Protective Efficacy of Orally Delivered Bovine Serum Immunoglobulin (BSIgG) Specific for the Colonization Factor CS6 Following Challenge With the CS6-expressing Enterotoxigenic E. Coli (ETEC) Strain B7A
Phase: PHASE1
Conditions: Healthy Volunteer
Sponsor: Johns Hopkins Bloomberg School of Public Health

 
 
Protective Efficacy of Orally Delivered Bovine Serum Immunoglobulin (BSIgG) 
Specific for the Colonization Factor CS6 Following Challenge With the CS6-
expressing Enterotoxigenic E. Coli (ETEC) Strain B7A   
 
 
NCT03040687 
 
 
November 22, 2016 

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Protective efficacy of orally delivered bovine serum immunoglobulin (BSIgG) specific for 
the colonization factor CS6 following challenge with the CS6-expressing Enterotoxigenic E. 
coli (ETEC) strain B7A 
 
 
Sponsor 
A. Louis Bourgeois, PhD, MPH 
Center for Immunization Research 
Department of International Health 
Johns Hopkins Bloomberg School of Public Health (JHBSPH) 
624 N. Broadway, HH, Rm 241 
Baltimore, MD 20215 
Telephone: 410-614-1860 
Email: lbourge1@jhu.edu 
Funding Agency 
Jointly funded by: Congressionally Directed Medical Research Program 
and Department of Defense  
Principal Investigator 
Kawsar R. Talaat, MD  
Center for Immunization Research 
Department of International Health (JHBSPH) 
624 North Broadway, Rm. 249  
Baltimore, MD, 21205  
Telephone: 410-502-9627  
Fax: 410-502-6898  
Email: ktalaat@jhu.edu  
Research Monitor 
Alexandra Singer, MD 
Malaria Department 
Naval Medical Research Center 
503 Robert Grant Avenue 
Silver Spring, MD 20910 
Telephone: 301-295-0007 
Fax: 301-295-8025 
E-mail: Alexandra.l.singer.mil@mail.mil 
NMRC Lead Investigator 
Christopher Duplessis, MD, MPH, MS  
Enteric Diseases Department (EDD) 
NMRC 
Telephone: 301-319-7312 
Fax: 301-319-7679  
E-mail: christopher.a.duplessis.mil@mail.mil 
Subinvestigators 
Ramiro L. Gutierrez, MD, MPH  
EDD, NMRC 
Telephone: 301-319-3193 
E-mail: ramiro.l.gutierrez.mil@mail.mil 
Mark S. Riddle, MD, DrPH  
EDD, NMRC 

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Telephone: 301-319-7686 
Fax: 301-319-7679 
E-mail:  mark.s.riddle10.mil@mail.mil 
Tida K. Lee, MD, PhD 
EDD, NMRC 
Telephone: 301-319-9260 
Fax: 301-319-7679 
E-mail: tida.k.lee.mil@mail.mil 
Scott C. Olson, MD 
EDD, NMRC 
Telephone: 301-319-9878 
Fax: 301-319-7679 
E-mail: scott.c.olson15.mil@mail.mil 
Chad Porter, PhD, MPH 
EDD, NMRC 
Telephone: 301-319-7505 
Fax: 301-319-7679 
E-mail: chad.k.porter2.civ@mail.mil 
Milton Maciel, PhD, PharmD 
EDD, NMRC 
Telephone: 301-319-7406 
Fax: 301-319-7679 
E-mail: milton.maciel.ctr@mail.mil 
Jane L. Halpern, MD, MPH, DrPH 
Department of International Health (JHBSPH) 
1219 Roundhill Road 
Baltimore, MD 21218 
Tel. 410-366-4823 
Email: jhjp@comcast.net 
Clinical Trial Sites 
Center for Immunization Research (CIR) Isolation Unit  
301 Building 
301 Mason Lord Drive Suite 4300  
Baltimore, MD 21224 
CIR Outpatient Clinic  
624 N. Broadway, Hampton House Rm. 117  
Baltimore, MD 21205 
Site Investigational Product 
Accountability 
CIR/GDEC Enterics Research Laboratory (JHSPH) 
615 N Wolfe St./W5620/5609/5614 
Baltimore, MD 21287 
Johns Hopkins Bayview Medical Center 
Department of Pharmacy Research 

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4940 Eastern Avenue, AA bldg., Rm 149 
Baltimore, MD 21224 
Phone: 410-550-2747 
Clinical laboratories 
Quest Diagnostics, Inc.  
Sulphur Spring Rd,  
Baltimore, MD 21227 
Johns Hopkins Hospital 
600 North Wolfe Street 
Baltimore, MD 21287 
Johns Hopkins Bayview 
4940 Eastern Ave. 
Baltimore, MD, 21224 
Core Lab of JHSPH 
615 N Wolfe St 
Baltimore, MD 21287 
Research laboratories 
Subhra Chakraborty, PhD, MS, MPH  
CIR Enteric Research Laboratory (JHSPH)  
615 N Wolfe Street / W5616  
Baltimore, MD 21205  
Telephone: 410-502-2510  
Email: schakr11@jhu.edu 
Milton Maciel, PhD, PharmD 
ETEC immunology laboratory (EDD, NMRC) 
Johns Hopkins Biological Repository 
Johns Hopkins Bloomberg School of Public Health 
Room W6620/ 615 North Wolfe St. 
Baltimore, MD 21205 
Telephone: 410-955-7203 
Email: JHSPH.jhbrlab@jhu.edu 
Data Management 
The EMMES Corporation 
401 N Washington Street, NW Suite 700 
Rockville, MD 20850 
Telephone: 301-251-1161 (Administrative Coordinator) 
Institutional Review Boards 
JHSPH IRB Office 
615 N. Wolfe Street 
Suite E1100 
Baltimore, Maryland 21205 
Phone: 410-955-3193 
Toll-Free: 1-888-262-3242 
Fax: 410-502-0584 
Email: JHSPH.irboffice@jhu.edu!

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NMRC IRB 
Research Services Directorate 
Office of Research Administration 
Code 025, Building 500, Room 004 
Silver Spring, MD 20910 
Telephone: 301-319-7276 
 
 

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Investigator’s Agreement 
 
 
Protective efficacy of orally delivered bovine serum immunoglobulin (BSIgG) specific for the 
colonization factor CS6 following challenge with the CS6-expressing Enterotoxigenic E. coli (ETEC) 
strain B7A 
 
 
“I have read this protocol and agree to conduct the study as outlined herein in accordance with 
International Conference on Harmonization Good Clinical Practice Guideline and FDA and DoD 
Regulations.” 
 
 
 
 
Kawsar R. Talaat, MD  
 
 
 
 
 
   Date 
 
Principal Investigator 
 
 
 

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TABLE OF CONTENTS 
Table of Contents .............................................................................................................................6 
List of Figures ..................................................................................................................................9 
List of Tables ...................................................................................................................................9 
Glossary of Abbreviations .............................................................................................................10 
Clinical Protocol Synopsis .............................................................................................................12 
1.0 
Background Information and Scientific Rationale ...................................................21 
1.1 
Background Information ...........................................................................................21 
1.1.1 Diarrhea in the Military ................................................................................22 
1.1.2 Pathogenicity of ETEC .................................................................................22 
1.1.3 ETEC Colonization Factors ..........................................................................23 
1.1.4 Class 5 Tip Adhesins ....................................................................................24 
1.1.5 Optimizing Protective Efficacy Exploiting Passive Oral Prophylaxis with 
BIgG Raised Against Tip Adhesins ..............................................................24 
1.1.6 Evidence for anti-CF Immunity ....................................................................24 
1.1.7 Understanding and Knowledge Gaps of CS6 as a Virulence Factor and 
Protective Antigen ........................................................................................25 
1.1.8 B7A Challenge Strain: CS6-expressing, O148:H28 (LT+/ST+) ..................25 
1.1.9 History of the ETEC Human Challenge Model ............................................26 
1.1.10 Active and Passive Immunoprophylactic Approaches .................................26 
1.1.11 Preclinical Model for Passive Prophylaxis ...................................................27 
1.1.12 Summary .......................................................................................................27 
1.2 
Rationale ...................................................................................................................27 
1.3 
Previous Human Experience with BIgG Products ....................................................28 
2.0 
Objectives .................................................................................................................31 
2.1 
Primary Objectives ....................................................................................................31 
2.2 
Secondary Objectives ................................................................................................31 
2.3 
Exploratory Objectives .............................................................................................31 
3.0 
Study Design .............................................................................................................32 
4.0 
Study Population .......................................................................................................32 
4.1 
Subject Inclusion Criteria .........................................................................................33 
4.2 
Subject Exclusion Criteria ........................................................................................33 
5.0 
Study Procedures ......................................................................................................34 
5.1 
Screening ...................................................................................................................34 
5.2 
Randomization ..........................................................................................................35 
5.3 
Group Assignment ....................................................................................................35 
5.4 
Blinding ....................................................................................................................35 
5.5 
Clinical Evaluations ..................................................................................................36 
5.5.1 Monitoring During Inpatient Phase ..............................................................36 
5.5.2 Monitoring During Outpatient Phase ............................................................37 
5.6 
Concomitant Medications/Treatments ......................................................................37 
5.7 
Laboratory Evaluations .............................................................................................38 
5.7.1 Specimen Preparation, Handling and Shipping ............................................38 
5.7.2 Clinical Laboratory Evaluations ...................................................................38 
5.8 
Outcome Measures ....................................................................................................38 
5.8.1 Clinical ..........................................................................................................38 

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5.8.2 Immunological ..............................................................................................39 
5.8.3 Microbiological .............................................................................................39 
5.8.4 Exploratory ...................................................................................................39 
5.8.5 Outcome Adjudication Committee ...............................................................40 
6.0 
Study Schedule ..........................................................................................................40 
6.1 
Screening (Day –90 to Day –5) ................................................................................41 
6.2 
Inpatient Phase (Day –3 to Day 8) ............................................................................41 
6.2.1 Admission (Study Day –3)............................................................................41 
6.2.2 Study Days –2 to 4 ........................................................................................42 
6.2.3 ETEC Challenge (Day 0) ..............................................................................42 
6.3 
Day 5-Discharge; Antibiotic Treatment ....................................................................43 
6.4 
Inpatient Discharge ...................................................................................................43 
6.5 
Outpatient Monitoring ..............................................................................................43 
6.6 
Early Termination .....................................................................................................44 
7.0 
Study Intervention/Investigational Product ..............................................................44 
7.1 
Study Products ..........................................................................................................44 
7.1.1 Antigens for Bovine Immunization ..............................................................44 
7.1.2 BSIgG Products ............................................................................................45 
7.1.3 Packaging of Final Product ...........................................................................46 
7.1.4 Product Storage .............................................................................................48 
7.1.5 Product Shipping ...........................................................................................48 
7.1.6 Dose Preparation ...........................................................................................48 
7.2 
ETEC Challenge Strain .............................................................................................48 
7.2.1 Challenge Inoculum: The CS6-Expressing ETEC B7A ...............................48 
7.2.2 Packaging and Labeling ................................................................................48 
7.2.3 Product Characterization ...............................................................................49 
7.2.4 Product Storage and Transfer ........................................................................49 
7.2.5 Product Preparation .......................................................................................49 
7.2.6 Product Administration .................................................................................49 
7.3 
Accountability Procedures for the Investigational Products .....................................50 
7.4 
Assessment of Subject Compliance with Investigational Products ..........................50 
8.0 
Assessment of Safety ................................................................................................50 
8.1 
Vital Signs .................................................................................................................51 
8.2 
Physical Examination ................................................................................................51 
8.3 
Laboratory Assessments ...........................................................................................51 
8.4 
IND Safety Reporting ...............................................................................................53 
8.4.1 Adverse Event or Suspected Adverse Reaction ............................................53 
8.5 
Serious Adverse Events ............................................................................................56 
8.5.1 Unexpected Adverse Event or Unexpected Suspected Adverse Reaction ....56 
8.5.2 Other Adverse Events ...................................................................................56 
8.6 
Relationship to Investigational Product (Assessment of Causality) .........................56 
8.6.1 Causality .......................................................................................................57 
8.7 
Recording of Adverse Events ...................................................................................57 
8.7.1 Methods / Timing for Assessing, Recording and Analyzing Safety 
Endpoints ......................................................................................................57 
8.7.2 Duration of Follow-up of Subjects after Adverse Events .............................58 

