A Phase 1 Dose Escalating Study of a Prototype CS6 Subunit Vaccine With a Modified Heat-labile Enterotoxin From Enterotoxigenic Escherichia Coli (ETEC)
Phase: PHASE1
Conditions: Diarrhea
Sponsor: PATH

CssBA Phase 1 Protocol 
 
 
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1. 
TITLE PAGE 
 
 
A Phase 1 Dose Escalating Study of a Prototype CS6 Subunit Vaccine with a Modified 
Heat-labile Enterotoxin from Enterotoxigenic Escherichia coli (ETEC)  
 
 
 
 
 
 
 
 
 
Version 7.0 
31 August 2018 
Sponsor 
PATH Vaccine Solutions (PVS) 
455 Massachusetts Ave NW 
Suite 1000 
Washington, DC 20001 
Sponsor’s Authorized Representative 
The EMMES Corporation 
401 N. Washington St., Suite 700 
Rockville, MD 20850 
Clinical Trial Registration # 
NCT03404674 
Funding Source 
PVS 
 
 

CssBA Phase 1 Protocol 
 
 
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INVESTIGATOR’S AGREEMENT 
 
A Phase 1 Dose Escalating Study of a Prototype CS6 Subunit Vaccine with a Modified Heat-
labile Enterotoxin from Enterotoxigenic Escherichia coli (ETEC) 
 
“I have read this protocol and agree to conduct the study as outlined herein in accordance with 
International Council on Harmonisation Good Clinical Practice Guideline and FDA, DoD 
Regulations.” 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Signature of Investigator  
 
 
 
 
 
 
 
Date 
 
Tida K. Lee, MD, PhD 
Principal Investigator 
Enteric Diseases Department 
Naval Medical Research Center 
 

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PROCEDURES IN CASE OF EMERGENCY 
 
Table 1: 
Contact Information 
Role in Study 
Name 
Address and Telephone number 
Principal Investigator 
Tida K. Lee, MD, PhD Naval Medical Research Center 
(NMRC) 
503 Robert Grant Avenue, Silver 
Spring, Maryland 20910-7500 
(301)-319-9260 
Tida.k.lee.mil@mail.mil 
Sub investigators 
 
Ramiro L. Gutierrez, 
MD, MPH 
 
NMRC 
(301)-319-3193 
Ramiro.l.gutierrez.mil@mail.mil 
Milton Maciel, PhD 
 
NMRC 
(301)-319-7406 
Milton.maciel.ctr@mail.mil 
Chad Porter, PhD, 
MPH 
 
NMRC 
(301)-319-7505 
Chad.k.porter2.civ@mail.mil 
Christopher Duplessis, 
MD, MPH, MS 
 
NMRC 
(301)-319-9407 
Christopher.a.duplessis.mil@mail
.mil 
Paige Waterman, MD 
Walter Reed Army Institute of 
Research (WRAIR)  
(301)-319-9312 
paige.e.waterman.mil@mail.mil 
Alison Lane, MD, MS 
Walter Reed National Military 
Medical Center 
(206)-354-4337 
Alison.b.lane.mil@mail.mil 
Mark Riddle, MD, 
DrPH 
Uniformed Services University of 
the Health Sciences 
(301)-295-9769 
mark.riddle@usuhs.edu 
Melinda Hamer, MD, 
MPH 
WRAIR 
(301)-319-3136 
melinda.j.hamer.mil@mail.mil 
Study Coordinator 
Kayla Jaep 
NMRC 
(301)-319-9609 
Kayla.m.jaep.ctr@mail.mil 
Research Monitor 
Michael Koren, MD 
 
WRAIR 
Telephone: 301-319-9904 
michael.a.koren2.mil@mail.mil  

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Role in Study 
Name 
Address and Telephone number 
PVS Medical Officer 
Rahsan Erdem, MD 
PATH 
455 Massachusetts Ave NW 
Suite 1000 
Washington, DC 20001 
Telephone: 202-540-4546 
Fax: 202-457-1466 
Email: rerdem@path.org 
Institutional Review Board 
Naval Medical 
Research Center, 
Institutional Review 
Board 
Research Services Directorate, 
Office of Research 
Administration 
Code 025 
500 Robert Grant Avenue, Rm 
004 
Silver Spring, MD 20910 
(301) 319-7276 
Clinical Trial Site 
Walter Reed Army 
Institute of Research 
(WRAIR) Clinical 
Trials Center (CTC) 
Walter Reed Army Institute of 
Research 
503 Robert Grant Avenue, Silver 
Spring, Maryland 20910-7500 
Clinical Laboratories 
Clinical Trials Center 
(CTC) 
Walter Reed Army 
Institute of Research 
503 Robert Grant Avenue, Silver 
Spring, Maryland 20910-7500 
Quest Diagnostics 
Incorporated 
Quest Diagnostics Incorporated 
1901 Sulphur Spring Road 
Baltimore, MD 21227-0580 
Research Laboratories 
Milton Maciel, PhD 
ETEC Immunology 
Laboratory 
Enteric Diseases 
Department, NMRC 
503 Robert Grant Avenue 
Silver Spring, MD 20910-7500 
(301)-319-7406 
Milton.maciel.ctr@mail.mil 
Data Management 
The EMMES 
Corporation 
401 N. Washington St., Suite 700 
Rockville, MD 20850 
 

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2. 
SYNOPSIS 
Name of Sponsor: PATH Vaccine Solutions (PVS)  
Name of Investigational Products: 
 spd_dsc16Bntd14CssBAB7A[His]6 (CssBA) - recombinant ETEC CssBA protein  
 LTR192G/L211A (dmLT) – E. coli double mutant heat labile toxin 
Title of Study: A Phase 1 Dose Escalating Study of a prototype CS6 subunit vaccine with a 
Modified Heat-labile Enterotoxin from Enterotoxigenic Escherichia coli (ETEC)  
Study center: Walter Reed Army Institute of Research (WRAIR) Clinical Trials Center 
(CTC)  
Principal Investigator: Tida K. Lee, MD, PhD 
Sub investigators: Ramiro Gutierrez, MD, MPH; Christopher Duplessis, MD, MPH, MS; 
Chad Porter, PhD, MPH; Milton Maciel, PhD; Paige Waterman, MD; Alison Lane, MD, MS; 
Mark Riddle, MD, DrPH; Melinda Hamer, MD, MPH  
Study Duration (per group): 
Screening (up to 90 days); Vaccination to final blood 
draw (70 days); long term follow-up (12 months); 
immunology analysis (12 weeks); data analysis and 
report writing (2 months).   
 
Phase of development: 1 
 
Trial Objectives:  
Primary Objective: 
 Evaluate the safety of CssBA ± dmLT given by IM injection  
Secondary Objectives: 
 Evaluate immune responses following IM vaccination with CssBA ± dmLT  
 Identify a safe and immunogenic dose and route of a CssBA-based vaccine to be used 
in a subsequent vaccination/challenge trial 
Study Design and Methodology: 
This is an open-label, Phase 1 clinical trial in which a total of 50 subjects will receive three 
injections of either CssBA alone, dmLT alone or CssBA + dmLT. The vaccine will be 
administered via IM injection to alternating deltoid regions on days 1, 22 and 43. Each 
subject will receive the same dose at each vaccination dependent upon group assignment. 
Group A is considered a pilot group in which all 3 doses will be administered and subjects 
monitored for safety 7 days after the third vaccination, prior to the enrollment of subjects in 
Group B.  
 
Each subject will receive 3 doses and all subjects within a group will be enrolled on the same 
day. Progression to subsequent groups will be dependent on safety measured in the first 7 
days after the third vaccination of the previous group.  
 
Study Design for a Phase 1 clinical trial of intramuscularly administered CssBA 
with/without dmLT (N=50) 

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Group 
N 
Route 
CssBA (µg) dmLT (ng) 
A 
5 
IM 
5  
0 
5 
IM 
0 
100 
B 
10 
IM 
5  
100 
C 
10 
IM 
5 
500 
D* 
10 
IM 
15 
100/500 
E* 
10 
IM 
45 
100/500 
*Plan to proceed with 500ng dose; however, if there is an aberrant safety signal in Group C, 
will proceed with the 100 ng dose (presuming no prior signal in Group B) 
Blood, saliva, and stool specimens will be collected at prescribed intervals to examine 
systemic and mucosal immune responses. Vaccine safety will be actively monitored during 
vaccination and for 28 days following the third vaccine dose. The decision to advance to the 
next group is based solely on the safety assessment through 7 days after the subjects in the 
prior group receive the third vaccination. All safety data will be summarized and reviewed by 
the Safety Review Committee (SRC) prior to dose-escalation. 
Estimated Number of Subjects Screened: 
Based on prior studies with similar study designs, sample collections and inclusion/exclusion 
criteria, we anticipate screening approximately 5 subjects for every one subject that is 
enrolled in the study.  Therefore we anticipate approximately 250 individuals will be 
screened. 
Maximum Number of Subjects Enrolled: 
A maximum of 50 subjects are planned for this study.  Up to four alternates per group will be 
selected as well that may be enrolled in the event a subject becomes ineligible prior to receipt 
of the investigational product. 
Main Criteria for Inclusion/Exclusion: 
Inclusion Criteria: 
 Healthy, adult, male or female, age 18 to 45 years (inclusive) at the time of 
enrollment. 
 Completion and review of comprehension test (achieved > 70% accuracy). 
 Signed informed consent document. 
 Available for the required follow-up period and scheduled clinic visits. 
 Women: Negative pregnancy test with understanding (through informed consent 
process) to not become pregnant during the study or within three (3) months 
following last vaccination. 

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Exclusion Criteria:  
General Health 
 Health problems (for example, intercurrent febrile illness, chronic medical conditions 
such as psychiatric conditions, diabetes mellitus, hypertension or any other condition 
that might place the subject at increased risk of adverse events) - study clinicians, in 
consultation with the PI, will use clinical judgment on a case-by-case basis to assess 
safety risks under this criterion. The PI will consult with the Research Monitor as 
appropriate. 
 Clinically significant abnormalities on physical examination. 
 Immunosuppressive drugs (use of systemic corticosteroids or chemotherapeutics that 
may influence antibody development) or illness (including IgA deficiency, defined by 
serum IgA <7mg/dL). 
 Women who are pregnant or planning to become pregnant during the study period 
plus 3 months beyond the last received dose and currently nursing women. 
 Participation in research involving another investigational product (defined as receipt 
of investigational product or exposure to invasive investigational device) 30 days 
before planned date of first vaccination or anytime through the last study safety visit. 
 Positive blood test for HBsAg, HCV, HIV-1/2. 
 Clinically significant abnormalities on basic laboratory screening. 
 
Research Specific 
 Exclusionary skin disease history/findings that would confound assessment or prevent 
appropriate local monitoring of AEs, or possibly increase the risk of a local AE. 
 History of chronic skin disease (clinician judgment). 
 Acute skin infection/eruptions on the upper arms including fungal infections, severe 
acne or active contact dermatitis. 
 Allergies that may increase the risk of AEs. 
 Regular use (weekly or more often) of antidiarrheal, anti-constipation, or antacid 
therapy. 
 Abnormal stool pattern (fewer than 3 stools per week or more than 3 stools per day) 
on a regular basis; loose or liquid stools on other than an occasional basis. 
 
Prior exposure to ETEC or Vibrio cholerae 
 History of microbiologically confirmed ETEC or cholera infection in the last 3 years 
 Travel to countries where ETEC or V. cholerae or other enteric infections are 
endemic (most of the developing world) within 3 years prior to dosing (clinician 
judgment). 
 Symptoms consistent with Travelers’ Diarrhea concurrent with travel to countries 
where ETEC infection is endemic (most of the developing world) within 3 years prior 
to dosing, OR planned travel to endemic countries during the length of the study. 
 Vaccination for or ingestion of ETEC, cholera, or E. coli heat labile toxin within 3 
years prior to dosing. 
 Occupation involving handling of ETEC or V. cholerae currently, or in the past 3 
years. 
Investigational product dosages and modes of administration: 

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The investigational products are CssBA and dmLT.  The initial group will assess the safety 
of the products independently with CssBA and dmLT alone. 
Following the initial group, escalating doses of CssBA (5µg, 15 µg, 45 µg) will be given 
with escalating doses of dmLT (100 ng or 500ng). All subjects will receive 3 doses of the 
investigational product 3 weeks apart (study days 1, 22, and 43). The dose of dmLT in 
Groups D and E will be determined from the safety data from previous groups. 
Duration of Treatment:  
Subjects will receive 3 vaccine doses, each 3 weeks apart (study days 1, 22, and 43). In 
person follow up will continue for 28 days after receipt of the last vaccine dose.  
Immunology: 
Primary Immunogenicity: 
 Serum samples will be assayed for antibody titers against LT and CS6. 
 Peripheral Blood Mononuclear Cells (PBMCs) will also be collected to determine 
Antibodies from Lymphocyte Supernatant (ALS) responses to CS6 and LT. 
Exploratory Immunogenicity:  
 Saliva and stool samples will also be collected to assess for secretory IgA responses 
to CS6 and LT. 
 Samples will be collected as outlined in the time and events schedule. In order to support 
additional future exploratory evaluations in systems biology, cells and serum samples 
will be collected for use in a variety of transcriptomic, proteomic, flow cytometry, 
memory B cells, and cytokine analysis. Any testing of samples for these exploratory 
outcomes will be performed under a modification to this protocol to further detail the 
assay(s) or performed under a separate research protocol. 
Statistical methods: 
With the early stage of the product concept/testing, the sample size for this study is designed 
to evaluate preliminary safety data but not designed to show statistically significant 
differences between groups.  Initially, a small number of subjects (n=5) will receive CssBA 
and dmLT independently at the lowest tested doses.  The small number of subjects in each of 
these groups is designed to minimize the risk of acute toxicity in this first-in-human study.  
Subsequent groups are planned with larger sizes (10 per group) for the purpose of safety and 
immunogenicity evaluation.   
 
Rates of all adverse events will be analyzed by Pearson’s Chi-square test (or Fisher’s exact 
test if assumptions are not met for Pearson’s Chi-square) to compare dose levels. Summary 
tables will be created which will indicate the number of subjects who experienced events. 
Vaccine-related events will be tabulated by study group. In addition, tables will be prepared 
to list each adverse event, the number of subjects in each treatment group who experienced 
an event at least once, and the rate of subjects with adverse event(s). Adverse events will be 
divided into defined severity grades (mild, moderate, and severe). The tables will also divide 
the adverse events by severity and relationship to the investigational product. All immunized 
subjects will be included in the safety analysis. 

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For immune responses, both qualitative and quantitative (log10 transformed values for non-
normally distributed samples) results will be analyzed. Geometric mean titers may be 
calculated along with their 95% confidence intervals. An immunological responder will be 
defined as those subjects with a ≥4 fold rise in reciprocal baseline titer. Between groups 
comparisons may be examined with nonparametric tests (Kruskal-Wallis for continuous data 
and Fisher’s exact test for categorical data) unless assumptions are fulfilled for Student’s t or 
χ2.  
Additional comparisons may include repeated measures analysis of variance with study 
group as the between subject factor and sample collection time-points as the repeated factor. 
Only subjects who receive at least 2 vaccine doses will be included in the immunology 
analysis. All statistical tests will be interpreted in a two-tailed fashion using an alpha = 0.05 
to represent statistical significance. 
 
