DC, 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 2 
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 2 trial, given that the legal age of consent in the United States is 18 years.
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 
number only, to maintain subject confidentiality. All computer data entry will be done by coded 
number only, and all databases will be secured with password-protected access systems.  
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
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Forms, lists, and any other documents that link subject identification numbers to other 
identifying information will be stored in a separate, locked file in an area with limited access.
Subjects’ study information will not be released without the written permission of the subject, 
except as necessary for monitoring by BCM and/or its designee, DMID, the GWU IRB and/or 
the FDA.
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 or CHHI will be administered.
In the event that the study is discontinued, all subjects who received CHHI and who have been 
and who have not been treated for hookworm will be offered albendazole (3 daily oral doses of 
400 mg). The subjects will be asked to follow up weekly for 2 weeks to ensure clearance of 
infection (see Section 7.7).
14.7 Future Use of Stored Specimens
Some of the biological samples collected from study subjects may be stored at the local site and 
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.  
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
80
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, parasitology and immunogenicity data).
15.4 Timing/Reports
A final clinical study report suitable for submission to the FDA in support of an application for 
licensure will be prepared following the last subject visit and upon completion of assays related 
to the efficacy and immunogenicity endpoints.
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 all products being tested in this trial. The Sponsor is required to inform 
the Principal Investigator as to when such documents need no longer be retained. Storage of all 
trial-related documents will be such that confidentiality will be strictly maintained. 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 
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
81
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 
required by the FDA. 
15.6 Protocol Deviations
No revisions to this protocol will be permitted without documented approval from the GWU IRB. 
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 of all such occurrences. Any change to the protocol will be submitted to the GWU IRB 
as a protocol amendment, and changes not affecting risk to subjects may be expedited, as 
appropriate.
A protocol deviation is any noncompliance with the study protocol, GCP, or protocol-specific 
requirements. The noncompliance may be either on the part of the subject, the PI, or other 
study personnel. As a result of deviations, corrective actions will be developed by the site and 
implemented promptly. It is the responsibility of the PI and other study personnel to use 
continuous vigilance to identify and report deviations within five working days of identification of 
the protocol deviation, or within five working days of the scheduled protocol-required activity.  All 
deviations must be promptly reported to the study Sponsor.
Any deviation from the IRB-approved protocol will be documented in study subject data 
collection forms, 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 GWU IRB according to 
their guidelines and reporting timelines. 
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
82
16
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 study subjects. 
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).
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
83
17
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IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
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IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
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85
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IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
86
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IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
87
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IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
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SUPPLEMENTS/APPENDICES
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
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APPENDIX A: SCHEDULE OF EVENTS – VACCINATION PHASE
1Completed within 90 days of first vaccination.
2CBC parameters to be assessed for safety: WBC, absolute neutrophil count, absolute eosinophil count, hemoglobin, and platelet count.
3Since Vaccination #2.
4Since Vaccination #3.
Study Day
Pre1
0
3
(±1)
7
(±2)
14
(±2)
28
(±4)
56
(±7)
59
(±1)3
63
(±2)3
70
(±2)3
84
(±4)3
112
(±7)3
115
(±1)4
119
(±2)4
126
(±2)4
Visit Number
Pre1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Blood 
Volume
Procedures
Complete History/Physical
X
Obtain Informed Consent
X
Interim Clinical Evaluation
X
X
X
X
X
X
X
X
X
Telephone call
X
X
X
X
X
Urinalysis
X
Urine pregnancy test (females)
X
X
X
X
2 mL
CBC2 
X
X
X
X
X
X
X
RF, anti-dsDNA
X
X
X
X
X
X
X
5 mL
Serum ferritin
X
ALT
X
X
X
X
X
X
X
5 mL
Creatinine
X
X
X
X
X
X
X
HCV testing
X
HBsAg testing
X
10 mL
HIV testing
X
Fecal sample collection
X
X
VACCINATION
1
2
3
10 mL
Anti-Na-GST-1 antibody assays 
X
X
X
X
X
X
50 mL
Innate & Cellular immunity assays
X
X
X
X
X
X
X
X
X
Blood Volume (mL)
22
72
50
72
72
50
72
72
50
72
Total Blood Volume (mL)
22
94
144 216
288
338
410
482
532
604
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025

Phase 2 Study to Assess the Safety, Efficacy and Immunogenicity of Na-GST-1/Alhydrogel® 
Version 6.0
Co-administered with Different Toll-Like Receptor Agonists in Hookworm-Naïve Adults
03 October 2025
90
APPENDIX A: SCHEDULE OF EVENTS – CHHI PHASE
Study Day 140
(±7)4
143
(±1)5
147
(±2)
154
(±2)
175
(±3)
182
(±3)
189
(±3)
196
(±3)
203
(±3)
210
(±3)
217
(±3)
224
(±3)
231
(±3)
238
(±3)
245
(±3)
252
(±3)
259
(±3)
266
(±3)
273
(±3)
280
(±3)
290
(±3)
294
(±3)
297
(±3)
320
(±14)
380
(±14)
Visit Number 15
16
17
18
19
20
21
22
23
24
25
26 27 28 29 30 31 32 33
34
35
36
37
38
39
Blood 
Volume
Procedures
Interim Clinical Evaluation
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Telephone Call
X
X
X
X
X
X
X
2 mL
CBC1 
X
X
X
X
X
X
X
X
X
X
X2
5 mL
ALT, Creatinine
X
X
5 mL
RF, anti-dsDNA
X
Urine pregnancy test
(females)
X
X
X
LARVAL INOCULATION
X
Fecal sample collection3
X
X
X
X
X
X
X
X
X
X
X
X
X
10 mL
Antibody assays 
X
X
X
X
X
X
X
X
X
X
50 mL
Innate & cellular immunity 
assays
X
X
X
X
X
X
X
Anthelminthic Rx
X
Blood Volume (mL)
67
10
67
62
62
2
2
62
2
2
62
62
15
10
Total Blood Volume (mL)
671
681 748 810
872
874
876
938
940
942
1004
1066 1081 1091
1CBC parameters to be assessed for safety: WBC, absolute neutrophil count, absolute eosinophil count, hemoglobin, and platelet count.
2Subjects with laboratory evidence of eosinophilia post-larval inoculation only.
3Subjects that withdraw from the study prior to study Day 280 will be strongly encouraged to provide post-treatment fecal samples to ensure cure of the infection. 
4Since Vaccination #3.
5Since Larval Inoculation.
IRB NUMBER: 121643
IRB APPROVAL DATE: 11/17/2025