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8.7.3 Safety Assessment ........................................................................................58 
8.8 
Reporting Adverse Events ........................................................................................59 
8.8.1 Reporting Serious and Unexpected Adverse Events ....................................60 
8.8.2 Immediately Reportable Events ....................................................................62 
8.8.3 IND Reporting ..............................................................................................63 
8.9 
Safety Criteria for Stopping Doses ...........................................................................63 
8.10 
Treatment of Adverse Events ....................................................................................64 
8.11 
Study Termination Criteria .......................................................................................64 
8.12 
Six Month Follow-up Safety Surveillance ................................................................64 
9.0 
Clinical Monitoring ...................................................................................................64 
10.0 
Statistical considerations ...........................................................................................65 
10.1 
Introduction ...............................................................................................................65 
10.2 
Sample Size Considerations ......................................................................................65 
10.3 
Analysis.....................................................................................................................66 
10.3.1 Safety ............................................................................................................66 
10.3.2 Protective Efficacy of anti-CS6 and anti-B7A Whole Cell Killed BSIgG 
Products.........................................................................................................66 
10.3.3 Immunogenicity ............................................................................................67 
11.0 
Data Management .....................................................................................................67 
12.0 
Record and Specimen Archival ................................................................................68 
13.0 
Obligations and Roles of the Sponsor, Investigator and Study Personnel ................68 
14.0 
Quality Control and Assurance .................................................................................68 
14.1 
QA/QC monitoring ...................................................................................................68 
14.2 
Protocol Deviation Management ..............................................................................69 
15.0 
Human Subjects Protections Considerations ............................................................69 
15.1 
Risks / Benefit ...........................................................................................................69 
15.1.1 Risks ..............................................................................................................69 
15.1.2 Risk Mitigation Strategies .............................................................................70 
15.1.3 Benefits .........................................................................................................71 
15.2 
Subject Compensation ..............................................................................................71 
15.3 
Research-Related Injury ............................................................................................72 
15.4 
Compensation for Investigators ................................................................................73 
15.5 
Fair and Equitable Selection of Subjects ..................................................................74 
15.6 
Informed Consent ......................................................................................................74 
15.7 
Recruitment ...............................................................................................................75 
16. 
Privacy and Confidentiality ......................................................................................75 
16.1. Storage of Data and Samples ....................................................................................75 
16.2. Provisions Protecting Privacy and Confidentiality ...................................................75 
16.3. Safeguards for Vulnerable Subjects ..........................................................................75 
17. 
Protocol Review Process ...........................................................................................75 
18. 
Publication Policy .....................................................................................................76 
19. 
References .................................................................................................................77 
 
 

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LIST OF FIGURES 
Figure 1. Conservative, Cumulative Estimates of Expected Global Coverage for an ETEC Vaccine or 
Immunoprophylactic ....................................................................................................................................23 
Figure 2. Label for Anti-CS6 BSIgG Product .............................................................................................47 
Figure 3. Label for Anti- WC B7A BSIgG Product ....................................................................................47 
Figure 4. Label for Negative Control Plasma Product .................................................................................47 
Figure 5. Power Curve for Sample Size Calculation ...................................................................................65 
 
LIST OF TABLES 
Table 1. Time and Events Schedule .............................................................................................................18 
Table 2. A summary of a Range of Clinical Trials Evaluating the Safety and Efficacy of BIgG 
Concentrates in Human Subjectsa ................................................................................................................29 
Table 3. Description of Study Groups .........................................................................................................32 
Table 4. Order of events on day of challenge ..............................................................................................42 
Table 5. Reference Ranges and Adverse Event Coding for Vital Signs Parameters ...................................51 
Table 6. Reference Ranges and Adverse Event Coding for Clinical Hematology Parameters ...................52 
Table 7. Reference Ranges and Adverse Event Coding for Blood Chemistry Parameters .........................53 
Table 8. Challenge Phase ETEC Infection Anticipated Adverse Event / Endpoint Assessments ...............59 
Table 9. Study Contacts for Reporting Serious Adverse Events .................................................................61 
Table 10. SAE Information to Be Reported to the Sponsor ........................................................................61 
Table 11. IRB Contact Information .............................................................................................................62 
 

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GLOSSARY OF ABBREVIATIONS 
Abbreviation 
Definition 
AE 
Adverse event 
ALS 
Antibody Lymphocyte Supernatant 
B7A 
CS6- expressing ETEC strain 
BIgG 
Bovine Immunoglobulin 
BP 
Blood Pressure 
BSIgG 
Bovine serum Immunoglobulin 
C 
Celsius 
CFR 
Code of Federal Regulations 
CBC 
Complete blood count 
CF 
Colonization Factor 
CFA 
Colonization factor antigen 
CfaB 
The major rod-forming subunit of CFA/I fimbriae 
CfaE 
Minor subunit (tip adhesin) of CFA/I fimbriae  
cfu 
Colony forming unit 
cGMP 
Current Good Manufacturing Practice 
CIR 
Center for Immunization Research 
CS 
Coli surface antigen  
CsbD 
Minor subunit (tip adhesin) of CS17 fimbriae 
CS17 
Coli surface antigen 17 
CS17-ETEC 
ETEC which have fimbriae composed of colonization factor CS17 
CsbA 
The major rod-forming subunit of CS17 fimbriae 
DoD 
Department of Defense 
DS 
Double strength 
eCRF 
Electronic case report form 
EDC 
Electronic data capture 
ELISA 
Enzyme-linked immunosorbent assay 
E. coli 
Escherichia coli 
ETEC 
Enterotoxigenic E. coli  
F 
Fahrenheit 
FDA 
US Food and Drug Administration 
FUP 
Fimbrial usher proteins 
F/U 
Follow Up 
GCP 
Good Clinical Practice 
GM1 
Anti-ganglioside M1 
H10407 
Escherichia coli strain H10407 
HBsAg 
Hepatitis B surface antigen 
HCV 
Hepatitis C virus 
HIV 
Human immunodeficiency virus 
ICH 
International Conference on Harmonization 
IgA 
Immunoglobulin A 
IgG 
Immunoglobulin G 
IND 
Investigational New Drug 
IP 
Investigational product 
IRB 
Institutional Review Board 

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JHBSPH 
Johns Hopkins Bloomberg School of Public Health 
JHH 
Johns Hopkins Hospital 
JHU 
Johns Hopkins University 
LPS 
Lipopolysaccharide 
LT 
Labile toxin 
CT 
Cholera toxin 
NMRC 
Naval Medical Research Center 
ORS 
Oral Rehydration Solution 
PBF 
WRAIR Pilot Bioproduction Facility 
PCB 
Production Cell Bank 
PBMC 
Peripheral blood mononuclear cell 
PVT 
Psychomotor vigilance testing 
PI 
Principal Investigator 
SAE 
Serious adverse event 
SAS 
Statistical Analysis System 
SOP 
Standard Operating Procedure 
SSP 
Study Specific Procedure 
tid 
Three times a day 
WBC 
White blood cell 
WRAIR 
Walter Reed Army Institute of Research 
!
!
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CLINICAL PROTOCOL SYNOPSIS 
!
 
PROTOCOL TITLE 
Protective efficacy of orally delivered bovine serum immunoglobulin (BSIgG) 
specific for the colonization factor CS6 following challenge with the CS6-
expressing Enterotoxigenic E. coli (ETEC) strain B7A 
IND NUMBER 
TBD 
INVESTIGATIONAL PRODUCTS 
1.! Anti-CS6 BSIgG (lot PD1601105CS) 
2.! Anti-B7A whole cell killed BSIgG (positive control) (PD1601132ET) 
3.! Non-hyperimmune BSIgG (negative control/placebo) (lot PD1601071NC) 
4.! CS6-expressing ETEC strain (B7A) (O148:H28- CS6+ LT+ST+) (Lot 
0481) 
 
SPONSOR 
A. Louis Bourgeois, PhD, MPH 
MANUFACTURERS 
ETEC strain B7A: Pilot Bioproduction Facility, Walter Reed Army Institute of 
Research, Silver Spring, MD. 
 
anti-CS6 and anti-B7A whole cell killed hyperimmune BSIgG products and non-
hyperimmune BSIgG: SAB Biotherapeutics, Sioux Falls, SD. 
PRINCIPAL INVESTIGATOR 
Kawsar Talaat, MD 
STUDY SITE 
Center for Immunization Research (CIR) Isolation Unit 
301 Building 
301 Mason Lord Drive Suite 4300  
Baltimore, MD 21224 
 
CIR Outpatient Clinic  
624 N. Broadway, Hampton House Rm. 117  
Baltimore, MD 21205 
 
LABORATORIES 
Quest Diagnostics Incorporated, Baltimore, MD 21227 
Johns Hopkins Hospital, Baltimore, MD 21287 
Johns Hopkins University School of Public Heath, Baltimore, MD 21205 
Johns Hopkins University School of Public Health, Baltimore, MD 21287 
Johns Hopkins Biological Repository, Baltimore, MD 21205 
Core Lab of Johns Hopkins School of Public Health, Baltimore, MD 21287 
Naval Medical Research Center, Silver Spring, MD 20910 
 
STUDY OBJECTIVES 
The primary objectives of this study are to assess the safety of serum-derived 
bovine immunoglobulins in healthy adult subjects when orally administered three 
times a day over 7 days and to estimate protective efficacy of those preparations 
against moderate-severe diarrhea upon challenge with B7A. The secondary 
objectives include assessments of a variety of clinical endpoints, measuring 
mucosal and systemic immune responses and obtaining and archiving samples for 
future proteomics and/or systems biology efforts. There are a variety of other 
exploratory clinical, immunological, and microbiological endpoints.  
 
STUDY DESIGN 
The study is a randomized, double-blind, placebo-controlled clinical trial in which 
up to 60 subjects (two inpatient periods of approximately 30 subjects) will receive 
one of the three investigational products (IP) three times daily following meals 
beginning 2 days prior to experimental challenge with B7A. Randomization and 
blinding will be utilized for the clinical study team. Subjects will be assigned to 
groups as per the Table below. The test articles/placebo will be administered for a 
total of 7 days, or until antibiotic treatment has been initiated. Subjects will be 

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assessed daily for adverse events and all stools will be collected to assess for the 
primary endpoint of moderate (4-5 loose stools in 24 hours or 401-800 g of loose 
stools in 24 hours) to severe (≥ 6 loose stools in 24 hours or >800g of loose stools 
in 24 hours) diarrhea post-inoculation. Any subject passing a grade 3-5 stool will 
be encouraged to start drinking oral rehydration solution (ORS) (an oral 
glucose/electrolyte solution to prevent dehydration) or Gatorade at a rate equal to 
their stool output. IV rehydration will be provided if pre-specified criteria are met. 
All subjects will be treated with ciprofloxacin (500 mg by mouth twice daily for 
three days) five days after ingesting the B7A unless early treatment criteria are 
met. Alternate antibiotic treatment to which the strain is susceptible may also be 
considered as clinically appropriate. Subjects will be discharged from the inpatient 
facility when clinical symptoms are resolved or resolving AND two consecutive 
stool cultures are negative for ETEC. 
 
Producta 
N 
Dose (approximate) 
Anti-CS6 BSIgG 
20 
1.0g three times 
daily (tid) 
Anti-B7A whole cell killed 
BSIgG 
20 
1.0 g tid 
Negative Control (Non-
hyperimmune) BSIgG 
20 
1.1 g protein total 
(equivalent) tid 
a All products will be given 3 times daily 
 
PRIMARY ENDPOINT 
Moderate to severe diarrhea defined as  
 
•!
Moderate diarrhea: 4 to 5 loose/liquid stools or 401-800 g of loose/liquid 
stool in any 24 hour period 
•!
Severe diarrhea: ≥6 loose/liquid stools or > 800 g of loose/liquid stool in 
any 24 hour period 
STUDY DURATION 
About thirty subjects per cohort: screening (85 days); inpatient (12 days); 
outpatient (28 days); immunologic assays (3 months); six-month phone check;  
Entire study, considering serial scheduling of cohorts of about 30 subjects, 
analysis and reporting after last clinic visit (2 months) – 1 to 1 ½ years 
 
ELIGIBILITY CRITERIA 
Potential subjects will be recruited by responding to IRB-approved 
advertisements, telephone calls, emails, and word of mouth. Subjects will be 
screened at the CIR. Up to 6 alternates per inpatient period will be recruited to 
replace anyone who does not report or is unable to participate at time of inpatient 
unit admission. 
 
Inclusion Criteria: 
1.! Male or female between 18 and 50 years of age, inclusive.  
2.! General good health, without significant medical illness, abnormal 
physical examination findings or clinical laboratory abnormalities as 
determined by principal investigator (PI) or PI in consultation with the 
research monitor and sponsor. 
3.! Demonstrate comprehension of the protocol procedures and knowledge of 
ETEC illness by passing a written examination (pass grade ≥ 70%) 
4.! Willing to participate after informed consent obtained. 
5.! Available for all planned follow-up visits. 
6.! Negative serum pregnancy test at screening and negative serum and/or 

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urine pregnancy test on the day of admittance to the inpatient phase for 
female subjects of childbearing potential. Females of childbearing 
potential must agree to use an efficacious hormonal or barrier method of 
birth control during the study. Abstinence is acceptable. Female subjects 
unable to bear children must have this documented (e.g., tubal ligation or 
hysterectomy). 
 
Exclusion Criteria: 
 
General health criteria 
1.! Presence of a significant medical condition, (e.g. psychiatric conditions or 
gastrointestinal disease, such as peptic ulcer, symptoms or evidence of 
active gastritis or gastroesophageal reflux disease, inflammatory bowel 
disease, alcohol or illicit drug abuse/dependency, or other laboratory 
abnormalities which in the opinion of the investigator precludes 
participation in the study. 
2.! Immunosuppressive illness or IgA deficiency (serum IgA < 7 mg/dL or 
below the limit of detection of assay) 
3.! Evidence of confirmed infection with HIV, HBsAg, or HCV, with 
confirmatory assays.  
4.! Use of any investigational product within 30 days preceding the receipt of 
the investigational products, or planned use during the active study period 
5.! Significant abnormalities in screening lab hematology or serum 
chemistries, as determined by PI or PI in consultation with the research 
monitor and sponsor. 
6.! Lactation or breastfeeding. 
 