 

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3. 
TABLE OF CONTENTS 
1. 
TITLE PAGE ................................................................................................................1 
2. 
SYNOPSIS ...................................................................................................................5 
3. 
TABLE OF CONTENTS ...........................................................................................10 
4. 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS.............................15 
5. 
INTRODUCTION ......................................................................................................18 
5.1. 
Background .................................................................................................................18 
5.1.1. 
Epidemiology ..............................................................................................................18 
5.1.2. 
CS6 Study Rationale ...................................................................................................18 
5.2. 
Nonclinical studies ......................................................................................................19 
5.2.1. 
Mouse Study ...............................................................................................................19 
5.2.1.1. 
Immunogenicity of CssBA in Mice ............................................................................19 
5.2.2. 
Nonhuman Primate Study ...........................................................................................20 
5.2.3. 
Preclinical GLP Toxicology of CssBA and dmLT in Sprague-Dawley Rats .............20 
5.2.3.1. 
Brief Summary ............................................................................................................20 
5.2.3.2. 
Methods ......................................................................................................................20 
5.2.3.3. 
Results.........................................................................................................................22 
5.2.3.4. 
Conclusions.................................................................................................................24 
5.3. 
dmLT ..........................................................................................................................24 
5.3.1. 
dmLT Clinical Experience ..........................................................................................24 
5.4. 
Vaccine Administration ..............................................................................................25 
6. 
TRIAL OBJECTIVES ................................................................................................26 
6.1. 
Primary Objective .......................................................................................................26 
6.2. 
Secondary Objectives .................................................................................................26 
7. 
INVESTIGATIONAL PLAN .....................................................................................27 
7.1. 
Study Design ...............................................................................................................27 
7.2. 
Number of Subjects ....................................................................................................27 
7.3. 
Screening and Study Schedule....................................................................................27 
7.4. 
Vaccination .................................................................................................................29 
7.4.1. 
Prior to Injection .........................................................................................................29 
7.4.2. 
Vaccine Administration ..............................................................................................29 
7.4.3. 
Post-Vaccination Observation Period .........................................................................30 
7.5. 
Post-Vaccination Follow-up .......................................................................................30 

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7.6. 
Dose Escalation and Down-Selection .........................................................................31 
7.7. 
Study Endpoints ..........................................................................................................31 
8. 
SELECTION AND WITHDRAWAL OF SUBJECTS ..............................................33 
8.1. 
Recruitment .................................................................................................................33 
8.2. 
Subject Inclusion Criteria ...........................................................................................33 
8.3. 
Subject Exclusion Criteria ..........................................................................................33 
8.4. 
Subject Withdrawal Criteria .......................................................................................34 
8.4.1. 
When and How to Withdraw Subjects ........................................................................35 
8.4.2. 
Data Collected for Withdrawn Subjects .....................................................................35 
8.4.3. 
Replacement of Subjects .............................................................................................35 
8.4.4. 
Follow-up for Withdrawn Subjects ............................................................................35 
9. 
TREATMENT OF SUBJECTS ..................................................................................36 
9.1. 
Description of Investigational Products ......................................................................36 
9.2. 
Concomitant Medications ...........................................................................................36 
9.3. 
Treatment Compliance ................................................................................................36 
10. 
INVESTIGATIONAL PRODUCTS AND MANAGEMENT ...................................37 
10.1. 
Investigational Products ..............................................................................................37 
10.2. 
Investigational Product Packaging and Labeling .......................................................37 
10.3. 
Investigational Product Storage ..................................................................................37 
10.4. 
Investigational Product Preparation ............................................................................38 
10.5. 
Investigational Product Accountability ......................................................................38 
11. 
LABORATORY AND CLINICAL ASSESSMENTS ...............................................39 
11.1. 
Sample Collection .......................................................................................................39 
11.1.1. 
Blood Sample Collection ............................................................................................39 
11.1.2. 
Stool Sample Collection .............................................................................................39 
11.1.3. 
Saliva Sample Collection ............................................................................................39 
11.2. 
Specification of Safety Endpoints ..............................................................................39 
11.2.1. 
Demographic/Medical History ...................................................................................39 
11.2.2. 
Vital Signs ..................................................................................................................39 
11.2.3. 
Physical Examination .................................................................................................40 
11.2.4. 
Laboratory Assessments .............................................................................................40 
11.2.4.1. 
Hematology .................................................................................................................40 
11.2.4.2. 
Blood Chemistry .........................................................................................................41 

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11.2.4.3. 
Virus Serology ............................................................................................................42 
11.2.4.4. 
Pregnancy Screen ........................................................................................................42 
11.2.5. 
Local Reactions ..........................................................................................................42 
11.3. 
Immunology ................................................................................................................43 
11.3.1. 
Primary Immunology ..................................................................................................43 
11.3.2. 
Exploratory Immunology ............................................................................................43 
12. 
ASSESSMENT OF SAFETY .....................................................................................44 
12.1. 
Safety Parameters .......................................................................................................44 
12.1.1. 
Safety Criteria for Dose Escalation or Stopping Doses ..............................................44 
12.1.2. 
Criteria for Study Termination ...................................................................................45 
12.2. 
Adverse and Serious Adverse Events .........................................................................45 
12.2.1. 
Definition of Adverse Events .....................................................................................45 
12.2.1.1. 
Adverse Event (AE) ....................................................................................................45 
12.2.1.2. 
Solicited Adverse Events ............................................................................................46 
12.2.1.3. 
Serious Adverse Event (SAE) or Serious Suspected Adverse Reaction ....................47 
12.2.1.4. 
Unexpected Adverse Event .........................................................................................47 
12.2.1.5. 
Serious and Unexpected Suspected Adverse Reactions (SUSARs) ...........................47 
12.2.1.6. 
Other Adverse Event (OAE) .......................................................................................48 
12.3. 
Relationship to Investigational Products ....................................................................48 
12.4. 
Recording Adverse Events .........................................................................................48 
12.4.1. 
Methods/Timing for Assessing, Recording, and Analyzing Safety Endpoints ..........48 
12.4.2. 
Duration of Follow-Up of Subjects after Adverse Events ..........................................49 
12.4.3. 
Severity Assessment ...................................................................................................49 
12.5. 
Reporting Adverse Events ..........................................................................................50 
12.5.1. 
Reporting Serious and Unexpected Adverse Events ..................................................50 
12.5.1.1. 
Reporting to the Sponsor ............................................................................................51 
12.5.1.2. 
Reporting to the IRB ...................................................................................................52 
12.5.2. 
Reporting Additional Immediately Reportable Events to the Sponsor’s 
Representative .............................................................................................................53 
12.5.2.1. 
Pregnancy ...................................................................................................................53 
12.5.2.2. 
AE-Related Withdrawal of Consent ...........................................................................54 
12.5.2.3. 
Pending Inspections/Issuance of Reports ...................................................................54 

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13. 
KNOWN AND POTENTIAL RISKS AND BENEFITS TO HUMAN 
SUBJECTS .................................................................................................................55 
13.1.1. 
Risks/Discomforts to Subjects and Precautions to Minimize Risk ............................55 
13.1.1.1. 
Local Reactions ..........................................................................................................55 
13.1.1.2. 
Systemic Reactions .....................................................................................................55 
13.1.1.3. 
Pregnancy ...................................................................................................................55 
13.1.1.4. 
Lactation .....................................................................................................................55 
13.1.1.5. 
Venipuncture ...............................................................................................................56 
13.1.1.6. 
Allergic Reaction ........................................................................................................56 
14. 
STATISTICS ..............................................................................................................57 
14.1. 
Data Analysis ..............................................................................................................57 
14.2. 
Planned Enrollment and Reason for Sample Size ......................................................58 
14.3. 
Level of Significance to be Used ................................................................................58 
14.4. 
Accounting for Missing, Unused, and Spurious Data ................................................58 
14.5. 
Procedures for Reporting Deviations from the Original Statistical Plan ....................58 
14.6. 
Selection of Subjects to be Included in Analyses .......................................................58 
15. 
DIRECT ACCESS TO SOURCE DATA/DOCUMENTS.........................................59 
15.1. 
Study Monitoring ........................................................................................................59 
15.2. 
Audits and Inspections ................................................................................................59 
15.3. 
Institutional Review Board (IRB) ...............................................................................59 
16. 
QUALITY CONTROL AND QUALITY ASSURANCE .........................................61 
17. 
ETHICS ......................................................................................................................62 
17.1. 
Ethics Review .............................................................................................................62 
17.1.1. 
Review/Approval of Study Protocol ...........................................................................62 
17.1.2. 
Protocol Modifications ...............................................................................................62 
17.1.3. 
Protocol Deviation Procedures ...................................................................................62 
17.2. 
Ethical Conduct of the Study ......................................................................................63 
17.2.1. 
Confidentiality ............................................................................................................63 
17.2.2. 
Compensation for Participation ..................................................................................63 
17.2.3. 
Medical Care for Research-Related Injuries ...............................................................65 
17.3. 
Written Informed Consent ..........................................................................................65 
18. 
DATA HANDLING AND RECORDKEEPING .......................................................67 
18.1. 
Inspection of Records .................................................................................................67 

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18.2. 
Retention of Records ..................................................................................................67 
19. 
PUBLICATION POLICY ..........................................................................................69 
20. 
LIST OF REFERENCES ............................................................................................70 
 

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4. 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
 
The following abbreviations and specialist terms are used in this study protocol. 
Table 2: 
Abbreviations and Specialist Terms  
Abbreviation or Specialist Term 
Explanation 
°C  
degrees Celsius  
Ab  
antibody  
Abs  
antibodies  
AEs  
adverse event  
Ag  
antigen  
ALS  
antibodies from lymphocyte supernatant  
ALT  
alanine aminotransferase  
ASC  
antibody-secreting cell  
AST  
aspartate aminotransferase  
B7A  
Enterotoxigenic Escherichia coli strain expressing 
colonization factor CS6  
BB-178B  
research lot of CssBA  
BP  
blood pressure  
BPR  
batch production record  
BUMED  
United States Navy Bureau of Medicine and Surgery  
BUN  
blood urea nitrogen  
CBC  
complete blood count  
CD4  
T cell cluster of differentiation 4  
CF  
colonization factor  
CFA  
colonization factor antigen  
CfaE  
colonization factor antigen E  
CfaEB  
colonization factor antigen EB  
CFR  
Code of Federal Regulations  
CFU  
colony forming units  
cGMP  
current good manufacturing practices  
Cl-  
Chloride  
CS3  
Coli surface antigen 3  
CS6  
Coli surface antigen 6  
CssA  
Structural subunit of CS6; component of CssBA  
CssB  
Structural subunit of CS6; component of CssBA  
CTC  
Clinical Trials Center  
dsc  
donor strand complemented  
ELISA  
Enzyme-Linked Immunosorbent Assay  
ELISPOT  
Enzyme-Linked ImmunoSpot  
ETEC  
enterotoxigenic Escherichia coli  
FBS  
fetal bovine serum  
FDA  
Food and Drug Administration  
g  
gram  

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Abbreviation or Specialist Term 
Explanation 
G  
gauge  
GI  
gastrointestinal  
GLA  
glucopyranosyl lipid adjuvant  
GLA-AF  
glucopyranosyl lipid adjuvant aqueous formulation  
GLA-SE  
glucopyranosyl lipid adjuvant stable emulsion  
GLP  
Good Laboratory Practice  
GST  
glutathione-S-transferase  
HAI  
hemagglutination inhibition  
HBsAg  
Hepatitis B surface antigen  
HCO3-  
bicarbonate  
HCV  
Hepatitis C virus  
HIB  
Haemophilus influenzae type B  
HIV-1  
human immunodeficiency virus 1  
HIV-2 
human immunodeficiency virus 2 
HR  
heart rate  
ICH  
International Council on Harmonisation  
ID  
Intradermal  
IDRI  
Infectious Disease Research Institute  
IgG  
immunoglobulin G  
IgG1  
Immunoglobulin G1  
IgG2  
Immunoglobulin G2  
IgG3  
Immunoglobulin G3  
IM  
Intramuscular  
IN  
Intranasal  
IND  
investigational new drug  
IRB  
Institutional Review Board  
K  
potassium  
K+  
potassium ion  
kg  
Kilogram  
LT  
Escherichia coli heat-labile enterotoxin  
LT(R192G/L211A) (dmLT)  
Escherichia coli double mutant heat labile toxin with 
two mutations at amino acids 192 and 211  
MD  
Maryland  
MD2  
myeloid differentiation factor 2  
mg  
milligram  
Min  
minutes  
mL  
milliliter  
mLT (LTR192G)  
Escherichia coli mutant heat labile toxin with a 
mutation at amino acid 192  
MOP 
Manual of Procedures 
N  
normal  
Na 
Sodium 
N/A  
not applicable  

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Version 7.0- 31 AUG 2018 
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Abbreviation or Specialist Term 
Explanation 
ND  
not done  
NFkB  
nuclear factor kappa-light-chain-enhancer of activated 
B cells  
NHP  
non-human primates  
NMRC  
Naval Medical Research Center  
OSP  
O-specific polysaccharide  
PBMCs  
peripheral blood mononuclear cells  
PBS  
phosphate-buffered saline  
PI  
Principal Investigator  
PM  
post meridiem (i.e., afternoon)  
PVS  
PATH Vaccine Solutions  
rLTB  
recombinant heat-labile toxin B subunit  
SAEs  
serious adverse events  
SC  
subcutaneous  
SD  
standard deviation  
SLA-SE  
second-generation glycopyranosyl lipid-A stable 
emulsion  
SOP  
standard operating procedure  
spd  
signal peptide 
SRC  
Safety Review Committee  
ST  
heat-stable enterotoxin  
SUSAR 
serious and unexpected adverse event 
T  
temperature  
TC  
transcutaneous  
TCI  
transcutaneous immunization  
TD  
travelers' diarrhea  
TH1  
T helper cell type 1  
TLR4  
toll-like receptor 4  
TLR4-MD2  
toll-like receptor 4-myeloid differentiation factor 2 
complex  
TLRs  
toll-like receptors  
WBC  
while blood cell  
WIRB 
Western Institutional Review Board 
WRAIR  
Walter Reed Army Institute of Research  
WRAIR PBF  
Walter Reed Army Institute of Research Pilot 
Bioproduction Facility  
β-hCG  
Beta human chorionic gonadotropin  
μg  
microgram  
 

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Version 7.0- 31 AUG 2018 
18 
5. 
INTRODUCTION 
5.1. 
Background 
5.1.1. 
Epidemiology 
Worldwide, diarrhea causes approximately 1.7 billion cases annually [1]. In developing 
countries, there is recognition of the disease burden and in many cases, significant efforts have 
been made to improve sanitation, nutrition, and treatment management. However, diarrhea 
related complications still result in approximately 760,000 deaths in children annually, with the 
highest numbers in those younger than two years of life [1, 2].  The recognized need for more 
early effective countermeasures has driven a number of research agendas, including the 
development of enteric vaccines. 
ETEC, one of several pathotypes of diarrheagenic E. coli, causes a secretory diarrhea that can 
range in presentation from mild discomfort to cholera-like purging. It is the most prevalent 
bacterial cause of childhood diarrhea in developing countries, and while the estimated number of 
ETEC episodes and deaths vary among studies, in one estimate, ETEC was thought to cause 210 
million cases of diarrhea and 380,000 deaths annually among infants and young children [3-7]. 
ETEC illness in the young has also been associated with growth faltering [8], and the repeated 
episodes caused by this infection are likely to lead to declines in both physical and cognitive 
development [9], which in turn are considered to have attendant macroeconomic consequences in 
countries and regions most heavily afflicted [10]. It is also the leading cause of travelers' 
diarrhea, etiologically implicated in 30-50% or more of cases [11-14], and this may be markedly 
underestimated due to the insensitivity of testing methods [15]. Its dual importance in global 
public health and military/travel medicine has galvanized policy makers in both sectors to 
develop a safe, effective ETEC vaccine, though such efforts remain under-resourced. 
5.1.2. 
CS6 Study Rationale 
ETEC express adhesive fimbriae [also known as colonization factors (CFs)], surface-exposed 
polymeric protein appendages that plays a vital role in the initial step of ETEC pathogenesis. CFs 
mediate initial ETEC adherence to, and colonization of, the small intestine, after which ETEC 
secrete one or both of two enterotoxins that induce fluid and electrolyte secretion resulting in 
watery diarrhea. The two enterotoxins produced by ETEC are heat-stable enterotoxin (ST) and 
heat-labile enterotoxin (LT). CFs have long been a prime target for vaccine research and 
development. Their role as protective antigens has been substantiated by a number of studies in 
populations naturally exposed to ETEC diarrhea as well as volunteer studies of experimentally 
induced diarrhea, as has the role of LT enterotoxin [16-20]. Evidence for the preventive role of 
anti-CF immunity also derives from studies showing that bovine milk antibody product with high 
antibody titers against ETEC, and more specifically, purified colonization factor antigens, 
provided protection to humans in challenge studies [21, 22]. To date, more than twenty-two 
serologically distinct CFs have been identified. 
CS6 is an atypical polymeric antigen that is highly prevalent among ETEC disease isolates from 
various geographic regions [20, 23, 24].  It is a heteropolymer composed of two structural 
subunits, CssA and CssB, in a ca. 1:1 ratio that has been confirmed with publication of the CssA 

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and CssB crystal structures [25]. Recent reports provide evidence that CS6 binds to the human 
intestinal cell lines Caco-2 and INT407 [26, 27], consistent with the adhesive role that CS6 is 
presumed to play in ETEC disease pathogenesis. Jansson et al. reported that purified CS6 and 
recombinant CssB fused to glutathione-S-transferase (GST) were both shown to bind to intestinal 
glycosphingolipid sulfatide by thin layer chromatography [28]. 
Given the relatively high global prevalence of CS6-ETEC, we view the development of a 
protective, recombinant derivative of CS6 as critical to our overall strategy to develop a broadly 
protective multivalent ETEC adhesin subunit vaccine. As such, with the current data known, 
development of the prototype CS6 adhesin-based vaccine began with the development and 
characterization of in cis donor strand complemented variants of CssA (dsc16CssA[His]6, 
referred to as dscCssA) and CssB (dsc16CssB[His]6, referred to as dscCssB).  Based on multiple 
lines of evidence, ntd14dsc16BCssBA (a variant of the original dscCssBA in which the N-
terminal 14 amino acids have been removed and a heterologous CssB-derived donor strand is 
used to complement the C-terminal CssA), here after termed CssBA, was selected as the lead 
vaccine prototype. This product was assessed serially in a mouse immunogenicity model and an 
A. nancymaae NHP vaccination-challenge model. We then scaled up fermentation and 
purification processes, in preparation for bioproduction under cGMP conditions. Subsequent 
mouse immunogenicity studies of the cGMP CssBA lot demonstrated similar anti-CS6 IgG 
response to that of the research grade lot. 
 