Research-related exclusions applicable to challenge 
7.! History of microbiologically confirmed ETEC or cholera infection in last 3 
years. 
8.! Occupation involving handling of ETEC or Vibrio cholerae currently, or 
in the past 3 years. 
9.! Travel to countries where ETEC or cholera infection is endemic (most of 
the developing world) within 3 years prior to dosing. 
10.!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.  
11.!Vaccination for or ingestion of ETEC, cholera, or E coli heat labile toxin 
within 3 years prior to dosing. 
12.!Any prior experimental infection with ETEC strain B7A. 
 
Study-specific Exclusion Criteria (potential increased risk or complicating 
outcome ascertainment) 
13.!Abnormal stool pattern (fewer than 3 per week or more than 3 per day). 
14.!History of diarrhea in the 2 weeks prior to planned inpatient phase. 
15.!Regular use of laxatives, antacids, or other agents to lower stomach acidity 
(regular defined as at least weekly). 
16.!Use of antibiotics during the 7 days before receipt of any investigational 

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product or proton pump inhibitors, H2 blockers, or antacids within 48 
hours of receipt of any investigational product. 
17.!Use of any medication known to affect the immune function (eg, systemic 
corticosteroids and others) within 30 days preceding the administration of 
challenge or planned use during the active study period. 
18.!Known allergy to fluoroquinolones.  
19.!Inability to tolerate 150 mL sodium bicarbonate buffer (based on 
requirement for frequent dosing). 
STUDY PROCEDURES 
RANDOMIZATION 
Subjects will be randomized in a 1:1:1 ratio to one of 3 treatment groups. An 
analyst at NMRC will prepare a randomization list, allocating volunteer 
identification numbers to the study groups using the PROC PLAN function of 
SAS v9.2 (Cary, NC). The randomization scheme will utilize block sizes of 6 in 
order to ensure comparable group sizes in the event that the targeted number of 60 
subjects is not reached.  NMRC staff will print the code, log and output of the SAS 
procedure, sign them, and store them under lock and key. A photocopy of the 
signed output will be e-mailed to the research pharmacist prior to the first BSIgG 
administration day.  
GROUP ASSIGNMENT 
Prior to the first dose of test article/placebo, subjects will be assigned a study 
number determining what IP they receive. Subjects will receive the test 
article/placebo in containers bearing their assigned identification numbers. This 
number will be linked to the randomization code list securely maintained 
throughout the clinical phase of the study by an unblinded NMRC analyst and the 
Johns Hopkins University (JHU) research pharmacist. Study identification 
numbers will identify all samples for laboratory analyses.  
BLINDING 
Investigators and subjects will remain blinded to group assignments until 
completion of the clinical phase of the trial and validation of the clinical and 
immunological data. Investigators may be unblinded prior to the 6 month follow 
up phone call. Each multi-dose test article/placebo bottle will be labeled with an 
open label. The research pharmacist will use the randomization list to prepare the 
IP. All mixing and administration of the test articles/placebo will be performed per 
formulation and product administration study specific procedures (SSPs). 
Administration will occur in a separate room from where the doses are formulated. 
 
Only in a medical emergency, when knowledge of the study treatment is essential 
for further management of subjects, will the randomization code be broken. In the 
event that this is necessary, the PI will provide the study identification number to 
the research pharmacist, who in turn will provide the investigator with the broken 
code for that subject. The investigator will notify the Sponsor immediately and 
document the event on the appropriate study documents.  
 
TEST ARTICLE DOSING 
BSIgG products will be administered starting day -2 and then continued for 6 
days.  
 
On Day 0, subjects will eat breakfast and then take their morning dose of IP about 
15 minutes later. Subjects will fast for 90 minutes and will drink 120 ml of sodium 
bicarbonate just prior to ingesting 30 ml of sodium bicarbonate containing the 
ETEC inoculum on day 0 of the study. Subjects will take the second daily dose of 
IP approximately 15 minutes after drinking the inoculum, and subjects will 
otherwise fast for 90 minutes after drinking the inoculum. Subjects will then be 
allowed to eat and will take the third daily dose of IP approximately 15 minutes 

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after dinner. 
 
Order of events on day of challenge 
Event 
Volume 
(approximate) 
Breakfast 
- 
1st daily dose of test article/placebo (range 10-25 
min) 
150 ml 
90 minutes fast  
- 
Bicarbonate buffer 
120 ml 
1 minute interval (up to 2 minutes) 
- 
Bicarbonate buffer + Challenge   
30 ml 
Interval of 15 minutes (range 10-25 min) 
- 
2nd daily dose of test article/placebo 
150 ml 
Fast at least 90 minutes from challenge 
- 
Lunch 
- 
Dinner 
- 
15 minutes after dinner complete (range 10-25 min) 
- 
3rd daily dose of test article/placebo 
150mL 
CLINICAL MONITORING 
Daily medical assessments with adverse event determination, vital signs three 
times daily, examination and weighing of all stools, stool culture work-up for 
ETEC study strain (at least once daily), and safety laboratory tests (refer to Time 
and Events Schedule). 
CLINICAL IMMUNOLOGY 
Immunology will be evaluated by measuring serum IgG and IgA responses to 
CS6, LPS type O148, and LT, and IgG and IgA antibody in lymphocyte 
supernatant (ALS) responses to CS6, LPS, and LT antigens. All immunological 
assessments to be carried out at NMRC Laboratories. 
 
CLINICAL MANAGEMENT 
Fluid Management  
Oral: Any subject passing a grade 3-5 stool will be encouraged to start drinking 
Gatorade or ORS at a rate equal to their stool output.  
 
Intravenous: A subject may be administered IV fluids (clinician discretion) for the 
following reasons:  
•!
Subject experiences abrupt onset of diarrhea defined by passage of an 
initial loose/liquid stool of > 300g or passage of > 400 g of loose/liquid 
stools over 2 hours. 
•!
Subject becomes hypovolemic. 
•!
It is determined necessary by the study physician, i.e., diarrhea with 
nausea/vomiting and unable to drink enough to keep up with output, or 
other reason. 
Antibiotic Treatment 
All subjects will be treated with ciprofloxacin (500 mg by mouth twice daily for 
three days). Alternate antibiotic treatment to which the strain is susceptible may 
also be considered as clinically appropriate. This ETEC strain is susceptible to 
ciprofloxacin and other common antibiotics. Administration of IV antibiotic 
treatment may be performed if warranted by the PI. The test article/placebo 
administration will be discontinued with initiation of treatment. 
 

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Early antibiotic treatment after challenge may commence when any of the 
following criteria are identified and a study physician considers it to be warranted:  
•!
Severe diarrhea (based on volume, 800 g in 24 hours)  
•!
Stool output consistent with moderate diarrhea for 48 hours  
•!
Mild or moderate diarrhea and 2 or more of the following symptoms: 
severe abdominal pain, severe abdominal cramps, severe nausea, severe 
headache, severe myalgias, any fever (≥ 38.0°C), or any vomiting.  
•!
A study physician determines that early treatment is warranted for any 
other reason. 
 
DISCHARGE PROCEDURES 
All subjects are scheduled for discharge from the inpatient ward approximately 8 
days after receipt of the challenge inoculum. The day of discharge may be earlier 
if the subject meets the criteria for discharge prior to day 8. Subjects will be 
discharged from the inpatient phase of the study when clinical symptoms are 
resolved or resolving AND two consecutive stool cultures are negative for ETEC. 
SAMPLE SIZE ESTIMATE/ 
ANALYSIS 
 
The hypotheses are that (1) anti-CS6 BSIgG confers ≥ 60% protective efficacy 
against moderate to severe diarrhea upon challenge with B7A (in comparison to 
the placebo group); and (2) anti-B7A whole cell killed BSIgG confers ≥ 60% 
protective efficacy against moderate to severe diarrhea upon challenge with B7A 
(in comparison to the placebo group).  
Assuming a two-sided alpha = 0.05 and an attack rate of 80% in the placebo 
group, the power (two group continuity adjusted chi-square) to detect a 
preliminary efficacy of ≥60% in the immunoprophylaxis groups is over 80% when 
each group contains 20 subjects. There will be no adjustment for multiple 
comparisons. 
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Table 1. Time and Events Schedule 
 
Screening 
(1-2 visitsa) 
Test Article Dosing and Challenge Phase (Inpatient) 
F/U Visit and Call 
Study Event 
-30 
-3b 
-2 
-1 
0 
1 
2 
3 
4 
5 
6 
7 
8c 
Outpatient 
f/u:  D28 
180d 
Compliance Range (study 
day) 
-90 to 
-31 
-30 
to -5 
-- 
-- 
-- 
-- 
-- 
-- 
-- 
-- 
-- 
-- 
6-8 
+/- 1d 
26-30 
+/- 1 
month 
Outpatient 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
Inpatient stay 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Comprehension test 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical interviewe 
X 
(X) 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
(X) 
X 
 
Focused physical examf 
(X) 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
(X) 
(X) 
 
Vital signsg 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
(X) 
X 
 
Serology (HIV, HBsAg, 
HCV), IgA level and 
blood typing 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CBC with differential 
X 
 
X 
 
 
 
 
 
 
 
X 
 
 
 
 
 
Serum chemistryh 
X 
 
X 
 
 
 
 
 
 
 
X 
 
 
 
 
 
Serum pregnancy test 
(females) 
X 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine pregnancy test 
(females) 
 
 
X!
 
 
 
 
 
 
 
 
 
 
 
X 
 
Drug screen (urine)i 
X 
(X) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Test article 
 
 
 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
Challenge 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
Start Antibiotic therapyj 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
Stool weighing/gradingk 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Stool bacteriology (CS6 
ETEC detection)l 
 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
(X) 
 
 
Stool collection for 
Microbiome 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
(X) 
X 
 
Stool Transcriptomics/ 
PCR 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
 

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Screening 
(1-2 visitsa) 
Test Article Dosing and Challenge Phase (Inpatient) 
F/U Visit and Call 
Study Event 
-30 
-3b 
-2 
-1 
0 
1 
2 
3 
4 
5 
6 
7 
8c 
Outpatient 
f/u:  D28 
180d 
Serology: IgG, IgA anti-
CS6, LT, LPS antigensm 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
X 
 
ALS: IgA, IgG anti-CS6, 
LT, LPS antigensm 
 
 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
 
Saliva and Fecal 
Immunology 
 
 
X 
 
 
 
 
 
 
 
 
 
X 
 
X 
 
Memory B Cells 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
X 
 
Flow Cytometry 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 Systems Biology 
X 
X 
 
 
 
X 
X 
 
 
X 
 
 
 
Cytokines PBMC 
 
 
X 
 
 
 
 
 
 
 
 
X 
 
Cognitive Study (PVT)n 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Functional Bowel 
Disorder Survey 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Discharge from inpatient 
phase b 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
Study completiono 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
6 Month phone follow-up 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Approximate blood 
volume (mL) by study 
dayp 
10 
49 
95 
0 
0 
0 
24 
8 
0 
0 
10 
0 
32 
0 
66 
0 
Note: (X) denotes optional event or procedure 
a Screening may consist of 1 to 2 visits. If within day -30 window, all screening activities may take place at one visit. After screening, subject continuing eligibility must be confirmed by reassessing 
relevant inclusion and exclusion criteria prior to first dose of IP, on either day of admission or day -2. 
b Baseline immunology specimens may be collected on Day-3 or Day-2.. 
c Criteria for discharge from the unit:  Subjects will be discharged from the inpatient phase of the study when they feel well enough, clinical symptoms have resolved or are resolving, have completed at 
least two doses of antibiotics, and have 2 consecutive negative stool cultures. Subjects will be required to complete their antibiotics as outpatients.  It is expected that most subjects will be discharged 
on days seven or eight.  If a subject does not fulfill criteria for discharge he/she may be required to stay on the unit until all criteria have been fulfilled. 
d Six-month (+/- 1 month) phone call will also be completed to inquire about new onset serious health events or hospitalizations. 
e  The medical interview occurring at baseline is to establish eligibility. During the inpatient and outpatient phases of the study, the interview will be used to update baseline medical history, monitor 
safety, and to confirm ongoing eligibility.  
f Physical Examination (PE) will include: HEENT (Head; Ears; Eyes; Nose; Throat), skin, respiratory (lung), cardiovascular (heart), abdomen, neurological and musculoskeletal system.  PE will be done 
at screening and on admission. During the inpatient period a symptom focused PE will be completed. 
g Vital Signs (VS) will include heart rate, blood pressure, and oral temperature. If a VS needs to be repeated, standard practice will be to repeat the VS within approximately 20 minutes of the original 
reading. Only the VS that needs to be repeated will be repeated. Both the original and repeat measurements will be recorded in the study source documents, however, only the repeat measurement will 
be recorded in the CRF field for that measurement if the PI or designee determines the repeat measurement to be more accurate, even though it may have been obtained several minutes later than the 
original VS.  