5.2. 
Nonclinical studies 
5.2.1. 
Mouse Study 
5.2.1.1. 
Immunogenicity of CssBA in Mice 
In Balb/C mice, CssBA (research grade) elicited strong IgG and IgA anti-CS6 serum titers after 
intradermal (ID) vaccination (admixed with mLT adjuvant), at levels that were similar or greater 
in magnitude compared to any of the other heterodimers. Three IN immunizations with 125 or 25 
µg of CssBA elicited levels of serum anti-CS6 Abs at day 42 comparable to the ID immunization 
with the same protein at 10 µg (see Table 3). A dose dependent drop in the levels of serum anti-
CS6 IgG and IgA Abs was observed when IN immunizations were performed with 2.5 and 0.25 
µg of CssBA. Similar patterns were observed with sera collected on day 28, after two 
immunizations (data not shown). 
Table 3. 
CssBA Mouse Immunogenicity Responses in Baseline Evaluation 
Protein 
Dose 
Route 
LT Component 
Anti-CS6 IgG titer (Log10) 
mean±SD 
Anti-CS6 IgA titer (Log10) 
mean±SD 
CssBA 
10 µg 
ID 
LTR192G (0.1 µg) 
5.3±0.24 
1.7±0.33 
CssBA 
125 µg 
IN 
LTR192G (1.5 µg) 
5.5±0.19 
4.1±0.4 
25 µg 
IN 
LTR192G (1.5 µg) 
5.6±0.18 
4.2±0.15 
2.5 µg 
IN 
LTR192G (1.5 µg) 
4.7±0.15 
3.1±0.92 

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0.25 
µg 
IN 
LTR192G (1.5 µg) 
2.6±1.3 
1.4±0 
 
5.2.2. 
Nonhuman Primate Study 
Following the mouse study, the vaccine (research grade) was tested in owl monkeys (A. 
nancymaae). The monkeys were vaccinated intradermally with CssBA plus dmLT (100ng), CS6 
plus dmLT or dmLT alone, while negative control monkeys were inoculated with PBS on days 0, 
14, 28, and 84. There were eight monkeys per group. The products were dosed: CssBA 100µg, 
dmLT 100ng, and CS6 100µg.  Animals vaccinated with CssBA or CS6 demonstrated high 
serum anti-CS6 IgA and IgG titers following the second vaccination, which remained high 
following the third and fourth vaccinations.  All animals that received dmLT demonstrated high 
serum anti-dmLT IgA and IgG titers following the second vaccination.  Following vaccination, 
the monkeys were challenged on study day 98 (two weeks after last vaccination dose) with a CS6 
expressing, LT+ ETEC strain (B7A). Monkeys vaccinated with CssBA and dmLT demonstrated 
100% protective efficacy, whereas vaccination with CS6 and dmLT provided 80% protective 
efficacy. Monkeys vaccinated with dmLT alone demonstrated a 40% protective efficacy, 
although this result was not statistically significant (see Table 4). 
 
Table 4. 
Protective Efficacy of Intradermal Vaccination Group Compared to PBS 
Control (N = 8/group) 
Group 
Regimen 
Diarrhea Attack 
Rate (%) 
Protective Efficacy 
(%) 
P-value 
1 
CS6 + dmLT 
12.5 
80 
0.12 
2 
CssBA + dmLT 
0 
100 
0.03 
3 
dmLT 
37.5 
40 
0.62 
4 
PBS 
62.5 
- 
- 
 
5.2.3. 
Preclinical GLP Toxicology of CssBA and dmLT in Sprague-Dawley Rats 
5.2.3.1. 
Brief Summary 
A repeat dose GLP-compliant toxicology study was performed in Sprague-Dawley rats to assess 
the toxicity and local site reactogenicity of subunit ETEC vaccine candidates, CssBA and dmLT 
by IM injection. The study also included investigation of the ID route of administration and with 
addition of SLA-SE via IM injection, although the ID route and SLA-SE will not be evaluated in 
this protocol. This study was conducted in compliance with GLP at Sinclair Research in 
Auxvasse, Missouri.  
5.2.3.2. 
Methods 
One-hundred and twenty male and 120 female rats were randomized into eight study groups by 
stratified body weights as shown in Table 5.  

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Version 7.0- 31 AUG 2018 
21 
Table 5. 
Preclinical Toxicology Study Design 
Group 
Dose 
Route 
Test Article 
(Dose/Inj.) 
Dose  
Vol. 
(mL/In) 
No. of Animals 
Main 
Recovery 
CssBA 
dmLT 
SLA-SE 
M 
F 
M 
F 
1 
ID 
NA a 
NA a 
NA a 
0.1 
10 
10 
5 
5 
2 
ID 
45 µg 
- 
- 
0.1 
10 
10 
5 
5 
3 
ID 
45 µg 
1 µg 
- 
0.1 
10 
10 
5 
5 
4 
IM 
NA a 
NA a 
NA a 
0.25 
10 
10 
5 
5 
5 
IM 
45 µg 
0.5 µg 
 
0.25 
10 
10 
5 
5 
6 
IM 
45 µg 
0.5 µg 
5ug 
0.25 
10 
10 
5 
5 
7 
IM 
45 µg 
1 µg 
- 
0.25 
10 
10 
5 
5 
8 
IM 
45 µg 
1 µg 
5 µg 
0.25 
10 
10 
5 
5 
Note: ID = Intradermal injection; IM = Intramuscular Injection; 
a Saline Control Group  
Animals were immunized a total of four times, by the ID or IM routes as shown in Table 5, with 
each dose administered three weeks apart on Study Days 1, 22, 43, and 64. Animals assigned to 
the main study cohort were sacrificed on Study Day 66, two days after the fourth and final 
vaccination. A second cohort of animals, designated as recovery, went through a 28-day 
treatment-free period prior to sacrifice on Study Day 92.   
Standard toxicology endpoints were evaluated, including body weights, food consumption, body 
temperatures at 6 and 24 hours post-dose, ophthalmology, detailed physicals, and daily clinical 
observations. Injection sites were evaluated and scored, according to a modified Draize (1965) 
method, daily at 24, 48, and 72 hours for all animals following each dose. If a positive score 
(> 0) was observed for an individual animal at 72 hours post dose, that animal’s dose site was 
evaluated daily until a score of zero for both erythema and edema was achieved. Injection sites 
were also evaluated daily as part of the clinical observations and included an evaluation of 
apparent pain and other signs of local tolerance or toxicity issues such as presence of a 
hematoma, ulceration, vesiculation, or eschar.  
To avoid sampling effects on hematology parameters, animals were assigned for evaluation of 
hematology, coagulation, or clinical chemistry during the in-life phase on Study Day 3 (all 
parameters) and Study Day 7 (coagulation and chemistry only). All parameters were evaluated 
on all animals at baseline and at sacrifice. Immunology was conducted to evaluate anti-CS6 and 
anti-LT serum IgG at baseline and at sacrifice.  
At each sacrifice the assigned animals underwent a full gross necropsy, including organ weights, 
and tissues were collected. Microscopic examination of all tissues was conducted on animals 
sacrificed on Study Day 66. Only injection sites and gross lesions (none present) were evaluated 
from animals sacrificed on Study Day 92. 
 

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22 
5.2.3.3. 
Results 
There were no significant effects on ophthalmology, body weight gains, food consumption, or 
body temperatures. Test article related changes in hematology, coagulation parameters, and 
clinical chemistry were observed, including increases in platelet count, fibrinogen, and increases 
of the globulin fraction of serum total protein. These findings were consistent with an acute 
inflammatory or immune-stimulatory effect of vaccination, short-lived, and non-adverse. 
Systemically, vaccination with CssBA and dmLT was well-tolerated and immunogenic. Test 
article related clinical findings were limited to the injection site.  
IM co-administration of 45 µg of CssBA and 0.5 µg of dmLT was associated with moderate to 
severe edema, including a more regional limb swelling, following the first injection. At 
subsequent doses the severity of this finding was substantially reduced. Erythema was minimal 
to moderate at the injection site. The incidence and severity of dose-site in-life findings in 
animals treated with saline (Group 4) or 45 µg CssBA and 0.5 µg dmLT (Group 5) is 
summarized in Table 6. 
Table 6: 
Dose Site Observations Following Vaccination 
Dose Administration Day 
Dose Day 1 
Dose Day 22 
Dose Day 43 
Dose Day 64 
Group 
4 
5 
4 
5 
4 
5 
4 
5 
CssBA (µg/mL) 
NA 
45 
NA 
45 
NA 
45 
NA 
45 
dmLT (µg/mL) 
NA 
0.5 
NA 
0.5 
NA 
0.5 
NA 
0.5 
N 
30 
30 
30 
30 
30 
30 
30 
30 
Limb 
Swelling 
% 
Distribution 
of Maximum 
Magnitude  
None 
100% 
7% 
100% 
73% 
100% 
47% 
90% 
37% 
Slight 
0% 
40% 
0% 
27% 
0% 
43% 
10% 
23% 
Mod 
0% 
53% 
0% 
0% 
0% 
7% 
0% 
40% 
Sev 
0% 
0% 
0% 
0% 
0% 
3% 
0% 
0% 
Duration 
Mean 
0 
3.03 
0 
0.37 
0 
1.2 
0.1 
0.87 
SD 
0 
1.35 
0 
0.67 
0 
1.3 
0.31 
0.78 
Erythem
a 
% 
Distribution 
of Maximum 
Score  
0 
100% 
87% 
100% 
93% 
100% 
97% 
100% 
100% 
Min 
0% 
3% 
0% 
7% 
0% 
3% 
0% 
0% 
Mild 
0% 
3% 
0% 
0% 
0% 
0% 
0% 
0% 
Mod 
0% 
3% 
0% 
0% 
0% 
0% 
0% 
0% 
Sev 
0% 
3% 
0% 
0% 
0% 
0% 
0% 
0% 
Duration 
Mean 
0 
0.13 
0 
0.07 
0 
0.07 
0 
0 
SD 
0 
0.35 
0 
0.25 
0 
0.37 
0 
0 

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Dose Administration Day 
Dose Day 1 
Dose Day 22 
Dose Day 43 
Dose Day 64 
Group 
4 
5 
4 
5 
4 
5 
4 
5 
CssBA (µg/mL) 
NA 
45 
NA 
45 
NA 
45 
NA 
45 
dmLT (µg/mL) 
NA 
0.5 
NA 
0.5 
NA 
0.5 
NA 
0.5 
N 
30 
30 
30 
30 
30 
30 
30 
30 
Edema 
% 
Distribution 
of Maximum 
Score  
0 
100% 
0% 
100% 
100% 
100% 
73% 
90% 
50% 
Min 
0% 
23% 
0% 
0% 
0% 
27% 
10% 
10% 
Mild 
0% 
53% 
0% 
0% 
0% 
0% 
0% 
37% 
Mod 
0% 
17% 
0% 
0% 
0% 
0% 
0% 
3% 
Sev 
0% 
7% 
0% 
0% 
0% 
0% 
0% 
0% 
Duration 
Mean 
0 
2.83 
0 
0 
0 
0.27 
0.1 
0.53 
SD 
0 
1.46 
0 
0 
0 
0.45 
0.31 
0.57 
Min = minimal; mod = moderate; sev = severe 
No manifestations of gross toxicity at the injection site such as hematoma, vesiculation, or ulcers 
were observed at this dose and route.  
At necropsy on Day 66, there were no gross observations noted for this treatment group nor 
differences in organ weights, either absolute or relative to body or brain weight. Microscopically, 
hyperplasia of the inguinal lymph node was observed in some animals, consistent with a post-
vaccination response. Injection site observations included minimal to mild inflammation of 
skeletal muscle and subcutis at all four injection sites, although at a higher incidence in the left 
biceps femoris (second dose; Day 22) and left gluteus (fourth dose, Day 64).  Following a 28-day 
recovery period, these findings had largely resolved and no chronic process was apparent. 
Overall, the vaccine induced an acute reactogenicity following the first dose, but was overall 
well-tolerated and non-adverse at 45 µg CssBA and 0.5 µg dmLT. 
When the dose of dmLT was increased to 1 µg with 45 µg CssBA IM, the incidence and severity 
of swelling increased. There were also observations of apparent pain in some dosed animals 
following the first dose. Changes in clinical pathology parameters associated with inflammation 
such as fibrinogen, platelet increases, changes in clotting time, and increase in the globulin 
fraction were higher in this group, consistent with the dose site clinical findings. As a result, the 
clinical trial design was modified to escalate to the 0.5 µg dmLT dose level and not exceed that 
dose.  
Addition of SLA-SE, regardless of the dose of dmLT (0.5 or 1 µg), via IM injection led to severe 
edema and limb swelling in 100% of vaccinated animals after the first dose. Animals in these 
groups (Groups 6 and 8) also exhibited apparent pain upon palpation of the dose site following 
the first dose; six animals were observed limping or favoring the injected limb and three animals 
were treated for pain with buprenorphine. Pain was also observed following the fourth dose. The 
incidence and severity overall decreased for these groups over the course of the study, but some 
severe edema and limb swelling was still observed after the fourth dose. Microscopically, 
minimal to marked inflammation of the skeletal muscle, subcutis, and/or dermis was observed at 

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the injection sites as well as hematoma, fibrin exudation, and subcutaneous edema. Severity and 
incidence was higher at dose sites 2 (left biceps femoris) and 4 (left gluteus), suggesting that the 
inflammatory response is more regional and related to the local draining lymph node. These 
findings are consistent with vascular damage related to reactogenicity. Due to the severity of 
local effects and microscopic findings, the clinical trial design was modified to not include SLA-
SE adjuvant. 
By the ID route, 45 µg CssBA was well-tolerated. Local site observations were similar to that 
seen in the saline ID route control animals (Group 1). Mild and transient changes in hematology, 
coagulation, and clinical chemistry were observed, consistent with an immune-stimulatory effect 
of vaccination. There were few test-article related findings microscopically, with just minimal 
residual inflammation observed at the injection site. Serum IgG anti-CS6 responses were low, 
even after four doses. All females, but not all males, seroconverted by Study Day 92. 
When 1 µg of dmLT was co-administered with 45 µg CssBA intradermally, an adverse effect 
was observed. As the study progressed, dose site reactogenicity, specifically erythema, was 
substantially increased in all animals assigned to this treatment group. By the fourth and final 
dose 25 of 30 animals were observed with an eschar at the injection site and were determined to 
exhibit apparent pain upon palpation of the injection site. Hematoma was also observed in 21 of 
30 animals and ulcers were observed in 7 of 30 animals. Microscopic evaluation revealed 
hematoma, inflammation of the dermis, subcutis, and underlying muscle, and fibrin exudation. 
These findings were especially pronounced at dose sites 2 (left biceps femoris) and 4 (left 
gluteus). These responses are consistent with reactogenicity. The clinical trial design was 
therefore modified to remove the ID route of administration. 
5.2.3.4. 
Conclusions  
In Sprague-Dawley rats, 45 µg CssBA and 0.5 µg dmLT by IM injection is well-tolerated and 
immunogenic. Mostly mild to moderate, short-lived edema and erythema as well as systemic 
inflammatory effects were observed, but non-adverse. This study supports the revised design of 
the Phase 1 study. 
A higher dose of dmLT (1 µg) with 45 µg CssBA IM is associated with increased inflammatory 
responses in rats, thus is not supported by current animal studies. Addition of 5 µg SLA-SE to 45 
µg CssBA and either 0.5 or 1 µg dmLT IM led to adverse effects such as limping and pain and 
an apparent immunotoxic reaction. Thus, a higher dose of dmLT and inclusion of SLA-SE are 
not supported by current animal toxicology data. 
ID administration of 45 µg CssBA was well-tolerated, but poorly immunogenic. Addition of 
1 µg of dmLT to 45 µg CssBA by ID injection led to substantial and escalating dose site 
reactogenicity and apparent immunotoxicity. This study supports the safe dosing of 45 µg 
CssBA by the ID route, but the inclusion of dmLT at any dose level is not supported. 
5.3. 
dmLT  
5.3.1. 
dmLT Clinical Experience 
Given the likely importance of anti-LT responses in any successful ETEC vaccine, CssBA will 
be co-administered with dmLT. To that end, a series of phase 1 trials with the CFA/I adhesin 
prototype candidate have recently been completed (Riddle and Gutierrez et al, unpublished data, 

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Version 7.0- 31 AUG 2018 
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NCT01382095, NCT01644565) in which the prototype vaccine was co-administered 
transcutaneously and intradermally with LTR192G, or mLT, an Escherichia coli mutant heat 
labile toxin with a mutation at amino acid 192. While 50 μg of LTR192G administered 
transcutaneously was safe and induced serum IgG and IgA responses in 75-100% of subjects, the 
magnitude of the responses was relatively low across all immune parameters. In contrast, anti-LT 
responses in subjects administered 100 ng of mLT intradermally was safe and demonstrated both 
a higher rate of serologic responses and ALS responses. Intradermal use of mLT was associated 
with transient mild to moderate induration, hyperpigmentation and erythema in all subjects that 
did not increase in intensity with subsequent doses. Local pruritus was also seen but somewhat 
less frequently, and mLT given alone at 100 ng gave similar local reactogenicity results to the 
mLT plus CFA/I adhesin prototype vaccine combination. Given the tolerable safety profile and 
immunogenicity results, the intradermal vaccine was moved into a Phase 2/2b challenge study in 
which a total of 56 subjects underwent vaccination and similar to the phase 1 trial there were no 
vaccine-attributable systemic adverse events while vaccine site reactions (rash) and pruritus were 
common. There is no previous human experience of dmLT given by the intradermal or 
intramuscular route; however, a first in man Phase 1 dose-escalation study evaluating the safety 
and tolerability of dmLT administered via the intradermal route is currently underway at 
CCHMC (DMID Protocol 13-0013; NCT02531685) and oral dmLT administered alone in a dose 
of up to 100 µg and together with an inactivated ETEC vaccine and live attenuated ETEC 
vaccine in a dose of up to 25 µg were all safe in their respective Phase 1 studies.  Finally, 
comparative preclinical data in mice indicate that dmLT may be less reactogenic at the injection 
site than mLT. 
5.4. 
Vaccine Administration 
The investigational products will be administered intramuscularly.  Ease and experience with IM 
vaccinations, without need for costly devices or additional training, would allow for more 
optimal transition to use in developing countries. Neither of these vaccine antigens has been 
administered intramuscularly so the anticipated immune response profile is uncertain.  
The purpose of the study is to determine if immunization with CssBA with dmLT is safe and 
immunogenic. If the vaccines are safe and induce robust humoral and mucosal immune 
responses, a vaccination/challenge study may occur to assess efficacy in a robust experimental 
ETEC challenge model. 