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The following VS are obtained and documented in the source documents:  
•! During the screening visit  
•! At least 3 times daily during in-patient period  
•! Before and after challenge  
•! At the day 28 visit 
A grade 1 bradycardia, or other grade 1 abnormalities will not be considered to be exclusionary at screening, unless judged to be clinically significant by the PI.  Clinically relevant and concurrent 
medical conditions or surgical procedures will be recorded as medical history if the onset is prior to administration of IP.  This includes pre-existing lab abnormalities, VS abnormalities, and 
symptoms associated with menses (e.g. cramps, headaches, etc.). Grade 2 abnormalities recorded after screening but prior to challenge administration will be determined on a case-by-case basis at PI 
discretion. Clinically significant abnormalities not on the toxicology table can be recorded on the MH if deemed necessary by the PI.  
The following VS will be captured in the electronic CRF:  
•! Screening 
•! Admission 
•! Before and after challenge 
•! At discharge  
•! At visit day 7 
•! At visit day 28 
•! In addition, any abnormal VS deemed to be clinically significant or clinically relevant may also be entered into the eCRF. 
h Serum chemistry will include: electrolytes (Na, K, creatinine, random glucose, and ALT (SGPT),. Follow-up samples may be taken if clinically significant abnormalities are seen.  Clinically relevant 
laboratory abnormalities will be recorded as medical history if obtained before day -2. 
i Urine Drug Screen will test for the presence of amphetamine, barbiturates, opiates, phencyclidine, cocaine, and benzodiazepine, methadone, and propoxyphene at screening and at the discretion of the 
study clinician. In addition, the study clinician may ask for a sample to test for the presence of antibiotics. 
j Subjects may begin antibiotic treatment early if one or more criteria are met. 
k During the inpatient period all stool samples are collected, weighed and graded. If a subject meets discharge criteria prior to day 7, no further stool samples will be collected. 
l Stool sample for bacteriology will begin the day after challenge, or prior to institution of early antibiotic therapy (whichever is sooner). If a stool sample is not obtained before 1300 hours, a rectal swab 
will be obtained. Swabs will be used only for bacteriology. Stool samples will be collected for assays as specified in the laboratory study of event schedule and as per written SSPs. A subset of these 
samples, during high shedding points, will be reserved for the later validation and development of bacteriological assays for shedding of ETEC and other organisms. 
m Blood for immunology endpoints will be collected as specified in the laboratory study of event schedule and as per written SSPs. Total approximate predefined blood volumes can be found in the 
laboratory study event schedule. 
n Exploratory Cognitive Assessment will be performed on all individuals during the inpatient phase (thrice daily) using PVT evaluation. 
o Study completion is defined as a subject completing all clinic visits. 
p Approximate total blood volume to be collected is 294 mL. 
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1.0!
BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE 
1.1!
Background Information 
Diarrhea is a significant medical problem globally yielding an estimated 1.3- 4.6 billion annual cases [1, 
2]. Infectious diarrhea causes significant acute morbidity (negatively impacting growth and cognitive 
development) and mortality in infants, young children, and vulnerable populations in resource-limited 
countries, and civilian and military travelers to these areas [3, 4].  According to the World Health 
Organization (WHO) diarrheal illness is the second leading cause of death in children under five years of 
age, accounting for 760,000 deaths per year in this age group attributed in part to malnutrition [1]. 
Travelers’ diarrhea (TD) affects up to 60% of travelers [5, 6].  TD incidence rates reach 0.5 episodes per 
person over 1 - 2 weeks of initial exposure in developing regions [7-11].  TD is commonly self-limiting, 
lasts 2-6 days [12], and resolves after a week in 90% of cases, with a minority of patients experiencing 
persistent or chronic diarrhea.  Although generally a self-limiting illness, about 20% of travelers who 
experience diarrhea are bedridden for some period and approximately 40% change their itinerary in some 
way because of the illness [6].  Diarrhea can vary in severity from mild discomfort to severe dehydration 
and dysentery.  Personal hygiene and field sanitation measures have been unsuccessful in eliminating the 
risk of TD [13-15].  For example, pre-travel education and counseling of individuals on reducing risk 
behaviors (e.g. avoid ice/tap water, undercooked meat, unwashed/unpeeled fruits/vegetables) is common 
practice, however, while this intuitively makes sense, multiple studies have failed to show any consistent 
reduction in disease incidence [16, 17]. Safe, efficacious preventive modalities are critically needed to 
minimize the impact of this common infectious disease threat. 
Bacterial enteropathogens comprise the majority of the pathogens identified in TD (civilian and military) 
encompassing upwards 80% of identified cases [18], with Enterotoxigenic Escherichia coli (ETEC) 
consistently the most identified.  Additionally, ETEC is the most common bacterial etiology of infectious 
diarrhea in endemic pediatric populations accounting for 30 - 50% of diarrheal episodes [9-11].  ETEC is 
culpable in an estimated 400 million cases and 160,000 deaths annually among infants and young children 
[19]. ETEC may be the first enteric illness encountered by infants [20] and the heavy burden of illness 
early in life contributes to malnutrition, which can then lead to growth stunting and diminished cognitive 
development [8].  In 2010 ETEC associated Disability-Adjusted Life Years (DALYs) were estimated at 
8.5 million (10 percent of all diarrhea DALYs), and Years Lived with Disability (YLDs) were estimated 
at one million (13 percent of all diarrhea YLDs) [21, 22]. 
ETEC exposures occur through ingestion of contaminated food and water, typically producing non-
invasive, watery diarrhea, although the diarrhea may manifest with a spectrum of disease presentations 
(based on strain virulence characteristics), ranging from mild diarrheal episodes to severe, cholera-like 
purging (even in immunocompetent hosts). Until vaccines become available, there is an urgent need for 
development of effective diarrhea prevention modalities suitable for use in different contingencies. A 
number of approaches have been taken to develop an ETEC vaccine, including killed whole-cell, live 
attenuated, and protein subunit vaccine strategies [23-28]. However, a licensed product is not expected in 
the near term, and this has revitalized interest in other approaches to ETEC diarrhea prevention.  
Development of an effective prophylactic agent to control ETEC diarrhea would offer a useful product for 
travelers and military personnel going to high-risk areas in Latin America, Africa and Asia. This study is 
part of an effort to fill this void by developing and advancing bovine serum immunoglobulins (BSIgG), 
targeting fimbriae (and their respective fimbrial tip adhesins) an investigational modality that has shown 
proof of principle as a safe, food-based anti-diarrheal supplement when constituted from bovine derived 
hyper-immunized serum targeting protective ETEC antigens.  

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In an effort to develop a product that will abrogate the effects of enterotoxigenic E. coli (ETEC)-
mediated diarrhea in military and civilian traveler populations, investigators at the Naval Medical 
Research Center (NMRC), Silver Spring MD had spearheaded a development program funded by the 
Peer-Reviewed Medical Research Program, and the U.S. Army Medical Material Development Activity. 
The objective of this program was to develop a passive oral immunoprophylaxis product composed of 
anti-adhesive hyperimmune BSIgG specific for the most prevalent class of ETEC colonization factors 
that would confer protection against ETEC diarrhea. The proposed clinical studies within this program 
will be conducted at the CIR, Johns Hopkins Bloomberg School of Public Health (JHBSPH). 
1.1.1! Diarrhea in the Military 
A unique subset of vulnerable travellers is the military.  Military associated diarrheal illness (essentially 
TD occurring in deployed military) has consistently been reported in deployed military personnel and 
remains the leading cause of disease non-battle injury (DNBI) accounting for a significant reduction in 
operational readiness, and mission capability [29] particularly for deployments to the developing world. 
Among military personnel mortality has decreased (compared to historical controls), however, there 
remains significant morbidity, and a clear impact on operational readiness [30].  For historical 
perspective, data suggests that during the U.S. Civil War, 21,000 military deaths were attributable directly 
to dysentery.  During the Korean War, approximately 80,000 duty-days were lost due to diarrhea and 
dysentery.  During the Vietnam War, hospital admission rates or confinement to quarters due to diarrheal 
illness was higher than malaria by a 4:1 ratio, making diarrhea the most burdensome disease of that 
conflict [31].  Up to 70% of deployed U.S. personnel in support of Operations Enduring Freedom and 
Iraqi Freedom reported diarrheal episodes and 30% had three or more episodes with some units 
experiencing a monthly incident rate of up to 60% [32, 33].  Forty percent of UK forces in Afghanistan 
suffered at least one episode of diarrhea during their tour contributing to significant operational impact 
[34] with up to 43,000 man-days lost to ‘no duty’ or ‘reduced performance’ during the six months 
between April and October  2009 [34].  UK military data from Kenya has shown up to a 60% attack rate 
over a 6 week exercising period [34].  Diarrheal disease continues to be of significant military relevance 
as large numbers of young service members are deployed to areas with high TD rates [29].  From a 
military public health standpoint, its acute impact on troop health is larger than any other infectious 
disease syndrome and is compounded by the chronic risk of significant post-infectious sequelae [33, 35-
37]. The most cost-effective response to this military readiness threat is to prevent the exposure leading to 
diarrhea.  The military has developed extensive capabilities for the provision of sanitation and hygiene, 
and clean food and water.  This strategy is reasonably effective when it is possible to develop the proper 
infrastructure, but it is often undermined during rapid deployments and during small scale and brief 
operations.  In large scale deployments conducted under strict security measures that prohibit routine 
exposure to indigenous food and water, diarrhea remains a serious problem.  During the joint 
multinational military exercise conducted in Egypt (Operation Bright Star ’01) under stringent security 
conditions, 9% of personnel reported developing diarrhea [37].  Controlling the base area infrastructure 
may be possible but patrolling patterns in high-risk areas often involves exposure to local pathogens. 
Therefore, development of countermeasures including a safe and effective vaccine is needed to reduce the 
impact of ETEC disease on deployed military personnel and has been deemed a high priority by the U.S. 
military, as ETEC diarrhea has the potential to curtail critical overseas missions.  
1.1.2! Pathogenicity of ETEC 
The pathogenesis of ETEC diarrhea involves the sequential steps of colonization (via colonization factors 
(CF) promoting intestinal adherence) followed by secretogenic toxin production.  CFs are surface-
exposed polymeric protein appendages that are vital to ETEC pathogenesis.  Colonization ensues via the 
proteinaceous adhesive fimbrial surface-exposed polymeric protein appendages (the CF) potentiating 

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microorganism attachment to the human intestinal epithelial cell contributing to infectivity and 
pathogenicity (interfering with intestinal physiology including motility) [38].  Upon colonization, ETEC 
secretes one or both of two enterotoxins that induce fluid and electrolyte secretion (by differing pathways) 
resulting in watery diarrhea. The two enterotoxins produced by ETEC are heat-stable enterotoxin (ST) 
and heat-labile enterotoxin (LT).   
1.1.3! ETEC Colonization Factors 
To date, more than two dozen serologically distinct CFs associated with ETEC and culpable in human 
disease have been identified.  The majority of CFs are fimbrial structures (bacterial surface appendages) 
composed of major and minor protein subunits, and some of these CF types are more prevalent in disease-
associated ETEC than others. Based on sequence analysis, ETEC CFs of relevance to human disease can 
be divided into 7 genetically distinct types, and more than a third of these (8) have been grouped as Class 
5 fimbriae.  Many of the most prevalent ETEC CFs fall into either the α- or γ3-clade [39]. 
CS3 and CS6 are both atypical fimbriae in the fimbrial usher proteins (FUP) γ3-clade, each with two 
major subunits and no tip-localized adhesin, and no sequence similarities between the CS3 and CS6 major 
subunits. Based on meta-analyses of all available reports on ETEC CF prevalence and distribution, very 
conservative estimates indicate that ETEC Class 5 fimbriae in the FUP α-clade along with CS3 and CS6 
(γ3-clade) are expressed by at least 70% of ETEC causing human disease [40]. The most commonly 
detected CFs of the α- and γ3-clade are CFA/I and CS6, respectively, which account for ~26% of all 
ETEC in travelers based on current evidence [40] (Figure 1). 
Figure 1. Conservative, Cumulative Estimates of Expected Global Coverage for an ETEC 
Vaccine or Immunoprophylactic 
  
!
!
!
!
!
!
!
!
!
!
!
!
!
 
CFA/IV includes the structurally indistinct CS6 expressed alone or with CS4 or CS5.  While human 
challenge studies with ETEC strains expressing CFA/I, CFA/II, CFA/IV as well as other CFs have 
demonstrated disease and induced CF-specific immune responses, the role of each CF in disease 
pathogenesis is less clear and rests largely on epidemiologic data showing their relative prevalence, 
distribution, and in some cases association with disease in case-control comparisons [41]. 
!!
0
10
20
30
40
50
60
70
80
90
100
Class/5a 
Class/5b 
Class/5c 
CS3 
CS6 
!
!
!
!
!
!
!
!
!
!
CFA/I 
CS4 
CS14 
CS1 
CS17 
CS19 
PCFO71 
CS2 
CS6 
CS3 
 

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1.1.4! Class 5 Tip Adhesins 
The tip-adhesin-based approach to ETEC prophylaxis is designed to grapple with the issue of 
multivalency using the epidemiologically important group of ETEC expressing Class 5 fimbriae. Tip 
adhesion antibodies may mediate homologous and heterologous protection (cross reactivity) to Class 5 
subclass fimbriae. For example, anti-CfaE antibodies may confer passive protection against ETEC that 
express any of the 3 subclass 5a fimbriae (i.e., CFA/I, CS4, and CS14). Thus, a multivalent vaccine 
containing three CF tip-adhesins could protect against a total of 8 CF types in class 5, thus greatly 
reducing the valency needed for an effective ETEC vaccine.  A final bovine IgG (BIgG) product with 
Class 5 fimbriae-wide protective coverage may afford clinically significant protection against ETEC 
diarrhea in real-life travel settings.  
1.1.5! Optimizing Protective Efficacy Exploiting Passive Oral Prophylaxis with 
BIgG Raised Against Tip Adhesins 
If antibodies to the fimbrial tip adhesins are responsible for inhibiting ETEC binding, then the drawback 
with administering CFA/I to cows to raise a BIgG product is that the tip adhesion accounts for only a 
small portion of the immunizing antigen, and the majority of antibodies are raised to the major subunit 
instead of the tip adhesion. If the formulation and dose of the product is not optimal and the degradation 
in the harsh environment of the stomach is high, the low concentrations of the important anti-CfaE 
antibodies may be reduced to below a crucial level for protection. One way to address this problem 
would be to immunize the cows with CfaE only, and to administer BIgG significantly enriched for 
antibodies to CfaE. A second and equally compelling potential advantage to using CfaE as the 
immunizing antigen would be that anti-CfaE antibodies would be more effective than antibodies to 
CFA/I in the inhibition of colonization against ETEC strains that express CFs other than CFA/I (i.e., tip 
adhesion antibodies potentiate superior heterologous cross-protection to that of the whole CF fimbriae or 
its stalk). If fewer bovine antigens are needed to produce a product with broadened coverage against 
ETEC, it would make such a multivalent product more practical and cost-efficient. A prior clinical study 
(NCT00435526) has compared the efficacy of anti-CfaE BIgG to that of anti-CFA/I BIgG as part of a 
larger product development plan to develop a multivalent BIgG anti-ETEC product finding them to be 
comparable thus supporting the assertion that a multivalent product is feasible. 
1.1.6! Evidence for anti-CF Immunity 
CFs are surface-exposed polymeric protein appendages that are vital to ETEC pathogenesis. Accumulated 
evidence suggests that protective immunity to ETEC may occur, attributable in part to B-cell responses 
against the surface-exposed CFs and labile toxin (LT) enterotoxin most probably in the gut mucosal 
compartment [42, 43]. In endemically exposed populations, there is an inverse relationship between age 
and ETEC disease incidence, consistent with the notion that protective immunity develops from repeated 
exposure to infection [19, 20, 44]. 
The role of CFs and LT enterotoxins 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 [42, 45-48].  Additionally, passive administration of bovine milk antibodies with activity against 
ETEC [expressing the CFs (CFA/I, CS17) and the respective tip-localized adhesins of CFA (CfaE)], has 
been shown to confer significant protection against diarrhea in controlled human challenge trials [49-54]. 
Based on this dual proof-of-principle indicating that two different !-clade ETEC fimbrial CFs  are 
protective antigens as measured by passive protection conferred by anti-fimbrial BIgG, it is reasonable to 
extrapolate that bovine immunization with a cocktail of !-fimbriae selected based on epidemiological 

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prevalence and cross-reactivities could be produced to cover a substantial proportion of naturally 
occurring, pathogenic ETEC. 
1.1.7! Understanding and Knowledge Gaps of CS6 as a Virulence Factor and 
Protective Antigen 
Despite the robust evidence supporting the class 5 CFs as protective antigens, there is a dearth of evidence 
on other CF types, such as CS6.  Since its first description in 1985 [55], CS6 has been the focus of 
considerable research, yet the generation of incontrovertible evidence as to its specific role in diarrhea 
pathogenesis and the role of antibodies towards it in protection against infection is lacking. One clear and 
consistent finding is that CS6, expressed most often alone but also with CS5 or CS4, is one of the most 
common CFs associated with symptomatic ETEC infection in both endemically exposed populations, as 
well as travelers [40]. This has driven the focus on CS6 as a target for many groups working in vaccine 
development [23, 56-59]. The crystal structure of the two CS6 major subunits have been solved [60], yet 
we have not defined its ultrastructural appearance on the bacterial surface [61] and have a limited 
understanding as to how CS6 might interact with the human intestinal surface as a CF [62-65]. 
The majority of individuals naturally infected with CS6-expressing ETEC exhibited mucosal and 
serological responses against CS6 [66] as well as CS6-specific B-cell memory responses [67], while naive 
subjects experimentally infected with CS6-expressing ETEC showed less robust mucosal and serological 
responses [68]. In limited investigations, however, serum anti-CS6 antibody titers did not show a 
protective relationship for subsequent CS6-expresssing ETEC diarrhea [69]. While these findings indicate 
that CS6 is recognized by the host during infection, we have little understanding regarding bacterial 
regulation of CS6 expression in vitro or in vivo [70].  Considering our current body of knowledge, the 
epidemiological importance of CS6 stands in sharp contrast with the absence of consistent, credible proof 
that CS6 serves as a virulent antigen--proof that is urgently needed to facilitate advances in vaccine 
development. The overarching goal of this study is to begin assessing the protective capacity of CS6 in 
passive prophylaxis studies by making use of the experimental human challenge model for ETEC strain 
B7A, which expresses this epidemiologically important CF.  The study proposed herein would serve the 
dual purpose of expanding the scientific basis for development of a multivalent anti-ETEC BIgG product 
while also putting a proven model to use in directly defining the role of CS6 as a virulent antigen. 
1.1.8! B7A Challenge Strain: CS6-expressing, O148:H28 (LT+/ST+) 
The one CS6-expressing ETEC strain that has been established as causing diarrhea in volunteer challenge 
studies is B7A, which expresses CS6, both LT and ST toxins, and is serotype O148:H28 [68, 71-74]. The 
B7A strain was originally isolated from a US military adult serving in Vietnam suffering from acute 
diarrhea. In the most recent volunteer challenge study for which the inoculum was prepared from a cell 
bank produced under current Good Manufacturing Practices (cGMP) conditions at the Walter Reed Army 
Institute of Research (WRAIR), this strain induced moderate-severe diarrhea attack rates of 37.5 and 
100% at doses of 109 and 1010 colony forming units (cfu) respectively. One concern regarding this and 
other ETEC challenge models is the high dose of inoculum required to induce high enough attack rates to 
facilitate evaluation of a vaccine in reasonable numbers of subjects. A B7A inoculum of 1010 cfu may not 
be reflective of the average inoculum in naturally acquired infection and may in turn skew efficacy results 
towards the null in an assessment of a protective intervention. In addressing this concern with another 
ETEC challenge strain, H10407 (CFA/I-expressing ETEC), a refinement of the model was instituted 
whereby implementation of an overnight fast (in place of the typical 90 minute fast before challenge) 
resulted in reproducible attack rates among subjects with inoculum doses 2 logs below previously 
required doses [75]. Prior to executing the investigation, under NMRC.2015.0007 we optimized the 
experimental infection model with B7A to identify the optimal fasting duration and dose required to 
achieve sufficient moderate-severe diarrhea attack rates. 

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1.1.9! History of the ETEC Human Challenge Model 
In a challenge model, a well characterized strain that has been associated with diarrhea and related 
gastrointestinal symptoms but is not resistant to antibiotics is selected. The strain is fed to inpatient 
subjects under supervision in a hospital at a dose that induces diarrhea. Once illness is induced the 
subject is treated with antibiotics, which has been universally effective in curing the infection in 1 to 2 
days. In the last four decades hundreds of subjects have participated in these challenge studies. There 
have been no deaths or long term side effects associated with these studies. Dehydration is the most 
serious potential side-effect, and that is routinely treated with proper oral rehydration therapy, and the 
occasional need for intravenous rehydration. Models for a number of different bacterial enteropathogens 
have been developed, including types of diarrheagenic E. coli [72, 76, 77], Shigella [78-80], Vibrio’s 
[81-83], and Campylobacter [84]. 
Over the past 40 years, the enterotoxigenic E. coli (ETEC) human challenge model has been used to 
elucidate the pathogenesis and immune responses associated with ETEC infection as well as to test the 
safety and efficacy of ETEC specific investigational drugs and vaccines.  The initial experimental 
infection, published in 1971, was a landmark study establishing ETEC as the organism responsible for 
causing acute, cholera-like illness in a U.S. soldier in Vietnam [72].  In this classic paper, researchers 
demonstrated that while porcine and human isolates of disease-causing E. coli were both capable of 
inducing fluid excretion in rabbit ileal loops, only human isolates were capable of causing disease in 
human subjects.  It was later discovered that the difference in the two strains was the species-specific 
tropism of the intestinal CF fimbriae.  One of the strains in that original study was B7A, a CS6-
expressing, LT+, ST+ strain of ETEC. 
Since that landmark study, over 700 naïve subjects have been administered ETEC in an experimental 
infection.  The majority of experience with ETEC strains is with a handful of challenge strains, including 
the B7A strain. In all prior studies, there have been no ‘related’ serious adverse events and all ‘related’ 
adverse events have been consistent with the acute diarrheal illness (with associated signs and symptoms) 
anticipated from an experimental infection with ETEC.  At least in one prior study, it was observed that 
initial experimental infection with the B7A strain protected subjects against re-challenge with the same 
organism approximately 9 weeks later [43].  The B7A strain is sensitive to most commonly used 
antibiotics, including ciprofloxacin, and is readily cleared following a routine 3-day course of antibiotics.   
1.1.10! Active and Passive Immunoprophylactic Approaches 
There are a number of approaches currently being taken to develop an active ETEC vaccine, including 
killed whole-cell, live attenuated, and adhesin based, protein subunit vaccine strategies [85-88]. While an 
increasingly robust effort has been mounted to develop an active ETEC vaccine over the past decade, 
none has yet to achieve licensure in the U.S., and the prospects for achievement of this goal in the next 
decade remains uncertain. 
A modality that has shown some success in the prevention of diarrhea is passive, oral administration of 
bovine milk IgG with specific activity against viral, bacterial and parasitic enteropathogens.  As ETEC 
infects the small intestine, however, the protective antibodies in colostrum must traverse the acid 
environment of the stomach intact and reach their site of action in adequate concentrations. For this 
reason, the effectiveness of passive immunization with bovine antibodies requires the ingestion of large 
quantities of antibodies or the co-administration of buffering agents, which paradoxically may increase 
susceptibility to various infections by reducing the efficacy of the gastric acid barrier.  Evidence for 
passive administration of bovine colostral antibodies in prophylaxis and treatment against a host of 
enteropathogens is discussed in section 1.3.  As expected for a food-based product, the safety record of 
BIgG preparations is excellent [89, 90].  While there is no anti-diarrheal BIgG product yet licensed in the 

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U.S., there are comparable veterinary products on the market indicated for protection of newborn farm 
animals against ETEC diarrhea [89]. Additionally, some evidence of protection was observed in 
randomized trials involving bovine colostrum extract from cows immunized against 14 ETEC strains, 
which is now commercially available in the U.S. as a dietary supplement (Travelan®) to prevent ETEC-
attributable travelers’ diarrhea [51]. 
1.1.11! Preclinical Model for Passive Prophylaxis 
There are no preclinical models in which the efficacy of passive prophylaxis for enterotoxigenic 
Escherichia coli (ETEC) can be assessed. While there has been an effort to develop the Aotus nancymaae 
model, modifying the model to enable passive oral prophylaxis before, during, and after challenge has 
proved difficult (S Savarino, personal communication).  The human challenge model has been (and 
remains) the most suitable model to assess the efficacy of active and passive prophylaxis [91].!
1.1.12! Summary 
Given the limited success of bovine milk IgG products, passive oral administration of BSIgG may protect 
against ETEC-mediated infectious diarrhea.  The hypothesized mechanism of protection stems from the 
passive administration of bovine anti-(tip adhesin or fimbriae) antibodies preventing their adherence in 
the human small intestine (the initial step in pathogenesis), thereby preventing downstream pathogenic 
processes and symptomatic illness.  Advances in technology and shifts in industry focus have led to a 
transition from the manufacturing of bovine colostral to serum antibody products for human use.  One 
potential goal is the development of a safe and efficacious commercially viable multi-valent anti-diarrheal 
BSIgG supplement [encompassing a multivalent cocktail of representative anti- fimbriae (and tip adhesin) 
antibodies] conferring broad protection against both homologous and heterologous populations of ETEC 
pathogens.  The study described herein will establish the foundation for evaluating BSIgG products 
against numerous ETEC CFs. 
Based on conclusive evidence that BIgG preparations against Class 5 fimbriae confer 90-100% protective 
efficacy against CF-homologous ETEC challenge, the critical next step is to demonstrate the passive 
protective efficacy of an anti-CS6 preparation in human subjects. This clinical investigation is designed to 
specifically identify 1) if the BSIgG products are safe and well tolerated and 2) if passive administration 
of bovine serum derived antibodies raised against CS6 and/or the CS6 expressing whole cell B7A ETEC 
protect against moderate-severe diarrhea following experimental infection with the CS6 expressing B7A 
ETEC challenge.  With proof-of-principle that CS6 can serve as a protective antigen, a multivalent BIgG 
product could be conceived for broad coverage against ETEC expressing the most common CFs. 
1.2!
Rationale 
Given the diversity of fimbrial types collectively expressed by ETEC disease isolates, one barrier to 
development of an affordable, effective BSIgG product for prevention of travelers' diarrhea due to ETEC 
is the potentially large number of antigens needed to produce a multivalent preparation that confers broad 
protection.  Since Class 5 fimbrial CFs are collectively expressed by as many as two-thirds of ETEC 
diarrhea case isolates in some areas [92], class-wide coverage by a multivalent BSIgG product would be 
expected to have a clinically significant impact.  If anti-adhesin-based BSIgG preparations are proven to 
be broadly protective, we postulate that a trivalent product containing BIgG with specificity for one 
representative adhesin from each of the three subclasses (i.e., anti-CfaE [5a], anti-CsbD [5b], and anti-
CotD [CS2 fimbrial adhesin, 5c] would be such a product, conferring class-wide protective efficacy [49]. 