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6. 
TRIAL OBJECTIVES 
6.1. 
Primary Objective 
 Evaluate the safety of CssBA ± dmLT given by IM injection  
6.2. 
Secondary Objectives 
 Evaluate immune responses following IM vaccination with CssBA ±dmLT 
 Identify a safe and immunogenic dose and route of a CssBA-based vaccine to be used in 
a subsequent vaccine and experimental challenge trial 

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Version 7.0- 31 AUG 2018 
27 
7. 
INVESTIGATIONAL PLAN 
7.1. 
Study Design 
This is an open-label Phase 1 clinical trial of CssBA±dmLT in which a total of 50 subjects will 
receive three vaccinations via IM injection on days 1, 22, and 43 (See Table 7). Dose escalation 
of CssBA from 5µg to 15µg to 45µg, and dmLT from 100 to 500ng will take place as outlined 
below. Group A is considered a pilot group in which CssBA and dmLT will be administered 
separately. All 3 doses will be administered and subjects monitored for safety 7 days after the 
third vaccination, prior to the enrollment of subjects in Group B.  Following Groups A, B, C, and 
D, a safety analysis will be completed and reviewed by the Safety Review Committee (SRC). 
Progression to subsequent groups will be dependent on safety measured in the first 7 days after 
the third vaccination of the previous group.  
Table 7. 
Study Design of Phase 1 Clinical Trial of Intramuscularly Administered 
CssBA with dmLT (N=50) 
Group 
N 
Route 
CssBA (µg) dmLT (ng) 
A 
5 
IM 
5  
0 
5 
IM 
0 
100 
B 
10 
IM 
5  
100 
C 
10 
IM 
5 
500 
D* 
10 
IM 
15 
100/500 
E* 
10 
IM 
45 
100/500 
*Plan to proceed with 500ng dose; however, if there is an aberrant safety signal in Group C, will 
proceed with the 100 ng dose (presuming no prior signal in Group B) 
7.2. 
Number of Subjects 
Based on prior studies with similar study designs, sample collections and inclusion/exclusion 
criteria, we anticipate screening approximately 5 subjects for every one subject that is enrolled in 
the study.  Therefore we anticipate approximately 250 individuals will be screened. A maximum 
of 50 subjects are planned for this study.  Up to four alternates per group will be selected as well 
that may be enrolled in the event a subject becomes ineligible prior to receipt of the 
investigational product. 
7.3. 
Screening and Study Schedule 
The screening visit is scheduled -90 to -2 days before the first vaccination. Fully informed, 
written consent must be obtained from each subject prior to conducting any study procedure. 

CssBA Phase 1 Protocol 
 
 
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Two consent forms will be signed; a consent form for study participation and a separate consent 
form for HIV testing. To assess understanding, a comprehension assessment will be administered 
after the subjects sign the study participation written consent. The following 
evaluations/ procedures will be carried out: 
 Oral and/or video presentation of clinical trial design, risks and study schedule 
 Review of informed consent document 
 One-on-one discussions with principal investigator (PI) or sub investigator 
 Signature of informed consent document 
 Comprehension test (minimum of 70% accuracy required for participation) 
 Medical history – between Days - 90 to -2 
 Physical examination – between Days -90 to -2 
 Vital Signs – between Days -90 to -2 
 Screening laboratory analysis (allowable time period for accepting test): 
− Complete blood count (between Days -90 to -2) 
− Serum transaminases (ALT/AST) (between Days -90 to -2) 
− Na, K+, CL-, HCO3, glucose, BUN and creatinine (between Days -90 to -2) 
− Pregnancy test (urine -hCG) for women, as well as 0-24 hours before 
each vaccination 
− Total serum IgA (between Days -90 to -2) 
Additionally, approximately 7 days prior to vaccination (allowable range: Days -14 to -2), 
subjects will have a follow-up medical history and brief physical exam to ensure ongoing 
eligibility. A verbal educational brief will also be provided to the subjects at this visit. The 
following screening laboratory analyses will be carried out: 
 Serum HIV 1/2 antibody (between Days -14 to -2) 
 Hepatitis B surface antigen (HBsAg) (between Days -14 to -2) 
 Hepatitis C virus (anti-HCV antibody) (between Days -14 to -2) 
If initial screening is within the -14-day window, it can count as both the initial screening and 
pre-vaccination visit. 
A screening test for any of the above assessments may be repeated (total screening blood 
volume not to exceed 30 mL) in the event of a laboratory error (i.e., hemolyzed sample) or in 
the event the screening physician believes: 1) the laboratory test has identified a normal variant 
of a healthy state and 2) the variant is no more than a Grade 1 abnormality.  This repeat 
screening applies to the initial screening tests [(executed between -90 to -2 days) and the repeat 
screening labs if executed (between days -14 to -2)].  
The Walter Reed Army Institute of Research Clinical Trials Center (WRAIR CTC) may use a 
screening protocol in recruiting subjects for this study. All screening procedures outlined in this 
section may be completed under this screening protocol. 
During this screening process, the laboratory assays will be reviewed to determine if any 
clinical laboratory abnormalities exist that would preclude study participation. Subjects who 
have only 1 mild (grade 1) abnormality may be included if the PI determines that their 

CssBA Phase 1 Protocol 
 
 
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participation will not present undue risk to the subject. Subjects with 2 or more mild 
abnormalities may be included in the study only with the consensus of the PI and the research 
monitor. Subjects with clinical laboratory abnormalities of greater than mild severity will not 
be eligible for this clinical trial. 
The clinical toxicity grading scale that will be used as a guideline is based on guidance from the 
Center for Biologics Evaluation and Research (Guidance for Industry: Toxicity Grading Scale 
for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials). 
Final grading determination will be made by the PI based on local normal lab value ranges for 
the specific lab and clinical symptoms. If any additional safety labs are performed, the FDA 
Guidance for Industry will be utilized. 
7.4. 
Vaccination 
After initial screening, eligible subjects will be vaccinated, dependent upon vaccine group 
assignment, with CssBA with or without dmLT via IM route to upper arms. The vaccine will be 
administered on days 1, 22, and 43 in accordance with formulation and vaccination procedures. 
IM injection of the vaccine will be given using a 1inch needle in the deltoid region (study 
clinician may choose another location on a case-by-case basis) by a study clinician. 
7.4.1. 
Prior to Injection 
Provisionally qualified subjects are scheduled to arrive at study site on the morning of Day 1 and 
will undergo the following procedures: 
 Interval medical history is obtained, including medications. 
 Vital signs are measured prior to dosing. 
 A directed physical exam is performed. 
 For female subjects, a urine pregnancy test performed. 
 Blood samples are obtained for immunological testing. 
 Pregnancy (if female of childbearing potential) test results from this visit and all other 
qualification-related laboratory test results from the screening visit(s) are reported and 
reviewed. 
 New health information is reviewed to determine qualification for enrollment. 
 Inclusion/Exclusion criteria are reviewed to assess continued eligibility. 
 The prospective injection site is examined visually and by palpation. 
7.4.2. 
Vaccine Administration 
Approximately 50 qualified subjects will be enrolled for vaccination; if an insufficient number of 
qualified subjects is available to complete enrollment of the group, partial enrollment may occur, 
with the balance of enrollment to occur on later date(s). The injection site will be observed prior 
to dose administration (as above). If a qualified subject selected for enrollment is disqualified 
prior to enrollment/randomization and dose administration (e.g., withdraws consent or the PI 
reconsiders and disqualifies for a documented reason), another qualified subject will be selected 
for enrollment in place of the disqualified subject.  

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7.4.3. 
Post-Vaccination Observation Period 
Subjects will remain at the site for at least 30 minutes post-injection. During this time, the 
subject will be provided with a Memory Aid, follow-up visit information, and contact telephone 
numbers for study staff; subjects will be instructed on the use of the materials. 
After at least 30 minutes, the following procedures will be done: 
 Vital signs will be measured. 
 The injection site will be assessed and any findings will be recorded. Any complaints or 
reactogenicity signs or symptoms will be assessed and documented. 
 The PI (or designee) may determine that a subject requires further on-site observation; 
additional site or clinical assessments may be completed as needed. 
When all study-related procedures are complete and the PI (or designee) determines that a 
subject’s condition is acceptable, the subject will be discharged from study site. 
Subsequent immunizations will be performed on the alternate upper arm from the previous 
vaccination. An exception may be needed if there is pain/tenderness, erythema, edema, pruritus, 
induration, or rash at the planned site of vaccination. In this case, the investigator may exercise 
the discretion to administer the vaccination on the same arm as the previous vaccination. The site 
of vaccine administration will be recorded in the source documents. 
7.5. 
Post-Vaccination Follow-up 
Subjects will return for follow-up approximately 24 hours and 7 days after the first vaccination 
for a clinical evaluation which includes collection of vital signs, assessment of AEs, and review 
of changes in medical history, concomitant medications, and targeted clinical assessment. For 
subsequent vaccinations, each subject will undergo these same clinical evaluations at 
approximately 24 hours and 7 days after the second and third vaccinations. A symptom memory 
aid will be used by the subjects to monitor local vaccine site findings and symptoms and this will 
be reviewed at the follow-up visits. 
Blood, saliva, and stool collections for safety and immunogenicity measures will be collected 
throughout the vaccination phase (See Study and Events Schedule; Table 8). For female subjects, 
a pregnancy test will be performed prior to each vaccination and on their last study visit. 
Study clinicians will review the memory aid with each subject and use the memory aid to 
complete visit source documents (clinical encounter forms, AE forms) and progress notes. The 
clinician will review entries with subject and any changes made to entries will be initialed and 
dated by the subject. 
Approximately 6 and 12 months from last vaccination, all subjects will be contacted via 
telephone to assess for a final safety assessment. In addition to soliciting history of any 
persistence of skin findings associated with vaccine sites, subjects will be asked about the 
diagnosis of any of the conditions included in the Medically Attended Adverse Events (MAAEs) 
Addendum. A history for any additional serious new diagnoses or hospitalizations will also be 
solicited. Identification of any of these conditions will be reported as an SAE per section 12.5.1. 

CssBA Phase 1 Protocol 
 
 
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Three attempts will be made and documented; after 3 unsuccessful attempts a certified letter 
and/or email will be used to request the subject to contact the study team. 
7.6. 
Dose Escalation and Down-Selection 
The decision to advance to the next group will be based solely on the safety assessment. A dose 
level with no occurrence of stopping criteria will prompt moving to the next higher level. All 
safety data will be summarized and reviewed with the SRC prior to advancing to dose escalation. 
Approximately one week after the first group (Group A) receives the third vaccination dose 
(Day 50), an interim Safety Report will be prepared by the PI and Study Statistician for review 
by the SRC. The content of the report will be agreed upon by the PI and the SRC and will 
include, but not be limited to, all adverse events (solicited, unsolicited, expected and unexpected) 
as well as relevant safety endpoints. Advancement to Group B will be based entirely on this 
safety assessment. The SRC’s concurrence to advance to the next group will be made and 
provided in written format. This process will be repeated after groups B, C, and D before 
enrollment for the next group. 
dmLT will be administered at the 500 ng dose in groups D and E if no significant reactogenicity 
is observed in Group C. 
7.7. 
Study Endpoints 
 Safety: Safety monitoring will be undertaken using in-person symptom surveillance, 
symptom memory aids, and targeted physical exams. AE monitoring will survey and 
specifically inquire about fever (oral temperature > 100.4oF), malaise, headache, rash, 
pain, and extremity pain, swelling, or local reactions (further addressed in 
section 12.2.1.2). Clinical definitions will be used to grade the severity of symptoms in 
accordance to the following severity scale: 
Mild 
Grade 1 
Does not interfere with routine activities 
Minimal level of discomfort  
Moderate 
Grade 2 
Interferes with routine activities 
Moderate level of discomfort  
Severe 
Grade 3 
Unable to perform routine activities 
Significant level of discomfort  
Potentially life-threatening Grade 4 
Hospitalization or ER visit for potentially 
life-threatening event  
 
 Immunology: Seroconversion to LT and CS6 will be defined as a ≥ four-fold increase in 
endpoint titer between pre-and post-vaccination samples. Mucosal responses will be 
assessed similarly after adjusting for total IgA. A positive ALS response will require a 
four-fold rise in antibody titers between pre and post vaccination samples. 

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Table 8. 
Time and Events Schedule 
The data will be locked following entry of the day 71 data. 
1 Chemistry includes serum electrolytes, glucose, BUN, creatinine, AST, and ALT  
2 Clinical checks: baseline physical exam and medical history at screening, pre- and post-vaccination medical history and targeted physical exams (subjects will be observed for 30 minutes post-vaccination). 
3 PBMCs include collection for Antibody Secreting Cells (ASC), Antibody-Lymphocyte Supernatant (ALS), Memory-B cells. These samples may also be stored and utilized in future expanded 
immunology to include transcriptomics, proteomics, flow cytometry, and cytokine analysis. 
4 Total approximate blood volume to be collected for cohorts A and B: 446 ml 
5 Total approximate blood volume to be collected for cohorts C, D, and E: 414 ml 
Study Event 
Screen 
-7 
1 
2 
8 
22 
23 
29 
43 
44 
50 
57 
71 
223 
403 
Compliance Ranges 
-90 to -2 
-14 
to -2 
NA 
NA 
7-9 d 
20-
24 d 
N/A 
27-
31 d 
41-
45 d 
N/A 
47-
53 d 
54-
60 d 
67-
75 d 
± 1 
mo 
± 1 
mo 
Study Briefing 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Comprehension Assessment 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Screening Medical History and 
Physical Examination 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CBC 
X 
 
 
 
X 
 
 
X 
 
 
 
X 
 
 
 
Chemistry1 
X 
 
 
 
X 
 
 
X 
 
 
 
X 
 
 
 
Anti-HIV-1/2 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
HBsAg 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Anti-HCV 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Serum IgA 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Vital signs (BP, HR, T) 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Urine pregnancy test 
X 
 
X 
 
 
X 
 
 
X 
 
 
 
X 
 
 
Vaccination 
 
 
X 
 
 
X 
 
 
X 
 
 
 
 
 
 
Clinical check2 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Serology 
 
 
X 
X 
 
X 
 
 
X 
 
 
 
X 
 
 
Saliva 
 
 
X 
 
X 
X 
 
X 
X 
 
X 
 
X 
 
 
Stool 
 
 
X 
 
X 
X 
 
X 
X  
 
X 
 
X 
 
 
Peripheral Blood Mononuclear 
Cells (PBMCs)3 
 
 
X 
 
X 
X 
 
X 
X 
 
X 
 
X 
 
 
Study completion 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
Post-study safety assessment 
(via telephone follow-up) 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
Approximate blood volume 
(ml) by study day4 
15 
25 
82 
 
34 
74 
 
34 
74 
 
24 
10 
74 
 
 
Approximate blood volume 
(ml) by study day5 
10 
15 
73 
4 
47 
49 
 
47 
49 
 
40 
7 
73 
 
 

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8. 
SELECTION AND WITHDRAWAL OF SUBJECTS 
8.1. 
Recruitment 
Healthy adult men and women, civilian and active-duty military, will be recruited from the 
Baltimore/Washington, DC, area through the WRAIR CTC by the use of advertisement in 
multiple media formats, to include but not limited to: newspapers, fliers, e-mails, the WRAIR 
CTC web site, public listservs, social media (such as Facebook), posters, bus ads, generic radio 
studies. E-mail announcements and web site postings will include information found on 
recruitment scripts (excluding any compensation information) or posters excluding any photos 
unless attached as a complete flyer. Recruitment may also include oral presentations at events, 
meetings, and briefings wherein the desired recruit population might reasonably be expected to 
attend. All forms of recruitment, printed media, handouts, and briefs will have IRB approval 
prior to being used. 
When a subject calls the WRAIR CTC recruitment office and discloses an interest in the study, 
the recruitment staff will discuss details of the trial from a prepared, IRB-approved script. If the 
subject is still interested, contact information will be obtained and an appointment for 
briefing/screening will be arranged. 
8.2. 
Subject Inclusion Criteria 
Subjects must meet all of the following criteria to be included in the study: 
 Healthy, adult, male or female, age 18 to 45 years (inclusive) at the time of enrollment. 
 Completion and review of comprehension test (achieved > 70% accuracy). 
 Signed informed consent document. 
 Available for the required follow-up period and scheduled clinic visits. 
 Women: Negative pregnancy test with understanding (through informed consent process) 
to not become pregnant during the study or within three (3) months following last 
vaccination. 
8.3. 
Subject Exclusion Criteria 
Subjects meeting any of the following criteria will be excluded from the study: 
General Health 
 Health problems (for example, intercurrent febrile illness, chronic medical conditions 
such as psychiatric conditions, diabetes mellitus, hypertension or any other conditions 
that might place the subject at increased risk of adverse events- study clinicians, in 
consultation with the PI, will use clinical judgment on a case-by-case basis to assess 
safety risks under this criterion. The PI will consult with the Research Monitor as 
appropriate. 
 Clinically significant abnormalities on physical examination. 
 Immunosuppressive drugs (use of systemic corticosteroids or chemotherapeutics that may 
influence antibody development) or illness (including IgA deficiency, defined by serum 
IgA <7 mg/dL). 

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 Women who are pregnant or planning to become pregnant during the study period plus 3 
months beyond the last vaccination and currently nursing women. 
 Participation in research involving another investigational product (defined as receipt of 
investigational product or exposure to invasive investigational device) 30 days before 
planned date of first vaccination or anytime through the last study safety visit. 
 Positive blood test for HBsAg, HCV, HIV-1/2. 
 Clinically significant abnormalities on basic laboratory screening. 
Research Specific 
 Exclusionary skin history/findings that would confound assessment or prevent 
appropriate local monitoring of AEs, or possibly increase the risk of an AE. 
 History of chronic skin disease (clinician judgment). 
 Acute skin infection/eruptions on the upper arms including fungal infections, severe acne 
or active contact dermatitis. 
 Allergies that may increase the risk of AEs. 
 Regular use (weekly or more often) of antidiarrheal, anti-constipation, or antacid therapy. 
 Abnormal stool pattern (fewer than 3 stools per week or more than 3 stools per day) on a 
regular basis; loose or liquid stools on other than an occasional basis. 
Prior exposure to ETEC or Vibrio cholerae 
 History of microbiologically confirmed ETEC or cholera infection in the last 3 years. 
 Travel to countries where ETEC or V. cholerae or other enteric infections are endemic 
(most of the developing world) within 3 years prior to dosing (clinician judgment). 
 Symptoms consistent with Travelers’ Diarrhea concurrent with travel to countries where 
ETEC infection is endemic (most of the developing world) within 3 years prior to dosing, 
OR planned travel to endemic countries during the length of the study. 
 Vaccination for or ingestion of ETEC, cholera, or E. coli heat labile toxin within 3 years 
prior to dosing. 
 Occupation involving handling of ETEC or V. cholerae currently, or in the past 3 years. 
8.4. 
Subject Withdrawal Criteria 
Each subject may withdraw consent at any time during the study without penalty.  Counseling 
about the subject's health will be provided if he/she decides to discontinue participation in the 
study.  Medical advice regarding what is in the best interest of the subject will be provided.  
The PI may discontinue the subject’s activity without the subject’s consent if any of these criteria 
are met: 
 A subject fails to comply with study procedures. 
 A subject’s safety or health may be compromised by further participation. 
 It is determined to be in the subject’s best interest. 

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8.4.1. 
When and How to Withdraw Subjects 
A subject may end his or her participation in the study at any time.  If a subject withdraws, the 
investigator will make a reasonable effort to determine the reason for the withdrawal from the 
study and to complete termination procedures as described in Section 8.  Telephone calls, 
registered letters, and email correspondence are considered reasonable effort.  For subjects 
leaving the study, a targeted examination may be performed, if medically indicated and if 
permitted by the subject. 
A subject may be withdrawn for an adverse event (AE) or serious adverse event (SAE) resulting 
in a safety concern or for noncompliance with protocol requirements. When a subject withdraws 
due to an AE or is withdrawn by the PI due to an AE, the Sponsor must be notified within 72 
hours. 
Investigators must follow specific policy at each institution regarding the timely reporting of 
AEs and SAEs to the local IRB (section 12.5). In all cases, the PI will make a reasonable effort 
to complete study termination procedures. 
If a subject meets withdrawal conditions for a concomitant medication violation or 
noncompliance, this should clearly be stated in the source document and the study termination 
eCRF. 
8.4.2. 
Data Collected for Withdrawn Subjects 
All data collected up to the time of withdrawal will be reported.  The study termination CRF will 
be completed, with the reason for withdrawal specified. 
8.4.3. 
Replacement of Subjects 
Up to four alternates may be recruited for every group and asked to come to the day of first 
vaccination for each group.  If an assigned subject does not present on the first day of 
vaccination, elects to withdraw, or is found to have met an exclusion criterion, an alternate will 
be enrolled. 
Subjects who withdraw or are withdrawn after the first vaccination will not be replaced. 
8.4.4. 
Follow-up for Withdrawn Subjects 
Attempts will be made to follow all subjects for safety up to 28-days after receipt of the last 
vaccine dose. Short term safety follow up 7-days post vaccination will also be attempted.  If 
unenrolled, attempts will be made to continue collecting specimens for immunological testing if 
subjects have received at least two doses of vaccine. Attempts will also be made to have the 6 
and 12 month calls if the subjects are willing. 

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9. 
TREATMENT OF SUBJECTS 
9.1. 
Description of Investigational Products 
Table 9. 
Investigational Products 
Product Name 
CssBA (Lot 1880) 
dmLT (Lot 001 08 16) 
Dosage Form 
5, 15, or 45 μg on days 
1, 22, 43 
100 or 500 ng on days  
1, 22, and 43 
Route of 
Administration 
IM 
IM 
Physical 
Description 
Clear to slightly hazy, 
homogenous liquid 
White to off-white 
uniform dense cake 
Manufacturer 
WRAIR PBF, Bldg 501, 
501 Robert Grant 
Avenue, Silver Spring, 
Maryland 20910 
IDT Biologika 
Corporation 
1405 Research 
Boulevard 
Rockville, MD 20850 
 
9.2. 
Concomitant Medications 
Subjects taking regular medication (i.e., birth control pills or multivitamins) prior to enrollment 
in the trial will be allowed to continue to take this medication unless it is specifically excluded as 
part of the inclusion/exclusion criteria for the trial.  Subjects needing to take non-approved or 
excluded medication will not be eligible for enrollment in this study.  Any medication ordered by 
the study physician during the course of the trial, such as a topical steroidal cream, will be 
documented on the concomitant medication page of the source documents.  Approved 
medications that were being taken prior to, as well as during the course of the trial will also be 
documented in this manner. 
9.3. 
Treatment Compliance 
All vaccines will be administered by a study investigator or research nurse. 

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10. 
INVESTIGATIONAL PRODUCTS AND MANAGEMENT 
10.1. 
Investigational Products 
The investigational products are CssBA (Lot 1880, manufactured at the WRAIR PBF) and 
dmLT, manufactured at IDT Biologika Corporation (comparable to lot being tested in the 
toxicology study and final lot # release Aug 2016).   Products have passed tests for sterility, 
purity, endotoxin content, appearance, pH, and protein concentration. In addition, all three have 
passed General Safety Tests in mice. Table 9 presents a summary description of the 
investigational products. 
 
10.2. 
Investigational Product Packaging and Labeling 
The investigational products will be covered under an Investigational New Drug (IND) 
application.  Each vial is labeled for human administration and includes the following statement: 
“New Drug  Limited by Federal Law to Investigational Use.”  IND product components are 
listed below: 
 CssBA (Lot 1880) vial label.  
 
 
 dmLT (comparable to lot being tested in the toxicology study and final lot # release 
Aug 2016): vials. 
 
o Reconstructed label from vial of dmLT: 
Recombinant double mutant Heat Labile 
Toxin (dmLT) 
LT (R192G/L211A) Expressed in E.coli 
Contents: 0.5 ml (1 mg/ml, Lyoph.) 
Storage ≤-10˚C 
Caution: New drug limited by Federal Law 
To investigational use only 
Manufactured by: IDT, Rockville, MD 20850 
IDT Biologika GmbH, Am Pharmpark, 06861, Dessau 
Roßlau 
Lot #: 001 08 16 
MFG Date: 08/2016 
 
 
10.3. 
Investigational Product Storage 
CssBA vials are stored at -80º±10ºC and dmLT vials are stored at <-10°C. 

CssBA Phase 1 Protocol 
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The day prior to vaccination, the required number of CssBA vials will be moved to overnight 
storage at the CTC.  The CssBA will be stored at 2-8oC to allow the frozen liquid to thaw. If it is 
not possible to move CssBA vials to 2-8oC for overnight storage prior to vaccination day, it is 
acceptable to rapid thaw the vials at room temperature. The dmLT will be maintained <-10°C 
until ready for use. 
10.4. 
Investigational Product Preparation 
On the day of vaccination, the products will be used to formulate the appropriate vaccine 
preparations for the clinical groups.  The vaccine will be used within 6 hours of preparation. 
Vaccine or open vials will not be saved for use on another study day. Any remaining vaccine 
product will be properly disposed of in accordance with Manual of Procedures (MOP). Vaccine 
formulation for each of the groups is described in detail in the MOP. 
10.5. 
Investigational Product Accountability 
Personnel in the Enteric Disease Department at the Naval Medical Research Center and the 
clinical coordinator staff at the CTC will be responsible for product accountability. The PI is 
responsible for and will maintain logs of investigational product receipt, storage, reconstitution, 
accountability by subject, and investigational product remaining before final disposition.  At the 
CTC, the logs will be maintained in the accountability files.  The PI may delegate, in writing, 
this responsibility to another individual, but the PI is ultimately responsible for the 
investigational product and its proper storage upon receipt at the study site. 
All unused or partially used investigational product and empty vials will be destroyed or used for 
non-clinical research purposes as per the MOP. 

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11. 
LABORATORY AND CLINICAL ASSESSMENTS 
Laboratory samples described below will be collected at times specified in Table 8. 
11.1. 
Sample Collection 
11.1.1. 
Blood Sample Collection 
Study serology and immunological analyses will be performed from blood. Peripheral Blood 
Mononuclear Cells (PBMCs) will also be collected to determine Antibody responses from 
Lymphocyte Supernatant. Blood samples for immunology will be separated into plasma and 
lymphocyte fractions using a FICOLL- HYPAQUE gradient technique with plasma stored at -
70°C (±10°C) and lymphocytes not used on the day of collection stored in vapor phase liquid 
nitrogen at NMRC until testing is initiated. 
11.1.2. 
Stool Sample Collection 
Stool will be collected from subjects for immunogenicity testing for fecal IgA.  Subjects will be 
provided stool hats to self-collect all stools which will be processed within two hours of receipt 
in the CTC and within 8 hours of collection by the subject, according to specific study 
procedures that have been utilized successfully for other enteric disease studies. 
11.1.3. 
Saliva Sample Collection 
Collection of saliva samples will be performed utilizing synthetic oral swabs (Salimetrics Oral 
Swab; SOS).  The subject will place a single swab in their mouth under the tongue to collect 
saliva for 10 minutes.  Subjects will be instructed not to eat or drink anything, including 
chewing gum, for 10 minutes prior to saliva sample collection. Subjects will be instructed to 
avoid drinking alcohol or using mouthwash for 24 hours and to avoid caffeinated beverages for 
12 hours prior to collecting the sample.  Saliva collection vials will be pre-loaded with 10uL of 
100X HALT Protease Inhibitor Cocktail. 
11.2. 
Specification of Safety Endpoints 
11.2.1. 
Demographic/Medical History 
During initial screening, a medical history is obtained before Study Day 1. Approximately 7 days 
prior to vaccination (allowable range: day -14 to -2), subjects will have a follow-up medical 
history. During subsequent vaccination and follow-up visits, a review for changes in medical 
history will be performed. 
11.2.2. 
Vital Signs 
Vital signs (oral temperature, blood pressure, and heart rate) will be obtained at each clinic visit. 

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Table 10. 
Reference Ranges and Adverse Event Coding for Vital Signs Parameters 
Vital Signs 
Mild  
 (Grade 1) 
Moderate 
 (Grade 2) 
Severe  
 (Grade 3) 
Potentially Life 
Threatening (Grade 4) 
Heart rate 
 
Tachycardia 
101–115 
116-130 
>130 
ER visit or hospitalization for 
arrhythmia 
 
Bradycardia 
50-54a 
45–49 
<45 
ER visit or hospitalization for 
arrhythmia 
Fever (°C)  
 (°F)  
>100.4 °F and 
<101.1°F 
(38.0-38.4°C) 
>101.1°F and 
<102.0°F (38.5-
38.9°C) 
>102.1 °F and 
<104 °F (39.0-
40.0 °C) 
 
> 104.0 °F; life threatening 
Blood Pressure 
 
Hypertension  
 (systolic, mm Hg) 
141–150 
151 - 155 
>155 
ER visit/hospitalization for 
malignant hypertension 
Hypertension  
(diastolic, mm Hg) 
91–95 
96 – 100 
>100 
ER visit/hospitalization for 
malignant hypertension 
Hypotension  
(systolic, mm Hg)b 
85–89 
80 – 84 
<80 
ER visit/hospitalization for 
hypotensive shock 
a Grade 1 bradycardia will not be considered an abnormality for this study unless judged to be clinically significant 
by the PI or the PI in consultation with the Research Monitor and sponsor. 
b If a subject has a baseline systolic BP in the 90’s then a decrease in BP < 10 without associated clinical symptoms 
will not be considered an abnormality for this study unless judges to be clinically significant by the PI. 
11.2.3. 
Physical Examination 
A complete physical exam will be conducted during the screening visit as part of the screening 
process. Subsequent focused clinical examinations will occur at each study visit with specific 
attention to the identification of local, systemic or other adverse reactions. 
11.2.4. 
Laboratory Assessments 
Venous blood samples will be collected for chemistry, hematology and immunological 
parameters. Hematology and Chemistry analyses (BUN, creatinine, AST, ALT, electrolytes, 
glucose) will occur at time times indicated in Table 8. Hematology and chemistry analyses will 
be performed by commercial laboratory (Quest, Incorporated).  Additional specimens may be 
collected to confirm and evaluate any abnormal values. 
The clinical toxicity grading scale that will be used as a guideline is based on the Guidance for 
Industry: Toxicity Grading Scale for Healthy Adult and Adolescent Subjects enrolled in 
Preventive Vaccine Clinical Trials.  Final grading determination will be made by the PI based on 
normal lab values for the specific lab and clinical symptoms. If any additional safety labs are 
performed, we will utilize the FDA Guidance for Industry. 
11.2.4.1. Hematology 
The following hematology parameters will be assessed: 
 White blood cell count 
 Red blood cell count 
 Platelet count 

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 Hemoglobin 
Table 11. 
Reference Ranges and Adverse Event Coding for Clinical Hematology 
Parameters 
Test 
Quest 
Normal 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially Life 
Threatening 
(Grade 4) 
Hemoglobin (g/dL) 
(for screening purposes only) 
M: LLN = 13.2 
F: LLN = 11.7 
M: 12.5-13.1 
F: 11.0-11.6 
M: 10.5-12.4 
F: 9.5-10.9 
M: 8.5-10.4 
F: 8.0-9.4 
M: <8.5 
F: <8.0 
Hemoglobin - decrease from 
lower limit of normal 
(used to grade toxicity) 
0.5-1.5 
1.6-2.0 
2.1-5.0 
> 5.0 
Neutrophils (cells/mm3) 
1,500-7,800 
1,225-1,499 
1,000-1,224 
776-999 
< 776 
Leukocytes (white blood 
cells) (cells/mm3) 
3,800-10,800 
Leukopenia 
2,500-3,799 
1,500-2,499 
1,000-1,499 
< 1,000 
Leukocytosis 
10,801-15,000 
15,001- 
20,000 
20,001- 
25,000 
> 25,000 
Lymphocytes (cells/mm3) 
850-3,900 
750-849 
500-749 
250-499 
< 250 
Eosinophils (cells/mm3) 
15-500 
551-1,500 
1,501-5,000 
> 5,000 
Hypereosinophilic 
Platelets decreased – 
103/mm3 
140-400 
125-139 
100-124 
25-99 
< 25 
 
11.2.4.2. Blood Chemistry 
The following clinical chemistry parameters will be assessed: 
 Serum electrolytes 
 Glucose 
 BUN 
 Creatinine 
 AST 
 ALT 
Table 12. 
Reference Ranges and Adverse Event Coding for Blood Chemistry 
Parameters 
Test 
Quest 
Normal 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially Life 
Threatening 
(Grade 4) 
Sodium 
135-146 
(mmol/L) 
Hyponatremia 
132-134 
130-131 
125-129 
< 125 
Hypernatremia 
147-148 
149-150 
151-152 
> 152 