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The purpose of this study is to determine if anti-CS6 BSIgG confers protection against oral challenge 
with B7A.  Hyperimmune anti-CS6 BSIgG will be tested in parallel to hyperimmune anti-B7A whole cell 
killed BSIgG to demonstrate the homologous protective effects of anti-CF BSIgG while corroborating the 
importance of CS6 as a protective immunogen.  We hypothesize that anti-CS6 BSIgG will confer 
protection against B7A mediated moderate to severe diarrhea upon challenge. 
1.3!
Previous Human Experience with BIgG Products 
Concentrates of immunoglobulin from bovine milk or colostrum have been evaluated in several human 
clinical trials in hundreds of subjects as outlined in Table 2., and these products have been very well 
tolerated. The products have been investigated as both a prophylactic and treatment for infectious 
diseases caused by organisms like ETEC or EPEC [50, 51, 93-96] (Savarino, unpublished), Rotavirus 
[97-100], Shigella [53], Cholera [101], Cryptosporidium parvum [102-105], Clostridium difficile [106, 
107] and Helicobacter pylori [108]. Depending on the target disease and population, the products have 
been tested for safety and efficacy in healthy adults, immuno-compromised adults and children, and 
healthy children or children hospitalized with diarrhea.!

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Table 2. A summary of a Range of Clinical Trials Evaluating the Safety and Efficacy of BIgG Concentrates in Human 
Subjectsa 
Reference 
Population and type of study 
Cow vaccine 
Daily dose, duration of treatment and 
controls used 
# subjects 
Results of safety and efficacy trials 
BIgG anti-E.coli 
Otto et al 
(2011) 
Healthy adults 
14 ETEC strains 
(including 
serogroup O78) 
400 mg BIgG taken thrice daily with 
bicarbonate buffer 
90 
90% protective efficacy against diarrhea. No 
difference when formulated with/without buffer. Of 
note, 200 mg BIgG conferred 58% efficacy 
compared to placebo. 
Tawfeek et al 
(2003) 
Healthy infants at a Child Health 
Center, Iraq, prophylactic field study 
EPEC (5 or 1 
serotype) 
0.5g BIgG/ kg body weight (supplement 
milk formula), 7 days.  
Control: milk formula only 
65 
Safe and well tolerated. Polyvalent BIgG reduced 
diarrhea, monovalent BIgG no effect 
Casswall et al 
(2000) 
Children, hospitalized with E. coli 
diarrhea, Bangladesh. Therapeutic 
study 
ETEC (14 
serotypes) or EPEC 
(15 serotypes) 
20g daily (5g x 4 doses), 4 days.  
Control: BIgG from non-immunized cows 
32 
Safe and well tolerated. No therapeutic effect 
Tacket et al 
(1999) 
Healthy adults. Inpatient 
prophylaxis/challenge study 
CFA/I, CS3, CS6 
2.07g (0.69g x 3 doses), 5 days.  
Control: BIgG from non-immunized cows 
10 
No report of side effects in publication. No efficacy. 
Savarino et al 
(unpublished) 
Healthy US soldiers deployed in Egypt 
CFA/I 
1:1:1 enrollment to AEMI, 2.07g (0.69g x3 
doses), uncoated granules or 50/50 mix of 
coated and uncoated granules or control. 10 
days intake 
200 
No difference in protective efficacy between control 
and interventional groups 
Freedman et al 
(1998) 
Healthy adults. Inpatient 
prophylaxis/challenge study 
CFA/I 
 
5.1g (1.7g x3 doses) or 1.3g (0.43g x 
3 doses), 7 days. 
Control: LactoFree infant formula 
15 
Safe and well tolerated. Protection from diarrhea. 
Tacket et al 
(1988) 
Healthy adults. Inpatient 
prophylaxis/challenge study. 
ETEC (14 
serotypes), CT, LT. 
10.65g (3.55g x3), for 7 days 
Control: BIgG anti-rotavirus 
10 
Generally well toleratedb0. Protection from diarrhea. 
Mietens et al 
(1979)b 
Infants hospitalized with EPEC 
diarrhea, Germany. Therapeutic study. 
EPEC (14 
serotypes). 
1g/kg body weight (distributed over meals), 
10 days. 
Control: children with EPEC of a serotype 
not present in cow vaccine treated with 
BIgG 
60 
No report of side effects in publication. Better 
clearance of infection. 
BIgG anti-Rotavirus 
Sarker et al 
(1998) 
Infants hospitalized with rotavirus 
diarrhea, Bangladesh. Therapeutic 
study. 
Rotavirus (4 
serotypes). 
10g (in 4 doses), 4 days 
Control: milk powder 
40 
Safe and well-tolerated. Reduced diarrhea. 
Mitra et al 
(1995) 
Infants hospitalized with rotavirus 
diarrhea, Bangladesh. Therapeutic 
study. 
Rotavirus (4 
serotypes). 
10g (in 3 doses), 3 days 
Control: normal colostrum 
35 
Safe and well-tolerated. Reduced diarrhea duration 
and output. 

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Brunser et al 
(1992) 
Children in Chile. Prophylactic field 
trial. 
Rotavirus + E. coli. 
1g BIgG product (supplementing milk 
formula), 6 months. 
Control: milk formula alone 
117 
Safe and well-tolerated 
No efficacy. 
Davidson et al 
(1989) 
Children hospitalized (not for diarrhea) 
in Australia. Prophylactic study. 
Rotavirus (4 
serotypes). 
50mL (concentration not reported), 10 days 
Control: infant formula 
55 
No report of side effects in publication. Prevented 
acquisition of symptomatic rotavirus infection. 
Hilpert et al 
(1987) 
Children hospitalized with rotavirus 
infection, Germany. Prophylactic study. 
Rotavirus (4 
serotypes), 3 
products with 
varying titers. 
2g/kg/day for a winter season. 
Control: No treatment 
75 
No report of side effects in publication. 
High-titer BIgG reduced excretion of virus and 
duration of diarrhea (latter non-significantly). Low-
titer BIgG no efficacy. 
Ebina et al 
(1985)e 
Children hospitalized with rotavirus 
infection, Japan. Therapeutic study. 
Rotavirus (1 
serotype). 
20-50mL (concentration not reported, 3 
days. 
Control: no BIgG treatment 
18 
Safe and well tolerated. No therapeutic effect. 
Ebina et al 
(1985)e 
Children in an orphanage in Japan. 
Prophylactic study (Rotavirus outbreak 
during the treatment phase) 
Rotavirus (1 
serotype). 
20mL (concentration not reported, approx 5 
weeks. Control: market milk 
6 
Safe and well tolerated. Reduced acquisition of 
rotavirus infection from outbreak. 
BIgG anti- Cryptosporidium parvum 
Okhuysen et al 
(1998) 
BB-IND-4122 
Healthy adults. Inpatient 
prophylaxis/challenge study. 
C. parvum. 
30 g (10g x3 doses), 5 days.  
Control: nonfat powdered milk 
10 
Safe and well tolerated. No reduction in infection or 
diarrhea. 
Greenberg & 
Cello (1996)c 
AIDS patient with chronic diarrhea 
from C. parvum. Case report. 
C. parvum. 
40g daily (10g x4 doses), 21 days 
23 
Generally safe and well tolerated. Reduction in 
diarrhea if product in powder form, not in capsule 
form. 
Ungar et al 
(1990)c 
AIDS patient with chronic diarrhea 
from C. parvum. Case report. 
C. parvum. 
Infusion by nasoduodenal tube, 20cm3/h for 
60h, concentration not reported. 
1 
No report of side effects in publication. Reduced 
diarrhea. 
Nord et al 
(1990)c 
AIDS patients with diarrhea from C. 
parvum. Case report. 
C. parvum. 
14g by infusion via nasogastric tube for 10 
days. 
3 
No report of side effects in publication. Reduced 
stool output and oocyst excretion in 1/3 patients. 2/2 
control patients (control colostrum) reduced stool 
output but not excretion. 
Tzipori et al 
(1987)c 
Immuno-deficient patients with 
diarrhea from C. parvum. Case 
report. 
C. parvum. 
200-500mL by nasogastric tube for 10-21 
days, concentration not reported 
3 
No report of side effects in publication. Resolved 
diarrhea in 3/3, resolved infection in 1/3. 
BIgG anti-Clostridium difficile 
Warny et al 
(1999)c 
Adults with end ileostomys, transit 
study. 
C. difficile toxoid 
(A). 
5g on 4 separate occasions. 
6 
No adverse experiences. Not an efficacy study. 
Kelly et al 
(1997)c 
Healthy adults, transit study. 
C. difficile toxoid (A 
and B). 
45g or 8g, one dose only. 
10 
No report of side effects in publication. Not an 
efficacy study. 
BIgG anti-Shigella flexneri 
Tacket et al 
(1992) 
Healthy adults. Inpatient 
prophylaxis/challenge study. 
Shigella flexneri. 
30g (10g x3 doses), 7 days. Control: sodium 
bicarbonate alone 
10 
Safe and well-tolerated. Protection from diarrhea. 
 

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!
2.0!
OBJECTIVES 
 
2.1!
Primary Objectives 
The primary objectives of this study are to assess the safety of serum-derived bovine immunoglobulins in 
healthy adult subjects when orally administered three times a day over 7 days and to estimate protective 
efficacy of those preparations against moderate-severe diarrhea upon challenge with B7A.  
The primary endpoint for this study is moderate-severe diarrhea defined as follows post-inoculation: 
•!
Moderate diarrhea: 4 to 5 loose/liquid stools or 401-800 g of loose/liquid stool in any 24-hour period 
•!
Severe diarrhea: ≥ 6 loose/liquid stools or > 800 g of loose/liquid stool in any 24-hour period 
2.2!
Secondary Objectives 
The secondary objectives include assessments of a variety of clinical endpoints, measuring mucosal and 
systemic immune responses and obtaining and archiving samples for future proteomics and/or systems 
biology efforts. 
1.! Measure mucosal and systemic immune responses to experimental infection  
2.! Obtain and archive samples for future proteomics, microbiome and/or systems biology efforts 
A number of secondary endpoints will be determined in this study.  Specific endpoints have been selected to 
support the primary outcome and are outlined below.  
1.! Maximum 24-hour stool output 
2.! Percent of subjects with severe diarrhea 
3.! Percent of subjects with diarrhea of any severity 
4.! Total weight of grade 3-5 stools passed per subject over 120-hour period 
5.! Number of grade 3-5 stools per subject  
6.! Percent of subjects with nausea, vomiting, anorexia, or abdominal pain/cramps rated as moderate to 
severe 
7.! Mean/Median time to onset of diarrhea 
8.! Number of subjects with moderate to severe ETEC illness 
9.! Number of cfu of the challenge strain per gram of stool 2 and 4 days after challenge 
10.!ETEC clinical severity score post-challenge 
2.3!
Exploratory Objectives 
1.! Exploratory immunology and systems biology analyses to include transcriptomics, proteomics, 
phosphoproteomics, cytokine secretion measurements, lymphocyte subpopulation characterizations, 
and antigen-specific memory B cell quantification. 
2.! Exploratory evaluation of the cognitive impact of acute diarrhea using psychomotor vigilance testing. 
3.! Evaluate the impact of both the B7A ETEC challenge and antibiotic exposure on short-term changes 
in host microbiota. 
4.! Explore the impact of the microbiome on disease susceptibility. 
5.! Evaluate the impact of the B7A ETEC challenge on short-term changes in intestinal 
inflammation/repair, epithelial barrier function, motility, and immune system modulation. 

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3.0!
STUDY DESIGN 
This is a Phase 1, randomized, double-blind, placebo-controlled study designed to investigate whether anti-
CS6 BSIgG protects subjects against diarrhea upon challenge with a CS6-expressing ETEC strain B7A, 
compared to the protective efficacy of the positive control (anti-B7A BSIgG) and negative control (non-
hyperimmune) BSIgG. The anti-B7A BSIgG will serve as a positive control and will not be directly compared 
to the anti-CS6 BSIgG. The study will also evaluate the safety and tolerability of these BSIgG products and 
describe the immune responses following challenge. The basic study design is depicted in Table 3. 
Table 3. Description of Study Groups 
Producta 
N 
Dose (approximate) 
Anti-CS6 BSIgG 
20 
1.0g three times 
daily (tid) 
Anti-B7A whole cell killed 
BSIgG 
20 
1.0 g tid 
Negative Control (Non-
hyperimmune) BSIgG 
20 
1.1 g protein total 
(equivalent) tid 
a All products will be given 3 times daily 
 
Subjects (N=60) will be randomized into three groups receiving anti-CS6 BSIgG, anti-B7A whole cell killed 
BSIgG, or a placebo control (non hyperimmune BSIgG).  Subjects will receive three doses a day of the test 
article 15 minutes (range 10 - 25 minutes) after each of their three daily meals (breakfast, lunch and dinner) 
for a period of 7 days (i.e., from study day -2 to study day 4). The study will be divided into two cohorts of 
approximately 30 subjects each. The target number of subjects to be challenged with ETEC is 60.  
Unit doses of the test article (as detailed in Table 3) will be administered according to the relevant SSP.  
Doses of the test articles/placebo will be prepared by the research pharmacist, and will start on study day -2. 
On study day 0, after receipt of test article/placebo, subjects will be given 120mL of sodium bicarbonate 
buffer to neutralize their stomach acidity. About 1 minute later they will ingest approximately 1 x 1010 cfu of 
CS6 expressing B7A-ETEC strain diluted in 30 ml sodium bicarbonate buffer.  Subjects will continue to 
receive three doses a day of the test articles/placebo until study day 4.  Subjects meeting pre-determined 
criteria for early antibiotic administration will be treated with antibiotics and test article/placebo 
administration will be discontinued with initiation of treatment. Subjects who do not receive early antibiotic 
treatment will start antibiotic treatment on study day 5.  Routine discharge is scheduled for day 8, when most 
subjects are expected to meet the discharge criteria of:  they feel well (clinical symptoms resolved or 
resolving) and have taken at least two doses of antibiotic and have 2 consecutive stool culture negative for the 
challenge strain. Subjects may be discharged earlier than day 8 on a case-by-case basis if they meet discharge 
criteria. For subjects who do not meet the discharge criteria on day 8 will remain on the unit until discharge 
criteria has been met. 
The duration of the active study period is approximately eleven months, encompassing up to 90 days of 
screening/enrollment, 4 weeks of the inpatient/outpatient phase when data and samples will be collected, 12 
weeks for immunology assays, and 2 months for analysis and report. Additionally, subjects will be contacted 
6 months after challenge to see if they are still well and to complete the functional bowel disorder survey. 
4.0!
STUDY POPULATION 
Subjects will be recruited from the Baltimore-Washington and surrounding areas via advertisements and word 
of mouth and screened at the CIR.  They will be healthy male and non-pregnant females, aged 18 to 50 
inclusive.  A sufficient number will be screened to provide 20 subjects in each of the three groups, with a 

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target of about 60 subjects enrolled.  Up to 6 alternates per cohort will be recruited to replace anyone who 
does not report or is unable to participate at the time of planned admission.  Two of these alternates may be 
admitted on Day-3 and stay overnight until the first dose of IP is given on Day -2 to replace subjects who 
become ineligible for continuation in the study on Day -2. Alternates will not be randomized unless they are 
replacing a study subject unable to participate in the study (prior to the first dose of test article/placebo). 
Alternates not replacing a subject will be discharged on Day-2 prior to IP receipt. 
4.1!
Subject Inclusion Criteria 
Inclusion Criteria: 
1.! Male or female between 18 and 50 years of age, inclusive.  
2.! General good health, without significant medical illness, abnormal physical examination findings or 
clinical laboratory abnormalities as determined by principal investigator (PI) or PI in consultation 
with the research monitor and sponsor. 
3.! Demonstrate comprehension of the protocol procedures and knowledge of ETEC illness by passing a 
written examination (pass grade ≥ 70%) 
4.! Willing to participate after informed consent obtained. 
5.! Available for all planned follow-up visits. 
6.! Negative serum pregnancy test at screening and negative serum and/or urine pregnancy tests on the 
day of admittance to the inpatient phase for female subjects of childbearing potential. Females of 
childbearing potential must agree to use an efficacious hormonal or barrier method of birth control 
during the study. Abstinence is acceptable. Female subjects unable to bear children must have this 
documented (e.g., tubal ligation or hysterectomy). 
4.2!
Subject Exclusion Criteria 
Exclusion Criteria: 
 
General health criteria 
1.! Presence of a significant medical condition, (e.g. psychiatric conditions or gastrointestinal disease, 
such as peptic ulcer, symptoms or evidence of active gastritis or gastroesophageal reflux disease, 
inflammatory bowel disease, alcohol or illicit drug abuse/dependency), or other laboratory 
abnormalities which in the opinion of the investigator precludes participation in the study. 
2.! Immunosuppressive illness or IgA deficiency (serum IgA < 7 mg/dL or below the limit of detection 
of assay ) 
3.! Evidence of confirmed infection with HIV, HBsAg, or HCV, with confirmatory assay.  
4.! Use of any investigational product within 30 days preceding the receipt of the investigational 
products, or planned use during the active study period 
5.! Significant abnormalities in screening lab hematology or serum chemistries, as determined by PI or PI 
in consultation with the research monitor and sponsor. 
6.! Lactation or breastfeeding. 
 
Research-related exclusions applicable to challenge 
7.! History of microbiologically confirmed ETEC or cholera infection in last 3 years. 
8.! Occupation involving handling of ETEC or Vibrio cholerae currently, or in the past 3 years. 
9.! Travel to countries where ETEC or cholera infection is endemic (most of the developing world) 
within 3 years prior to dosing. 

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10.!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.  
11.!Vaccination for or ingestion of ETEC, cholera, or E coli heat labile toxin within 3 years prior to 
dosing. 
12.!Any prior experimental infection with ETEC strain B7A. 
 
Study-specific Exclusion Criteria (potential increased risk or complicating outcome ascertainment) 
13.!Abnormal stool pattern (fewer than 3 per week or more than 3 per day). 
14.!History of diarrhea in the 2 weeks prior to planned inpatient phase. 
15.!Regular use of laxatives, antacids, or other agents to lower stomach acidity (regular defined as at least 
weekly). 
16.!Use of antibiotics during the 7 days before receipt of any investigational product or proton pump 
inhibitors, H2 blockers, or antacids within 48 hours of receipt of any investigational product. 
17.! Use of any medication known to affect the immune function (eg, systemic corticosteroids and others) 
within 30 days preceding the administration of challenge or planned use during the active study 
period. 
18.!Known allergy to fluoroquinolones.  
19.!Inability to tolerate 150 mL sodium bicarbonate buffer (based on requirement for frequent 
dosing). 
 
5.0!
STUDY PROCEDURES 
5.1!
Screening 
The CIR may use a screening protocol approved by the Johns Hopkins School of Public Health (JHSPH) 
Institutional Review Board (IRB) in recruiting subjects for this study. The screening protocol is entitled 
“Screening of adult volunteers for eligibility to participate in clinical studies evaluating investigational 
vaccines, antimicrobial agents, or disease prevention measures or the pathogenesis of infectious agents” 
JHSPH IRB 200, JHSPH IRB H.22.04.02.19.A2. Subjects will be made aware that the screening process may 
take several visits to complete. Using this screening protocol, a medical history/exam and a series of clinical 
laboratory tests may be completed to rule out occult illness and pregnancy. These laboratory tests may 
include, but are not limited to complete blood count (CBC), serum chemistries, hepatitis B antigen, hepatitis 
C antibody, HIV-1 antibody, IgA levels, serum HCG (for females of childbearing potential), and urine 
toxicology (drug screening). (Confirmatory testing will be performed on subjects who test positive for 
hepatitis B, hepatitis C, or HIV-1 antigens.) Subjects who have ≤ 2 mild (grade 1) non-hematologic 
abnormalities may be included if the PI determines that their participation will not present undue risk to the 
subject. Subjects with > 2 mild abnormalities will not be included in the study. Subjects with clinical 
laboratory abnormalities of greater than mild severity will not participate in this clinical trial. The clinical 
toxicity grading scale that will be used as a guideline is based on the scale used by the Division of AIDS 
(DAIDS) for adverse events and the guidance from the US Food and Drug Administration (FDA) Center for 
Biologics Evaluation and Research. If any additional safety labs are performed, either scale may be utilized. 
Potential subjects will be given a complete description of the study. To ensure comprehension of the study, all 
subjects will have to pass a written examination before inclusion in the study. Subjects who meet all inclusion 
criteria and none of the exclusion criteria, pass the comprehension test, and sign the study Informed Consent 
Document (ICD) may be eligible for the study.  

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Informed consent is an ongoing process which includes the informed consent document. Subjects will receive 
an oral presentation of the study. Each prospective subject will be given the written, IRB-approved informed 
consent, allowed ample time to read the consent, allowed to ask questions about the study, have his/her 
questions answered, and given time to decide if he/she would like to participate in the study. To document 
subjects’ understanding of informed consent, immediately before the consent is signed, the person obtaining 
consent will administer a brief quiz or comprehension test. Incorrect answers will be discussed with subjects 
to reinforce the consent. A final acceptable test score is 70% or more answered correctly. Subjects who fail 
the comprehension test on the first attempt may retake the comprehension test on the same day, or they may 
come back on a separate visit to retake the test. Subjects failing after two attempts are not eligible for study 
enrollment. No coercion or influence is allowed in obtaining subjects’ consent. Before subjects participate in 
the study, consent forms will be signed and dated by subjects as well as by the PI or designee. Subjects will 
receive copies of the signed consent prior to participation. As part of the consent process, subjects will also be 
asked to read and sign additional IRB approved forms including but not inclusive, a Medical Records/Lab 
Results Release, alternate information form, inpatient guidelines, with an opportunity to ask questions, if 
relevant.  
Subjects will be asked to drink about 150 mL of sodium bicarbonate buffer to ensure tolerability for frequent 
dosing. Subjects will be asked to complete a Functional Bowel Disorder Survey (Rome III) to establish a 
baseline of general GI health for subsequent surveys (survey either taken by subjects or administered by study 
staff). 
Additionally, samples for future ABO and RH blood typing may be collected following recent data suggesting 
correlation between ABO typing and susceptibility to moderate to severe diarrhea following challenge with 
ETEC strain H10407 (Fleckenstein, unpublished). RH typing has been previously reported to affect clinical 
outcomes in subjects infected with other enteric pathogens and as such similar associations will be assessed as 
part of this study [109-111]. 
5.2!
Randomization 
Subjects will be randomized in a 1:1:1 ratio to one of 3 treatment groups. An investigator at NMRC not 
involved in outcome assessment will prepare a randomization list, allocating volunteer identification numbers 
to the study groups using the PROC PLAN function of SAS v9.2 (Cary, NC). The randomization scheme will 
utilize block sizes of 6 in order to ensure comparable group sizes in the event that the targeted number of 60 
subjects is not reached.  NMRC staff will print the code, log and output of the SAS procedure, sign them, and 
store them under lock and key. A photocopy of the signed output will be provided to the research pharmacist 
prior to the first BSIgG administration day.  
5.3!
Group Assignment 
Prior to the first dose of test article/placebo, subjects will be assigned a study number. Subjects will receive 
the test article/placebo in containers bearing their assigned identification numbers.  This number will be 
linked to the randomization code list securely maintained throughout the clinical phase of the study by the 
designated NMRC staff and the JHU research pharmacist.  Study identification numbers will identify all 
samples for laboratory analyses. 
5.4!
Blinding 
Investigators and subjects will remain blinded as to group until completion of the clinical phase of the trial 
and validation of the clinical and immunological data.  Each test article/placebo bottle is labeled with an open 
label as described in section 7.0. The research pharmacist will use the randomization list to prepare the IP. IP 
bottles will be sent to the research pharmacy prior to administration.  All mixing and administration of the test 