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Potassium 
3.5-5.5 
(mmol/L) 
Hypokalemia 
3.3-3.4 
3.1-3.2 
2.9-3.0 
< 2.9 
Hyperkalemia 
5.6-5.7 
5.8-5.9 
6.0-6.1 
> 6.2 
Glucose, Random 
65-139 
(mg/dL) 
Hyperglycemia 
140-155 
156-200 
> 200 
Insulin requirements 
or hyperosmolar coma 
Hypoglycemia 
60-64 
55-59 
45-54 
< 45 
SGOT/AST (elevation) M: 10-40 U/L 
F: 10-30 U/L 
M: 41-100 
F: 31-75 
M: 101-200 
F: 76-150 
M: 201-400 
F: 151-300 
M: > 400 
F: > 300 
SGPT/ALT (elevation) 
M: 9-60 U/L 
F: 6-40 U/L 
M: 61-150 
F: 41-100 
M: 151-300 
F: 101-200 
M: 301-600 
F: 201-400 
M: > 600 
F: > 400 
BUN (elevation) 
7-25 
26-28 
29-31 
> 31 
Requires dialysis 
Creatinine (elevation) 
M: 0.7-1.4 
F: 0.5-1.1 
M: 1.5-1.7 
F: 1.2-1.7 
M: 1.8-2.0 
F: 1.8-2.0 
M: 2.1-2.5 
F: 2.1-2.5 
M: >2.5 
F: >2.5 or requires 
dialysis 
11.2.4.3. Virus Serology 
Serologic evidence of chronic HIV-1/2, HCV and HBV infections will be obtained during the 
screening process no earlier than 14 days before initial vaccination. Evidence of infection would 
make a subject ineligible. Additional testing will not be performed as part of this study. 
11.2.4.4. Pregnancy Screen 
A urine sample for pregnancy testing will be collected at the screening visit for female subjects; 
Pregnancy tests will also be obtained on each day of vaccination, and at the final study visit. A 
positive pregnancy test prior to vaccination will result in no additional study vaccinations being 
provided. 
Any subjects who become pregnant during the study will have follow-up safety labs (as 
appropriate) prior to being removed from the study. The subject will then be removed from the 
study and followed until the end of their pregnancy. Procedures to be followed in the event a 
study participant becomes pregnant during the study period are outlined below in Section 
12.5.2.1. 
11.2.5. 
Local Reactions 
Symptoms and signs will be specifically surveyed for at each visit as well as by patient self-
report using a symptom memory aid tool. Specifically, the following symptoms will be solicited: 
local pain, pruritus, induration, erythema and other rash/skin lesions as well as any systemic 
signs and symptoms. Local reactions will be coded for severity based on the severity scale 
outlined in Section 12.4.3.  

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Photograph and Measuring Tool: A baseline photograph of the vaccination site may be 
obtained prior to vaccination, one day post-vaccination and, in the event of a skin rash, every 7 
days until resolution or study completion. The subject may be photographed in order to record 
and follow any vaccine site or other local reactions. These photographs will not be part of 
statistical analysis of study endpoints but rather may serve for internal monitoring and follow up 
of any reactions. Photograph files will be maintained at the Enterics Department. 
 
11.3. 
Immunology 
11.3.1. 
Primary Immunology 
Serum samples will be assayed for antibody IgG and IgA titers against LT and CS6 using ELISA 
methods previously established in the NMRC Immunology Laboratory.  Previously established 
high-titer specimens will be included on each plate to track day to day interassay variation.  For 
each antigen, pre- and post-vaccination serum samples will be assayed concurrently.  The 
antibody titer assigned to each sample will represent the geometric mean of duplicate tests 
performed on two different days.  Reciprocal endpoint titers < 5 will be assigned a value of 2.5 
for computational purposes.  Seroconversion will be defined as a ≥ 4-fold increase in endpoint 
titer between pre-and post-vaccination samples AND a post-vaccination reciprocal titer >10. 
Peripheral Blood Mononuclear Cells (PBMCs) will be collected to determine antibody 
responses from Lymphocyte Supernatant against CS6 and LT. Antibody in Lymphocyte 
Supernatant (ALS) is an indirect quantification of antibody secreting cells (ASC) activated in 
the mucosa that circulate in the peripheral blood about seven days post-mucosal 
immunization/infection. This method has been shown to be a replacement for ELISPOT 
methodology.  PBMCs are incubated without stimulation and the supernatant is later assayed for 
antigen-specific IgG and IgA Abs by ELISA. A positive ALS response will require a four-fold 
rise in antibody titers between pre and post vaccination samples. For each antigen, pre-and post-
challenge samples will be tested concurrently. 
11.3.2. 
Exploratory Immunology 
Stool and saliva samples will be collected to explore the antigen-specific IgA response at the 
mucosal level by measuring IgA antibody titers against CS6 and LT. Immunologic responders 
will be defined as subjects with a > two-fold increase in reciprocal endpoint titer. 
 
Samples will be collected as outlined in the time and events schedule. In order to support additional 
future exploratory evaluations in systems biology, cells and serum samples will be collected for use 
in a variety of transcriptomic, proteomic, flow cytometry, memory B cells, and cytokine analysis. 
Any testing of samples for these exploratory outcomes will be performed under a modification to this 
protocol to further detail the assay(s) or performed under a separate research protocol. 
 

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12. 
ASSESSMENT OF SAFETY 
Safety monitoring will be conducted throughout the study; therefore, safety concerns will be 
identified by continuous review of the data by the PI, clinic staff, clinical monitor, Research 
Monitor, and SRC. 
The SRC will be comprised of one Independent Safety Monitor (ISM) and a committee of 
outside members with expertise in early phase 1 clinical trials. The SRC will be established 
under a charter and function to offer their expertise, complete expedited review of safety reports, 
review safety data prior to dose escalation, make recommendations about as to the study 
continuation, and advise the study sponsor regarding trial safety.  
Study Safety Management: The IRBs, Research Monitor, SRC, and PI will review any safety 
concerns. 
Research Monitor: The Research Monitor will function as an independent safety advocate for 
subjects per AR 70-25, DoD Instruction 3216.2. An independent Research Monitor is required to 
review all unanticipated problems involving risk to subjects or others, SAEs, and all subject 
deaths associated with the protocol and provide an unbiased written report of the event. At a 
minimum, the Research Monitor should comment on the outcomes of the event or problem and, 
in the case of a SAE or death, comment on the relationship to participation in the study. The 
Research Monitor should also indicate whether he/she concurs with the details of the report 
provided by the study investigator. Reports for events determined by either the investigator or 
Research Monitor to be possibly or definitely related to participation and reports of events 
resulting in death should be promptly forwarded to the IRB and the Sponsor. 
12.1. 
Safety Parameters 
12.1.1. 
Safety Criteria for Dose Escalation or Stopping Doses 
The study will use a dose -escalation design to evaluate increasing doses of CssBA in 
combination with increasing doses of dmLT. 
The PI, along with the Research Monitor and SRC, may determine a subject’s local site 
reactions warrant discontinuation of receiving subsequent vaccinations. If any of the following 
events occurs, administration of investigational product will be discontinued until a thorough 
review of the events is undertaken by the investigators and the SRC: 
 
The occurrence of one or more serious adverse events determined to be related to the 
investigational vaccine (definitely, probably or possibly) within the 72 hour period post-
vaccination. 
 
Severe diarrhea, defined as six or more liquid stools within 24 hours of vaccination.  
 
A severe local rash, defined as one that makes the subject unable to perform normal daily 
activities and which is not attributable to another cause. 
 
Systemic rash, including but not limited to generalized urticaria, generalized petechiae, 
or erythema multiforme, occurring in two or more subjects in a group will result in 
stopping further vaccination in subjects groups. 

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 
One vaccine-related serious or unexpected AE evaluated by the PI, Research Monitor, 
and sponsor’s representative and determined to be an unacceptable risk to the health and 
safety of other investigational product recipients. 
Further vaccination, in accordance with the protocol, may be resumed with the concurrence of 
the SRC, sponsor, PI, and the US Food and Drug Administration (FDA). 
If it is determined that a subject will not continue with future vaccine doses, they will remain 
enrolled in the study for follow-up. 
An interim safety analysis for Group A will be prepared and reviewed to include all safety data 
through 7 days after the third vaccination dose for Group A. This report will be provided to the 
SRC after Day 50 for their review to determine whether the study can continue to enroll for 
Group B or whether the study should be stopped. An interim safety analysis for Group B, C, 
and D will be prepared and reviewed to include all safety data through 7 days after the third 
vaccination dose. This report will be provided to the SRC after Day 50 for their review to 
determine whether the study can continue to enroll for the next, dose-escalating group or 
whether the study should be stopped. In addition to the interim safety analyses, the PI, along 
with the SRC will stop the study until further review by the SRC if any of the following criteria 
that are thought to be related to the investigational vaccine are identified: 
 
One or more grade 3 laboratory abnormalities. 
 
Two or more grade 2 laboratory abnormalities. 
 
Any serious adverse event (SAE) occurring at any time during the study duration and 
assessed as at least possibly related to study vaccination.  
 
Any non-serious unexpected AE related to study vaccination and determined to be an 
unacceptable risk to the health and safety of other investigational product recipients. 
 
The same or similar severe (grade 3 or higher) solicited or unsolicited AEs assessed as at 
least possibly related to study vaccination reported by 10% or more subjects in the 7 
days following vaccination. 
If the study were to be stopped, subjects would be followed for the safety assessments but no 
additional vaccinations would be given (unless otherwise specified by the SRC). 
12.1.2. 
Criteria for Study Termination 
The PI, the Research Monitor, the SRC, IRB, sponsor, or the FDA may stop or suspend the use 
of the products at any time. 
12.2. 
Adverse and Serious Adverse Events 
12.2.1. 
Definition of Adverse Events 
12.2.1.1. Adverse Event (AE) 
An AE, as defined by the ICH guideline for GCP, is: 
“Adverse event means any untoward medical occurrence associated with the use of a drug in 
humans, whether or not considered drug related. 

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Suspected adverse reaction means any adverse event for which there is a reasonable possibility 
that the drug caused the adverse event. For the purposes of IND safety reporting," reasonable 
possibility means there is evidence to suggest a causal relationship between the drug and the 
adverse event. Suspected adverse reaction implies a lesser degree of certainty about causality 
than adverse reaction, which means any adverse event caused by a drug." 
An AE is considered to be any adverse change or exacerbation from a baseline condition which 
occurs following the initial administration of an investigational product whether or not the event 
is considered to be related to the investigational product. Examples of this include but are not 
limited to the following: 
 Adverse changes including new signs and symptoms, intercurrent illness modifying 
the clinical course, or the worsening of a baseline condition including the increased 
frequency of an event or an increased intensity of a condition. 
 Concomitant disease with onset or increased severity after the start of study product 
administration. 
 A new pattern in a pre-existing condition, occurring after the receipt of 
investigational product that may signal a clinically meaningful change. 
 Clinically significant changes in laboratory values. 
Medically attended adverse events (MAAEs) are adverse events that include potential 
immune-mediated medical conditions as listed in the MAAEs Addendum. 
12.2.1.2. Solicited Adverse Events 
A solicited AE is a predetermined event, identified in the Investigator’s Brochure, which may 
reflect safety concerns related to the investigational product. The solicited AEs for this study 
include: 
1. Vaccine Site Pain 
2. Vaccine Site Pruritus 
3. Vaccine Site Rash/eruption 
4. Vaccine Site Swelling (As reported by subject) 
5. Vaccine Site Tenderness 
6. Fever (subjective or objective) 
7. Headache 
8. Loose Stools 
9. Arthralgia 
10. Myalgia 
11. Malaise 
12. Nausea 
13. Vomiting 
Cutaneous evidence of systemic allergic reactions such as urticaria or angioedema will be also 
documented. Subjects will also be evaluated for development of regional lymphadenopathy. A 
photograph may be taken of local vaccine site reactions for internal use. Systemic signs and 
symptoms will also be documented in detail and evaluated as clinically appropriate. 

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Abnormalities will be described in detail in order to monitor progress.  This will include the 
nature of the adverse event, the date and time of onset and offset (duration), severity, and PI 
determination of relationship to the investigational vaccine. 
12.2.1.3. Serious Adverse Event (SAE) or Serious Suspected Adverse Reaction 
An adverse event or suspected adverse reaction is considered “serious” if, in the view of either 
the investigator or sponsor, it results in any of the following outcomes: 
 Death. 
 Life-threatening adverse event. 
 Inpatient hospitalization or prolongation of existing hospitalization. 
 Persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions. 
 Congenital anomaly/birth defect. 
An adverse event or suspected adverse reaction is considered “life-threatening” if, in the view of 
either the investigator or sponsor, its occurrence places the patient or subject at immediate risk of 
death. It does not include an adverse event or suspected adverse reaction that, had it occurred in a 
more severe form, might have caused death. 
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered serious when, based upon appropriate medical judgment, they 
may jeopardize the subject and may require medical or surgical intervention to prevent one of the 
outcomes listed in this definition. Examples of such medical events include allergic 
bronchospasm requiring intensive treatment in an emergency room or at home, blood dyscrasias 
or convulsions that do not result in inpatient hospitalization, or the development of drug 
dependency or drug abuse. 
12.2.1.4. Unexpected Adverse Event 
An adverse event or suspected adverse reaction is considered “unexpected” if it is not listed in 
the investigator brochure or is not listed at the specificity or severity that has been observed; or, 
if an investigator brochure is not required or available, is not consistent with the risk information 
described in the general investigational plan or elsewhere in the current application, as amended. 
For example, under this definition, hepatic necrosis would be unexpected (by virtue of greater 
severity) if the investigator brochure referred only to elevated hepatic enzymes or hepatitis. 
Similarly, cerebral thromboembolism and cerebral vasculitis would be unexpected (by virtue of 
greater specificity) if the investigator brochure listed only cerebral vascular accidents. 
“Unexpected,” as used in this definition, also refers to adverse events or suspected adverse 
reactions that are mentioned in the investigator brochure as occurring with a class of drugs or as 
anticipated from the pharmacological properties of the drug, but are not specifically mentioned 
as occurring with the particular drug under investigation. 
12.2.1.5. Serious and Unexpected Suspected Adverse Reactions (SUSARs) 
 

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Adverse events which are Serious and Unexpected, and which are felt to be at least possibly 
related to the investigational agent(s) or procedures may be of particular importance for 
reporting. 
12.2.1.6. Other Adverse Event (OAE) 
Other adverse events will be identified by the PI during the evaluation of safety data. Significant 
adverse events of particular clinical importance, other than SAEs and those AEs leading to 
discontinuation of the subject from the study, will be classified as other adverse events. For each, 
a narrative may be written and included in the clinical study report. 
12.3. 
Relationship to Investigational Products 
The PI must assign a relationship of each AE to the receipt of the investigational product.  The 
investigator will use clinical judgment in conjunction with the assessment of a plausible biologic 
mechanism, a temporal relationship between the onset of the event in relation to receipt of the 
investigational product, and identification of possible alternate etiologies including underlying 
disease, concurrent illness or concomitant medications.  The following guidelines will be used by 
investigators to assess the relationship of an AE to study product administration. 
 Not related: No relationship to investigational product. Applies to those events for which 
evidence exists that there is an alternate etiology. 
 Unlikely related: Likely unrelated to the investigational product. Likely to be related to 
factors other than investigational product, but cannot be ruled out with certainty. 
 Possibly related: An association between the event and the administration of 
investigational product cannot be ruled out. There is a reasonable temporal association, 
but there may also be an alternative etiology such as the subject's clinical status or 
underlying factors including other therapy. 
 Probably related: There is a high degree of certainty that a relationship to the 
investigational product exists. There is a reasonable temporal association, and the event 
cannot be explained by known characteristics of the subject's clinical state or factors 
including other therapy. 
 Definitely related: An association exists between the receipt of investigational product 
and the event. An association to other factors has been ruled out. 
12.4. 
Recording Adverse Events 
12.4.1. 
Methods/Timing for Assessing, Recording, and Analyzing Safety Endpoints 
AEs, solicited AEs, and SAEs will be assessed at all study visits, documented in the source 
records, and recorded on the eCRFs using accepted medical terms and/or the diagnoses that 
accurately characterize the event. When a diagnosis is known, the AE term recorded on the 
eCRF will be the diagnosis rather than a constellation of symptoms. The investigator will assess 
all AEs for seriousness, relationship to investigational product, severity, and other possible 
etiologies. When an event has not resolved by the prescribed reporting period, it will be 
documented on the AE eCRF as “unknown”. 

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The timeframe for the collection of AEs and SAEs begins at the first administration of 
investigational product through 28 days after the last dose of investigational product is 
administered.  Additionally, subjects will be contacted by telephone approximately 6 and 12 
months after their final study vaccination to assess for any autoimmunity associated SUSARs 
and new onset SAEs. During the long-term follow-up phone call, subjects will be asked if they 
have been diagnosed with any of the medical conditions listed in the MAAEs Addendum. 
12.4.2. 
Duration of Follow-Up of Subjects after Adverse Events 
Investigators are required to follow AEs and SAEs to resolution, even if this extends beyond the 
prescribed reporting period. Resolution is the return to baseline status or stabilization of the 
condition with the probability that it will become chronic. The SAE outcomes will be reported to 
the sponsor’s representative (see below) using the Supplemental SAE Report Forms.  
PATH MEDICAL 
OFFICER: 
Rahsan Erdem, MD 
Senior Medical Officer, Center for Vaccine Innovation & Access 
455 Massachusetts Avenue NW, Suite 1000 
Washington, DC 20001 
Email: rerdem@path.org, T: 202.540.4546  
 
Investigators are not obligated to actively seek SAEs in former subjects; however, if a SAE, 
considered to be related to the investigational products is brought to the attention of the 
investigator at any time following completion of the study, the event will be reported to the 
sponsor’s representative as defined in section 12.5.1.1. 
12.4.3. 
Severity Assessment 
All AEs will be assessed for severity by the investigator. Inherent in this assessment is the 
medical and clinical consideration of all information surrounding the event including any 
medical intervention required. Each event will be assigned one of the following categories: mild, 
moderate, severe, or life-threatening. See section 11.2.2 for vital signs and section 11.2.4 for 
Laboratory values for further guidance in the assignment of severity.  The criteria below may be 
used for any symptom not included in the grading scale. Any grade 4 (life-threatening) AE must 
be reported as an SAE. The eCRF for AEs will reflect only the highest severity for continuous 
days an event occurred. 
Mild 
Grade 1 
Does not interfere with routine activities 
Minimal level of discomfort  
Moderate 
Grade 2 
Interferes with routine activities 
Moderate level of discomfort  
Severe 
Grade 3 
Unable to perform routine activities 
Significant level of discomfort  
Potentially life-threatening Grade 4 
Hospitalization or ER visit for potentially 
life-threatening event  
 
FDA guidelines for toxicity will be followed; however, if a subject is evaluated in an emergency 
room for nonlife threatening illness or symptoms (i.e., visits emergency department on weekend 

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for mild problems because the physician’s office is closed), the information from that visit will 
be reviewed and severity of the adverse event will be assessed according to the subject’s clinical 
signs and symptoms. 
As defined by the ICH guideline for GCP, the term “severe” is often used to describe intensity 
(severity) of a specific event (as in mild, moderate, or severe myocardial infarction); the event 
itself however, may be of relatively minor medical significance (such as severe headache). This 
is not the same as “serious”, which is based on subject/event outcome or action criteria usually 
associated with events that pose a threat to a subject’s life or functioning. Seriousness (not 
severity) serves as a guide for defining regulatory reporting obligations. 
12.5. 
Reporting Adverse Events 
The PI will report all AEs to the sponsor’s representative and the local IRB in the appropriate 
safety, annual, and/or final reports. The investigators will prepare annual and final reports to the 
FDA in consultation with the sponsor’s representative. 
12.5.1. 
Reporting Serious and Unexpected Adverse Events 
Contact information for reporting SAEs is provided in Table 13. 

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12.5.1.1. Reporting to the Sponsor 
Table 13. 
Study Contacts for Reporting Serious Adverse Events 
Sponsor’s Representative 
 
 
The EMMES Corporation 
401 N. Washington St., Suite 700 
Rockville, MD 20850 
Sponsor’s Medical Officer 
Rahsan Erdem, MD 
455 Massachusetts Ave NW 
Suite 1000 
Washington, DC 20001 
Telephone: 202-540-4546 
Fax: 202-457-1466 
Email: rerdem@path.org 
Institutional Review Board 
Naval Medical Research Center 
Research Services Directorate, 
Office of Research Administration 
Code 025, Building 500, Rm 004 
Silver Spring, MD 
Telephone: 301-319-7276 
Fax:  301-319-7277 
Research Monitor 
Michael Koren, MD 
Walter Reed Army Institute of Research 
503 Robert Grant Avenue 
Silver Spring, MD 20910 
Telephone: 301-319-9904 
michael.a.koren2.mil@mail.mil 
 
Table 14. 
SAE Information to be Reported to the Sponsor's Representative 
Notification 
Method 
Information to be Provided 
Email or 
Telephone (within 
72 hours) 
IND number, sponsor study number, name of the investigational 
product, and investigator name and contact number 
 
Subject identification number 
 
SAE, onset date, event term, date of investigational product 
administration, severity, relationship, and subject’s current status 
AND 
 
Email or Fax 
Cover sheet or letter 
 
Adverse event case report form 
 
Supplemental SAE report forms  
 
Concomitant medication case report form or a list of concomitant 
medications 

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Notification 
Method 
Information to be Provided 
 
Medical record progress notes including pertinent laboratory/diagnostic 
test results 
NOTE: When submitting SAE reports via email, the subject line of each email notification will 
read as follows: 
SAFETY REPORT – IND # _____, Sponsor Study #_____, Subject# _____, Event term: 
_____ 
 
In order to comply with regulations mandating sponsor notification of specified SAEs to the 
FDA within 7 calendar days, investigators must submit additional information as soon as it is 
available. The sponsor’s representative will report unexpected SAEs associated with the use of 
the drug to the FDA as specified at 21 CFR 312.32 (c). 
Investigators must follow all relevant regulatory requirements as well as specific policy at each 
institution regarding the timely reporting of SAEs to the local IRB, Research Monitor, and other 
applicable regulatory agencies. 
Reporting to the sponsor’s representative does not fulfill the investigator’s duty to report all 
unanticipated problems involving risk to human subjects or others to the IRB. The PI will notify 
the local IRB, the Research Monitor, and other applicable groups, if necessary. 
12.5.1.2. Reporting to the IRB 
It is NMRC's policy to report to the IRB within 24 hours after determination of the event. A 
formal written report is due within 5 days of AE reporting. 
Following AEs should be considered as unanticipated problems that must be reported to the 
NMRC IRB: 
 Single occurrence of a serious, unexpected, and uncommon event that is strongly 
associated with investigational products exposure. 
 A single or small number of a serious, unexpected event that is not commonly associated 
with investigational products exposure but is uncommon in the study population. 
 Multiple occurrences of an AE that, based on aggregate analysis, is determined to be an 
unanticipated problem. There should be a determination that the series of AEs represents 
a signal that the AEs were not just isolated occurrences and involve risk to human 
subjects. 
 An AE that is described in the investigator’s brochure, protocol, or informed consent 
documents, but occurs at a specificity or severity that is inconsistent with prior 
observations. 
 A serious AE that is described in the investigator’s brochure, protocol, or informed 
consent documents, but for which there is a clinically significant increase in the expected 
rate of occurrence. 

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 Any other AE or safety finding, including those based on animal or epidemiologic data 
that would cause the sponsor to modify the investigator’s brochure, protocol, or informed 
consent documents, or would prompt other action by the IRB to ensure the protection of 
human subjects. 
All SAEs will be reported to the Western Institutional Review Board (WRIB) by the sponsor 
according to the WIRB guidelines and using the WIRB Promptly Reportable Information Form. 
WIRB Phone: 800-562-4789, Fax: 360-252-2498. 
PVS is required to report SAEs that fit the following criteria within 5 working days of the time of 
becoming aware of them: 
 New or increased risk  
 Protocol deviation that harmed a subject or placed subject at risk of harm  
 Protocol deviation made without prior IRB approval to eliminate an immediate hazard to 
a subject  
 Audit, inspection, or inquiry by a federal agency  
 Written reports of federal agencies (e.g., FDA Form 483)  
 Allegation of Noncompliance or Finding of Noncompliance  
 Breach of confidentiality  
 Unresolved subject complaint  
 Suspension or premature termination by the sponsor, investigator, or institution 
 Incarceration of a subject in a research study not approved to involve prisoners  
 Adverse events or IND safety reports that require a change to the protocol or consent  
 State medical board actions  
 Unanticipated adverse device effect  
 Information where the sponsor requires prompt reporting to the IRB 
12.5.2. 
Reporting Additional Immediately Reportable Events to the Sponsor’s 
Representative 
12.5.2.1. Pregnancy 
Each pregnancy must be reported immediately (within 72 hours of identification) by email or 
fax to the sponsor’s representative and the NMRC IRB. 
Subjects who become pregnant within 3 months after their last dose of investigational product 
will be followed to the end of their pregnancy, and information will be gathered for outcome, 
date of delivery, health status of the mother and child including the child’s gender, height and 
weight. Complications and/or abnormalities should be reported, including any premature 
terminations. A pregnancy is reported as an AE or SAE only when there is suspicion that the 
investigational product may have interfered with the effectiveness of contraception or there was 
a serious complication in the pregnancy including a spontaneous abortion or an elective 
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12.5.2.2. AE-Related Withdrawal of Consent 
Any AE-related withdrawal of consent during the study must be reported immediately (within 
72 hours of identification) by email or fax to the sponsor’s representative and NMRC IRB. 
12.5.2.3. Pending Inspections/Issuance of Reports 
The knowledge of any pending compliance inspection/visit by the FDA, Office for Human 
Research Protections (Department of Health and Human Services), or other government agency 
concerning clinical investigation or research, the issuance of Inspection Reports, FDA Form 483, 
warning letters, or actions taken by any regulatory agency including legal or medical actions and 
any instances of serious or continuing noncompliance with the regulations or requirements will 
be reported immediately to the sponsor’s representative. 

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13. 
KNOWN AND POTENTIAL RISKS AND BENEFITS TO HUMAN 
SUBJECTS 
13.1.1. 
Risks/Discomforts to Subjects and Precautions to Minimize Risk 
Outlined as follows are anticipated and unexpected adverse reactions and a brief description of 
procedures to ameliorate risks and symptoms. All known risks and precautions described here 
are explained in detail in the informed consent. In general, dose escalation is being performed to 
ensure an acceptable safety record for IM administration of vaccine products in this study. Prior 
to proceeding with the next dose in the next cohort of subjects, all safety data will be reviewed 
by the SRC, and a determination will be made as to whether the study can continue to enroll (at 
the dose escalation planned) based on the observed safety record from the preceding dose level. 
In addition to this overall safeguard, individual risks will be closely monitored and any AEs will 
be quickly identified and managed. 
13.1.1.1. Local Reactions 
Local reactions at the vaccination site are expected from the IM immunization.  IM vaccination, 
in general, has been shown to be a safe and effective route of administration. There may be some 
mild discomfort and irritation from the initial injection. These are typically mild and transient in 
nature. In addition, there is the possibility of local erythema, pruritus, swelling, and/or induration 
that appears over time. In addition, subjects may develop temporary swelling in the lymph nodes 
under the arm where vaccination occurred. 
13.1.1.2. Systemic Reactions 
Any vaccine may be associated with a wide range of systemic reactions, such as fevers, 
constitutional symptoms (fatigue, malaise, appetite change), and gastrointestinal symptoms 
(diarrhea, abdominal pain) as well as others. The frequency and type of systemic symptoms will 
be assessed and analyzed with respect to study products. 
13.1.1.3. Pregnancy 
During initial screening and prior to each vaccination, a urine pregnancy test will be performed. 
Subjects with a positive test will be excluded from the study. A final urine pregnancy test will be 
conducted during the last clinic visit. While female subjects will not be required to disclose their 
method of birth control, they will be counseled during the initial screening on acceptable forms 
of birth control required for study enrollment. At each visit, female subjects will be reminded 
that they must agree to use effective contraception to remain in the study. Female subjects should 
not become pregnant during the study and for at least 3 months after the last vaccine dose. 
13.1.1.4. Lactation 
Risks to nursing infants are unknown at this time; breastfeeding females will be excluded from 
this study. Lactating females must agree not to breastfeed during the study and for 3 months after 
the last dose of study vaccination/dose of investigational product. 

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13.1.1.5. Venipuncture 
Blood sampling carries a minimal risk of minor discomfort and the possibility of minor bruising 
at the site of the needle puncture and, rarely, the possibility of infection at the needle puncture 
site. 
13.1.1.6. Allergic Reaction 
As with any Investigational New Drug (IND) product administration and no matter what 
precautions are taken, there is always the risk of a serious, or even life-threatening, allergic 
reaction. Although anaphylactic reactions are not anticipated, as with any vaccination, the rare 
possibility exists. For this reason, all subjects will be observed at the study site WRAIR CTC or a 
minimum of 30 minutes following vaccine dosing. Appropriate emergency equipment (e.g., 
automated external defibrillator, air-shields manual breathing unit bag) and medication [e.g., 
antihistamines and adrenaline (epinephrine)] for initial treatment of an allergic reaction will be 
available at the WRAIR CTC whenever immunizations are given. This equipment is available to 
handle emergencies such as anaphylaxis, angioedema, bronchospasm, and laryngospasm. 

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14. 
STATISTICS 
Detailed statistical procedures, listings, table shells and figures will be provided in a separate 
statistical analysis plan (SAP). The following key statistical components will be considered and a 
detailed description will be documented in the SAP: 
 Primary and secondary endpoints and how they will be measured,  
 Statistical methods and tests that will be used to analyze the endpoints,  
 Strategy that will be used if the statistical test assumptions are not satisfied,  
 Indication of whether the comparisons will be one-tailed or two-tailed (with 
justification of the choice) and the level of significance to be used,  
 Identification of whether any adjustments to the significance level or the overall 
p value will be made to account for any planned or unplanned subgroup analyses or 
multiple testing,  
 Specification of potential adjusted analyses and a statement with which covariates or 
factors will be included,  
 Planned exploratory analyses and justification of their importance, and  
 Any subgroup effects with biological justification and support from within and 
outside the study. 
14.1. 
Data Analysis 
The primary study objective is safety.  All subjects who receive vaccination will be included in 
the safety analysis.  Adverse event data will be listed individually and summarized by body 
system and preferred terms within a body system for each treatment group. Serious and/or 
unexpected AEs will also be discussed on a case-by-case basis. For the tabulation of the AEs by 
body system, a subject will be counted only once in a given body system. For example, a subject 
reporting nausea and diarrhea will be reported as one subject, but the symptoms will be listed as 
two separate AEs within the class. Therefore, the total number of AEs reported within a body 
system may exceed the number of subjects within the body system reporting AEs. 
Rates of all adverse events will be analyzed by Pearson’s Chi-square test (or Fisher’s exact test if 
assumptions are not met for Pearson’s Chi-square) to compare dose levels. Summary tables will 
be created which will indicate the number of subjects who experienced events. Vaccine-related 
events (probably or possibly related) will be tabulated by study group.  In addition, tables will be 
prepared to list each adverse event, the number of subjects in each treatment group who 
experienced an event at least once, and the rate of subjects with adverse event(s). Adverse events 
will be divided into defined severity grades (mild, moderate, severe). The tables will also divide 
the adverse events by severity and relationship to the investigational product.  All immunized 
subjects will be included in the safety analysis. 
Rates of all MAAEs identified during the long term telephone follow-up calls will be analyzed 
by Pearson's Chi-square test (or Fisher's exact test if assumptions are not met for Pearson's Chi-
square) to compare dose levels.  MAAEs data will be listed individually and summarized by type 
of disorder. Summary tables will be prepared to list each MAAE, the number of subjects in each 
treatment group who experienced an event, and the rate of subjects with MAAE(s).  All 
immunized subjects who complete the long term follow up will be included in the analysis. 

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For immunology responses, both qualitative (responder rates) and quantitative (log10 transformed 
values) results will be analyzed.  Geometric mean titers will be calculated along with their 95% 
confidence intervals.  Between groups comparisons will be examined with nonparametric tests 
(Kruskal-Wallis for continuous data and Fisher’s exact test for categorical data) unless 
assumptions are fulfilled for Student’s t or 2. 
Additional comparisons may be made using repeated measures analysis of variance with study 
group as the between subject factor and sample collection time-points as the repeated factor.  
Only subjects who receive at least 2 vaccine doses will be included in the immunology analysis.  
All statistical tests will be interpreted in a two-tailed fashion using P < .05 to represent statistical 
significance. 
14.2. 
Planned Enrollment and Reason for Sample Size 
The sample size for this study is limited by the early stage of the product concept/testing and is 
designed to evaluate preliminary safety data but not designed to show statistically significant 
differences between groups.  Given the small number of subjects per group, the precision of our 
estimate for adverse events is limited.  For example, using binomial probability formulae for no 
observed adverse events within the 8 subjects yields a 95% exact confidence interval of 0-31%.  
Follow-on studies evaluating seemingly safe and immunogenic doses will be required with larger 
numbers of subjects in order to better define the safety profile. 
14.3. 
Level of Significance to be Used 
All statistical analysis will be made using a two-tailed alpha=0.05. 
14.4. 
Accounting for Missing, Unused, and Spurious Data 
Non-analyzable data will be documented in the deviations. 
14.5. 
Procedures for Reporting Deviations from the Original Statistical Plan 
Any deviation(s) from the original statistical plan as indicated in the protocol will be described in 
an amendment to the protocol and the SAP. Deviations from the SAP will be documented in 
accordance with NMRC SOPs.  
14.6. 
Selection of Subjects to be Included in Analyses 
All subjects who receive one or more doses of the investigational product(s) will be included in 
the safety analyses. Subjects who receive at least 2 doses of the investigational products will be 
included in the immunological analyses. 

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15. 
DIRECT ACCESS TO SOURCE DATA/DOCUMENTS 
The investigator(s)/institution(s) will permit trial-related monitoring, audits, IRB review, and 
regulatory inspection(s) by providing direct access to source data/documents. 
Subjects will be identified on eCRFs by a unique subject identification number and on source 
documents by name and date of birth. No personal identifier will be used in any publication or 
communication used to support this research study. The subject identification number will be 
used if it becomes necessary to identify data specific to a single subject. Representatives of 
PATH, the sponsor’s representative, the local IRB, and the FDA are eligible to review medical 
and research records related to this study as a part of their responsibility to protect human 
subjects in clinical research. Personal identifiers will be removed from photocopied medical and 
research records. 
15.1. 
Study Monitoring 
Study monitoring will be the responsibility of The EMMES Corporation. Upon successful 
approval of the protocol and establishment of the regulatory file, the clinical monitor will 
establish a clinical monitoring plan. To ensure that the investigator and the study staff understand 
and accept their defined responsibilities, the clinical monitor will maintain regular 
correspondence with the site and may be present during the course of the study to verify the 
acceptability of the facilities, compliance with the investigational plan and relevant regulations, 
and the maintenance of complete records. 
Monitoring visits by a sponsor’s representative-designated clinical monitor will be scheduled to 
take place at the initiation of the study, during the study at appropriate intervals, and after the last 
subject has completed the study. A report of monitoring observations will be provided to the PI 
(for corrective actions), PATH, and the product manager. 
15.2. 
Audits and Inspections 
Authorized representatives of the sponsor, the FDA, or Institutional Review Board may visit the 
site to perform audits or inspections, including source data verification. The purpose of the audit 
or inspection is to systematically and independently examine all study-related activities and 
documents to determine whether these activities were conducted, and data were recorded, 
analyzed, and accurately reported according to the protocol, ICH GCP, and any applicable 
regulatory requirements. 
The investigator should contact the IRB and the sponsor’s representative immediately if 
contacted by a regulatory agency about an inspection. 
15.3. 
Institutional Review Board (IRB) 
The PI must obtain IRB approval for the study. Initial IRB approval, and all materials approved 
by the IRB for this study, including the subject consent form and recruitment materials, must be 
maintained by the investigator and made available for inspection. 

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The PI will be responsible for preparing and submitting continuing review reports per institution 
and IRB policies. The PI or a designee will submit the approved continuing review reports and 
the local IRB approval notifications to HRPO as soon as the documents are available. The PI or a 
designee will transmit the approved final study report and the local IRB approval notification to 
the sponsor as soon as the documents are available. 

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16. 
QUALITY CONTROL AND QUALITY ASSURANCE 
To ensure compliance with GCP and all applicable regulatory requirements, the sponsor’s 
representative may conduct quality assurance audits. Refer to section 15.2 for more details 
regarding the audit process. 
Auditing of the clinical trial may be conducted at any time during the study to ensure continued 
compliance with regulations, policies and procedures. Auditing will be undertaken, as needed, by 
independent personnel designated by the sponsor. Audit findings will be documented in a formal 
audit report that will detail the conduct of the audit and summarize the observations noted. 
WIRB will provide sponsor level review for PVS for this study. PVS will submit the AE/SAE to 
WIRB only if it is considered unanticipated and related to study product(s) according to the 
WIRB defined procedures. 

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17. 
ETHICS 
17.1. 
Ethics Review 
The study is based on adequately performed laboratory and animal experimentation; the study 
will be conducted under a protocol reviewed by Naval Medical Research Center IRB; the study 
is to be conducted by scientifically and medically qualified persons; the benefits of the study are 
in proportion to the risks; the rights and welfare of the subjects will be respected; the physicians 
conducting the study will ensure that the hazards do not outweigh the potential benefits; the 
results to be reported will be accurate; subjects will give their informed consent and will be 
competent to do so and not under duress; and all study staff will comply with the ethical 
principles in 21 CFR Part 50 and the Belmont Principles. 
17.1.1. 
Review/Approval of Study Protocol 
Before a clinical study can be initiated, the study protocol and other required documents will be 
submitted to the following departments for review and/or approval, with the final review by the 
FDA.   
 NMRC Scientific Review Board  
 NMRC IRB 
 WIRB (Sponsor level review) 
 Sponsor’s Representative  
 WRAIR Commander’s Implementation Authorization. 
Enrollment in this protocol may not begin until all approvals have been obtained and the formal 
authorization letter is received by the PI from the sponsor’s representative. 
17.1.2. 
Protocol Modifications 
All modifications to the protocol and supporting documents (informed consent, study-specific 
procedures, SOPs, recruitment materials, etc.) must be reviewed and approved by the NMRC 
IRB prior to implementation except where necessary to eliminate apparent immediate hazards to 
subjects. The informed consent document must be revised to concur with any amendment as 
appropriate and must be reviewed and approved with the amendment. Any subject already 
enrolled in the study will be informed about the revision and asked to sign the revised informed 
consent document if the modification directly affects the individual’s participation in the study. 
A copy of the revised, signed, and dated informed consent document will be given to the subject. 
All original versions of the informed consent document will be retained in the protocol 
regulatory file, and a copy will be retained in the clinic medical record. 
17.1.3. 
Protocol Deviation Procedures 
All subject-specific deviations from the protocol (e.g., failure to return for follow-up visits or 
blood collection within the time indicated in the protocol) are to be documented. The PI or 
designee will be responsible for identifying and reporting all deviations, which are defined as 
isolated occurrences involving a procedure that did not follow the study protocol or study-
specific procedure. Deviations will be reported annually in the continuing review report to the 

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IRB, if appropriate. Action taken in response to the deviation, and the impact of the deviation 
will be assessed by the PI or Sub investigator. 
If a protocol deviation jeopardizes the safety or rights of a subject or scientific integrity of the 
study, the deviation will be reported immediately to the sponsor’s representative and the IRB. 
17.2. 
Ethical Conduct of the Study 
This study will be conducted in accordance with 21 CFR Part 50 and the Belmont Principles of 
respect for persons, beneficence, and justice. 
The procedures set out in this study are designed to ensure that the sponsor’s representative and 
all study personnel abide by the principles of the ICH GCP Guideline and the CFR.  The PI 
confirms this by signing this study protocol and FDA Form 1572. 
17.2.1. 
Confidentiality 
In this research, the subject’s health information will be collected and used to conduct the study; 
to monitor the subject’s health status; to measure effects of the study product; to determine 
research results, and possibly to develop new tests, procedures, and commercial products. Health 
information is used to report results of research to the sponsor’s representative and Federal 
regulators and may be reviewed during study audits for compliance with study plans, regulations, 
and research policies. After the study ends, each subject has the right to see and receive a copy of 
his/her information. 
No personal identifier will be used in any publication or communication used to support this 
research study. The subject’s identification number will be used in the event it becomes 
necessary to identify data specific to a single subject. 
17.2.2. 
Compensation for Participation 
All study-related medical care will be provided to subjects without cost.  Should a subject be 
injured as a direct result of participating in this research project, s/he will be provided medical 
care by the staff at the Walter Reed National Military Medical Center (or other Army-affiliated 
medical center), at no cost to the subjects, for that injury.  The subjects will not receive any 
injury compensation, only medical care.  The subjects will not be compensated for care if s/he 
chooses to seek care from his/her own physician.  Compensation will occur at the time of each 
designated visit (see Time and Event Schedule). Compensation will be provided only for 
completed study procedures designated for compensatory payment. Subjects will only be eligible 
for compensation outlined in the Informed Consent Document at the time their consent is 
obtained. If a subject is eligible to participate in the investigational protocol after screening, s/he 
will receive the following compensation: 
Civilian/off-duty military compensation: 
 Screening: $25 
 D-7: $100 
 D1: $150 
 D2: $75 

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 D8: $125 +$25 (if subject return and complete memory aid) 
 D22: $150 
 D23: $50 
 D29: $125 +$25 (if subject return and complete memory aid) 
 D43: $150 
 D44: $50 
 D50: $125+$25 (if subject return and complete memory aid) 
 D57: $125  
 D71: $125 
 D223: $25 
 D403: $25 
 Total: $1500 
If subjects do not complete the study, their compensation will be less in proportion to the 
amount they did not complete. 
Federal Employee/on-duty military compensation: 
By regulation, active duty personnel and federal employees can be compensated only for visits 
in which blood draws occur, and then only $50 per visit, unless the visits occur during off-duty 
hours or when they are on leave. If the volunteer is off-duty or on leave, he or she will be paid 
the same as non-military/non-federal personnel. The total amount of compensation may vary 
depending on the number of visits completed. 
 Screening: $25 
 D-7: $50 
 D1: $50 
 D2: $50 
 D8: $50 
 D22: $50 
 D23: $0 
 D29: $50  
 D43: $50 
 D44: $0 
 D50: $50 
 D57: $50  
 D71: $50 
 D223: $0 
 D403: $0 
 Total: $525 
Compensation for visits that are not specifically planned (scheduled) in the protocol, such as 
may be required to repeat labs to verify/clarify results or labs drawn to better evaluate 
abnormal lab values or adverse events may be compensated at the discretion of the PI relative 
to the severity of the event and scope of evaluations. For unplanned visits for active duty 
subjects, compensation limitations as previously described will apply. Civilian and military 
subjects who have completed the screening process for this study will be given the opportunity 

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to receive compensation for assisting in recruitment of additional subjects. Subjects will be 
asked to direct interested persons to contact the CTC. Subjects will receive $25 for each 
referred person who then attends a screening session and meets all inclusion and none of the 
exclusion criteria. Compensation will be independent of the referred person's decision to enroll. 
Referral compensation for active duty personnel and federal employees is subject to the same 
restrictions described earlier. Final authority over dispensation of referral compensation will lie 
with the CTC director. Subjects asked to come to the clinic as an alternate on the first 
vaccination day of a cohort may be compensated $50 if they are not enrolled in that cohort. 
17.2.3. 
Medical Care for Research-Related Injuries 
If a subject is injured because of participation in this research and is a DoD healthcare 
beneficiary (e.g., active duty in the military, military spouse or dependent), the subject is entitled 
to medical care for that injury within the DoD healthcare system, as long as the subject remains a 
DoD healthcare beneficiary. This care includes, but is not limited to, free medical care at Army 
hospitals or clinics. 
If a subject is injured because of participation in this research and is not a DoD healthcare 
beneficiary, the subject is entitled to free medical care for that injury at an Army hospital or 
clinic. It cannot be determined in advance which Army hospital or clinic will provide care. If the 
subject receives care for research-related injuries outside of an Army hospital or clinic, the 
subject or the subject’s medical insurance will be responsible for medical expenses. 
For all subjects: Transportation to and from Army hospitals or clinics will not be provided. No 
reimbursement is available if the subject incurs medical expenses to treat research-related 
injuries. No compensation is available for research-related injuries. The subject is not waiving 
any legal rights. The subject should contact the PI if the subject believes he or she has sustained 
a research-related injury. The subject should contact the PI for any questions. 
Requests for other benefits, such as compensation for lost time from work, are processed 
independently of this protocol. Military members retain the right to pursue military disability 
benefits, and Federal civilian employees retain the right to pursue relief through established 
workers compensation processes, but neither military disability benefits nor workers 
compensation benefits are guaranteed. The right of other parties to seek redress against the 
United States Government is limited to that set forth by existing agency regulations and the 
Federal Tort Claims Act. The subject should understand that this does not constitute a waiver or 
release of legal rights. This issue is addressed in the informed consent and will be discussed with 
the subject by the investigator or designee before the subject signs the informed consent to 
participate in the study. 
17.3. 
Written Informed Consent 
The informed consent process and document will be reviewed and approved by the IRB and 
sponsor’s representative prior to initiation of the study. The consent document contains a full 
explanation of the possible risks, advantages, and alternate treatment options, and availability of 
treatment in the case of injury, in accordance with 21 CFR 50. The consent document indicates 
that by signature, the subject, or where appropriate, legal guardian, permits access to relevant 

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medical records by the sponsor’s representative and by representatives of the FDA. The 
sponsor’s representative will submit a copy of the initial IRB- and sponsor’s representative-
approved consent form to the FDA and will maintain copies of revised consent documents that 
have been reviewed and approved by the IRB/ethics committee. 
A written informed consent document, in compliance with 21 CFR Part 50, 32 CFR Part 219, 
and the Belmont Principles will be signed by the subject before any study-related procedures are 
initiated for that subject. This consent document must be retained by the investigator as part of 
the study records. The investigators or their designees will present the protocol in lay terms to 
individual subjects. Questions on the purpose of the protocol, protocol procedures, and risks to 
the subjects will then be solicited. Any question that cannot be answered will be referred to the 
PI.  The subject will be allowed to take the consent document home to consider and discuss it 
with others and return to the CTC at a later time to sign it.  The subject should understand that 
the study product is an investigational drug and is not licensed by the FDA for commercial use, 
but is permitted to be used in this clinical research. Informed consent includes the principle that 
it is critical the subject be informed about the principal potential risks and benefits. This 
information will allow the subject to make a personal risk versus benefit decision and understand 
the following: 
 Participation is entirely voluntary,  
 Subjects may withdraw from participation at any time, 
 Refusal to participate involves no penalty, and  
 The individual is free to ask any questions that will allow him/her to understand 
the nature of the protocol. 
A description of this clinical trial will be available on http://www.ClinicalTrials.gov, as required 
by US law. 
Should the protocol be modified, the subject consent document must be revised to reflect the 
changes to the protocol. If a previously enrolled subject is directly affected by the change, the 
subject will receive a copy of the revised informed consent document. The approved revision 
will be read, signed, and dated by the subject. 
Military personnel will be recruited by CTC personnel. No recruitment of military personnel will 
occur in the presence of his/her supervisor.  There is no benefit to military personnel for 
participating in this study.  There will be no coercion or disciplinary actions for not participating 
or withdrawing if enrolled. 

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18. 
DATA HANDLING AND RECORDKEEPING 
The primary source document for this study will be the subject's record. If separate research 
records are maintained by the investigator(s), the medical record and the research records will be 
considered the source documents for the purposes of auditing the study. The source documents 
will be retained at the site. 
For this study, an EDC database system will be used for the collection of the study data in an 
electronic format. The EDC database system will be designed based on the protocol 
requirements, the approved eCRF layouts and specifications, and in accordance with 21 CRF 
Part 11. The eCRF layouts and specifications define and identify the applicable source data that 
will be collected and captured into the EDC database system. The applicable source data will be 
electronically transcribed by the site designee onto the eCRF (data entry screens) in the EDC 
database system. The investigator is ultimately responsible for the accuracy of the data 
transcribed on the eCRF. Data monitoring and management will be performed in the EDC 
database system by the study monitor and the designated Data Management group. 
A detailed data management plan will be written and approved by the study team and the PI prior 
to study start. All updates to the data management plan must be approved before study close-out 
and database lock. 
18.1. 
Inspection of Records 
The sponsor’s representative or designee will be allowed to conduct site visits to the 
investigation facilities for the purpose of monitoring any aspect of the study. The investigator 
agrees to allow the monitor to inspect the drug storage area, investigational product stocks, drug 
accountability records, subject charts, study source documents, and other records relative to 
study conduct. 
Subjects’ health information is used to report results of research to the sponsor’s representative 
and Federal regulators and may be reviewed during study audits for compliance with study plans, 
regulations, and research policies. The consent document indicates that by signature, the subject 
permits access to relevant medical records by the sponsor’s representative and by representatives 
of the FDA. 
Upon a subject’s termination from the trial, completed eCRFs will be ready and available for on-
site review by the sponsor’s representative or the designated representative within 14 days of 
receipt of study data. 
18.2. 
Retention of Records 
The PI will maintain all documentation relating to the study for a period of 2 years after the date 
a marketing application is approved for the investigational product or for 2 years following the 
discontinuance of the investigational product for investigation. The records will be kept in a 
secure area with access by authorized personnel only. If it becomes necessary for the sponsor’s 
representative or designee or the FDA to review any documentation relating to the study, the 
investigator must permit access to such records. 

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Completed, monitored eCRFs will be stored in a secure location by the sponsor’s representative 
or designee. A copy of each completed eCRF will be retained by the investigator. 
The PI will be responsible for retaining sufficient information about each subject, i.e., name, 
address, telephone number, and subject identifier in the study, so that the sponsor’s 
representative, the local IRB, the FDA or other regulatory authorities may have access to this 
information should the need arise. 

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19. 
PUBLICATION POLICY 
All data collected during this study will be used to support this IND.  All publications and 
presentations are governed by the standards and norms detailed in NAVMEDRSCHCENINST 
5721.1. All authors will submit the proposed publication/presentation at least 30 days prior to the 
submission date.  Prior to submission, the directorate will conduct a substantive scientific and 
professional review. The document is routed to the Office of Research Administration (ORA) for 
review and routing for Command review and approval, ultimately by the NMRC Public Affairs 
Officer (PAO). Once it is cleared at NMRC, it will be forwarded to BUMED through NMSC, if 
appropriate.  Prior to publication, an author must have a completed Publication Clearance 
Request Submission Form with signatures from all approving and reviewing authorities. 

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20. 
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