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articles/placebo will be performed per formulation and product administration SSPs. Administration will 
occur in a separate room from where the doses are prepared. 
Only in a medical emergency, when knowledge of the study treatment is essential for further management of 
subjects, will the randomization code be broken.  In the event that this is necessary, the PI will provide the 
study identification number to the 24-hour pharmacy, who in turn will break the seal on that subject’s 
envelope and provide the investigator with the broken code for that subject.  The investigator will notify the 
Sponsor immediately and document the event on the appropriate Source Documents and electronic Case 
Report Forms (eCRF). 
5.5!
Clinical Evaluations 
5.5.1! Monitoring During Inpatient Phase 
Subjects will be monitored daily while inpatient for general, gastrointestinal, and systemic signs and 
symptoms, have medical conditions reviewed, and adverse effects noted. This will include examination by a 
study physician/nurse practitioner and solicitation of daily progress reports. Additionally, subjects will be 
examined for symptoms and signs of dehydration, including thirst, dizziness on standing, decreased skin 
turgor, and dryness of mucous membranes. Vital signs will be recorded three times daily, and more often 
when subjects are ill.  If subjects develop moderate or severe diarrhea, postural blood pressure and pulse will 
be measured as necessary for clinical management according to the judgment of the physician/nurse 
practitioner. 
Johns Hopkins Bayview Medical Center inpatient research facility is a self-contained unit, suited for 
conducting live-in studies.   
Subjects will remain at the inpatient facility under clinical observation. Vital Signs will be assessed at least 3 
times each day, once in the morning, in the afternoon and at bedtime.  On challenge day, vital signs will be 
assessed 4 times, once prior to challenge, once about 30 minutes after challenge, and then 2 additional times 
this day. A clinician will conduct a daily medical interview and focused physical exam to assess health status, 
follow-up, monitor, and treat as indicated. All stools will be collected for weighing and grading. Following 
ETEC B7A challenge, up to 3 stool samples will be collected daily for culture as per SSP starting the day 
after challenge. If a subject is unable to provide a stool sample by 1300 hours, s/he will be asked to obtain a 
rectal swab. Swabs will be used starting the day after challenge. 
Subjects will perform 5-minute psychomotor vigilance testing (PVT) at least three times a day during the 
inpatient phase. As an exploratory assessment, performance of the three PVTs per day will be predicated on 
the subject not undergoing other procedures or primary study related events. Missed PVTs will not be 
considered protocol deviations. Similarly, management of symptoms associated with ETEC or other illness 
will have priority over completion of PVTs.   
Treatment for severe nausea or vomiting may be needed. Subjects who experience severe nausea or vomiting 
may be given ondansetron (Zofran) ODT or ondansetron IV. 
5.5.1.1!
Rehydration Procedures 
Subjects passing grade 3-5 stools post-challenge will be offered ORS or Gatorade to prevent dehydration, at 
the same volume as their stool output. For documentation purposes of concomitant medications, ORS will not 
be considered a concomitant medication while IV fluids will. 
A subject may be administered IV fluids (clinician discretion) for the following reasons:  

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•!
Subject experiences abrupt onset of diarrhea defined by passage of an initial loose/liquid stool of > 
300g or passage of > 400 g of loose/liquid stools over 2 hours. 
•!
Subject becomes hypovolemic. 
•!
It is determined necessary by the study physician, i.e., diarrhea with nausea/vomiting and unable to 
drink enough to keep up with output, or other reason. 
Hypovolemia is a significant decrease in blood volume, characterized by: 
•!
Orthostatic hypotension, confirmed systolic blood pressure (BP) < 90 mmHg and associated 
symptoms 
•!
Or significant lightheadedness on standing with a confirmed postural change in BP or pulse. Postural 
vital signs will be measured lying and 2 minutes after standing. A significant change will be either of 
the following: decrease in systolic BP of > 20 mmHg, or diastolic BP of > 10 mmHg or increase in 
pulse of > 30 beats/min. 
5.5.1.2!
Routine Discharge 
Routine discharge is scheduled for study day 8. Two consecutive negative stool cultures for B7A are required 
before discharge (can be collected on the same study day). If the subject has not completed antibiotics, then 
the remaining doses of antibiotic will be given to the subject for self-administration. Vital signs will be 
collected.  
5.5.1.3!
Early Discharge 
Early discharge is permitted in cases where early antibiotic treatment has been initiated. The subject needs 2 
consecutive stool cultures negative for B7A and to have taken two doses of antibiotic with resolved or 
resolving clinical symptoms before discharge. Remaining doses of antibiotic will be given to the subject for 
self-administration. Subjects discharged before study day 7 will return on day 7 and provide the requisite 
samples (stool, blood) as delineated in Table 1.   
5.5.2! Monitoring During Outpatient Phase 
On study day 28 (+/- 2 day), subjects will return to the clinic for a follow up visit as described in section 6.5. 
Some subjects may also be outpatients on day 7.  In addition, subjects will also have a single phone follow-
up on day 180 (+/- 1 month). Clinic visits during follow-up will include vital signs assessment, clinical 
checks, including concomitant medications and AEs, and sample collection for immunogenicity and 
exploratory outcome evaluation. 
5.6!
Concomitant Medications/Treatments 
Only concomitant medications approved by the study physician will be used during the study. Subjects 
needing to take unapproved or excluded medication will not be eligible for enrollment in this study.  As the 
subjects will stay in the inpatient facility after challenge until treatment, this should not be an issue.  Subjects 
taking regular medication (i.e., birth control pills) prior to enrollment will be allowed to continue unless it is 
specifically excluded as part of the inclusion/exclusion criteria. Any medication ordered during the trial (i.e., 
Tylenol or ciprofloxacin or alternative antibiotics) will be documented in the subject’s study chart and on the 
appropriate page of the eCRFs.  Approved medications that were being taken prior to, as well as during the 
course of the trial will also be documented in this manner. 

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5.7!
Laboratory Evaluations 
5.7.1! Specimen Preparation, Handling and Shipping 
Research microbiology, including the preparation of live inoculum and culturing of specimens, will be carried 
out in the laboratory of the CIR in the JHBSPH.  Immunologic assays will be carried out at the Enteric 
Diseases Department at NMRC or the Core Lab of the JHBSPH.  Samples collected under this protocol will 
be used to conduct protocol-related safety and immunogenicity evaluations.  Samples for immunogenicity will 
be collected at the CIR and maintained at the CIR or core lab until transport to NMRC. Storage at NMRC of 
these biological samples will be handled according to appropriate procedures. Any study for the future use of 
these biological samples will have IRB approval. All subjects will consent for the future use of their 
specimens. 
5.7.2! Clinical Laboratory Evaluations 
Standard clinical laboratory tests for the purpose of inclusion and exclusion of potential subjects and for 
safety monitoring will be carried out at JHH, JH Bayview Medical Center, or Quest Diagnostics in Baltimore 
City. Microbiology tests will be done in the CIR bacteriology laboratory. Study related samples will be 
labeled according to the relevant SSP. 
5.8!
Outcome Measures 
5.8.1! Clinical 
The primary endpoint of this study is moderate to severe diarrhea according to the following definitions post-
inoculation:  
Severe diarrhea: ≥ 6 grade 3-5 stools in 24 hours, or > 800 g of grade 3-5 stools in 24 hours and,  
Moderate diarrhea: 4-5 grade 3-5 stools in 24 hours or 401-800 g of loose/liquid stool in any 24-hour 
period 
Stool will be graded based on a standard stool grading scale as follows:  
Grade 1 = Fully formed (normal)  
Grade 2 = Soft (normal)  
Grade 3 = Thick liquid (diarrheal)  
Grade 4 = Opaque watery (diarrheal)  
Grade 5 = Rice-water (diarrheal) 
Additional secondary endpoints have been selected as follows:  
•!
Maximum 24-hour stool output  
•!
Percent of subjects with severe diarrhea  
•!
Percent of subjects with diarrhea of any severity  
•!
Total weight of grade 3-5 stools passed per subject  
•!
Number of grade 3-5 stools per subject  
•!
Percent of subjects with nausea, vomiting, anorexia, or abdominal pain/cramps rated as moderate to 
severe  
•!
Mean/median time to diarrhea onset 
•!
Number of subjects with moderate to severe ‘ETEC illness’ 
•!
Number of cfu of the challenge strain per gram of stool 2 and 4 days after challenge  

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•!
ETEC systemic and diarrhea severity score post-challenge with B7A [112] 
An exploratory assessment of the cognitive impact of ETEC challenge will be conducted with the use of PVT 
monitoring. The outcomes are exploratory in nature and will not be utilized as part of the regulatory, safety, 
immunogenicity, or efficacy evaluation of the study product. Subjects will use PVT device while inpatients, 
and PVT is a measure of a subject’s ability to respond to a visual prompt by pushing a button.  Three PVT 5-
min tests per day will be performed by each subject up until discharge from the treatment facility or as 
outlined in the SSP. Comparisons will be made between symptom presence/severity and adjusted for other 
confounding variables. 
5.8.2! Immunological 
Blood will be collected per the Time and Events Schedule from subjects to assess for ETEC challenge 
antigen-specific serum IgA and IgG responses.   
The serum will be processed at the CIR laboratory, transferred to the NMRC laboratory, and assayed for IgG 
and IgA antibody titers against LT using anti-ganglioside M1 (GM1)-enzyme-linked immunosorbent assay 
(ELISA), and against CS6 and LPS using methods previously described [113, 114]. For all antigens, pre- and 
post-dosing serum samples from the same individual will be tested side by side.  The antibody titer ascribed 
to each sample will represent the geometric mean of duplicate determinations. Reciprocal endpoint titers <50 
will be assigned a value of 25 for computations.  Seroconversion is defined as ≥ four-fold increase in endpoint 
titer between pre- and post-challenge specimens AND a post-challenge reciprocal titer > 100. Exploratory 
immunological assays may include memory B cell evaluation, flow cytometric assays, and systems biological 
assays (transcriptomics, proteomics, phosphoproteomics, if funds are obtained) as outlined in the Time and 
Events Schedule.  
Qualitative (responder rates) and quantitative assessments (log transformed values) will be made in addition 
to evaluation of the kinetics of the immune response.  Median increases (fold-rises) of anti-ETEC (i.e., CS6, 
LT, and LPS) antibody concentrations and seroconversion rates will be calculated.  Geometric mean titers will 
also be determined.   
PBMCs will be assayed to determine antigen specific (CS6, LPS, and LT) ALS responses.  ALS is a 
methodology that has been shown to be a replacement for enzyme-linked immunospot assay (ASC) 
methodology. PBMCs are incubated without stimulation and the supernatant is later assayed for antigen-
specific IgG and IgA antibodies by ELISA. A positive ALS response will require a two-fold rise in antibody 
titers between pre and post challenge samples.  
5.8.3! Microbiological 
During the inpatient study [post-challenge day 0 to day 8 (or day of discharge)], stool samples (at least 1per 
subject per day post-inoculation) or rectal swab (if necessary) will be screened for the presence of B7A. 
Samples will be collected, processed and shipped as per the SSP, for qualitative cultures. Up to 10 E. coli-like 
colonies from MacConkey selective media will be subcultured onto CFA without bile salts agar and then 
screened for agglutination with challenge strain-specific antiserum using a slide agglutination technique.  
Additional culture-independent methods may be used to quantitate B7A shedding. 
5.8.4! Exploratory 
Exploratory and expanded immunological assessments will be planned for this study. Among these, serum 
and PBMC samples may be collected for transcriptomic, cytokine, proteomic, and other systems biology 

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analyses to identify molecular signatures associated with ETEC infection. The cytokine analyses will 
encompass representation from multiple pathways including pro-and anti-inflammatory, and regulatory 
pathways.  
Antigen specific memory B cell quantification may be performed with purified PBMCs to investigate the 
response generated following oral challenge. Briefly, following an in vitro stimulation/expansion to activate 
memory B cells, they are finally quantified as Ag-specific antibody-secreting cells.  
Fecal and salivary IgA samples will be obtained to assess for mucosal IgA (including but not limited to total 
and anti-CS6, anti-LPS, and anti-LT) (Table 1).  Subjects will be provided collection containers to collect all 
stools which will be processed per SSP. 
Collection of a sublingual saliva sample 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 (only the 
lingual area—not from the parotid) for several (approximately 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.  Immunologic responders will be defined as subjects with a ≥ two-fold 
increase in reciprocal endpoint titer. 
In addition, stool samples will be obtained to assess for exploratory endpoints to include microbiome 
characterization, culture-independent methods to quantitate B7A shedding, and PCR and transcriptomics (on 
the microbiome).  This testing is subject to change as advances in research occur during the time that the stool 
is archived. These samples will be collected per SSP. 
5.8.5! Outcome Adjudication Committee 
In an effort to obtain an unbiased determination of the efficacy outcomes, an independent outcome 
adjudication committee, the members of which will be blinded as to the treatment regimens of the subjects, 
will evaluate challenge outcome data after completion of the inpatient phase of the study. 
The committee will be comprised of at least 3 individuals, independent of the study sponsor and investigative 
team, who are experts on diarrheal illness case identification and pathogen diagnosis. The committee will also 
include a statistician/data analyst who will lead and coordinate the committee but will not have a voting role 
in deliberations. 
The committee voting members will review all potential efficacy-related cases and endpoint data. Among the 
committee’s responsibilities, they will (1) review and confirm all primary endpoint cases; (2) review all 
protocol-specified entry criteria, adherence, and compliance issues to ascertain classification in the per-
protocol and other study populations; and (3) provide guidance regarding secondary and other endpoint 
classifications to include agreement on objective criteria for classification of endpoints. Specific duties and 
responsibilities will be outlined by charter prior to the start of the study. 
6.0!
STUDY SCHEDULE 
The Time and Events Schedule (Table 1) details the study schedule. Subjects will receive unique, individual, 
study identification numbers either at screening or upon admission. 

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6.1!
Screening (Day –90 to Day –5) 
The following procedures will be completed at the JH200 and/or study-specific screening visit, between days 
-90 to -5, to determine and confirm study eligibility.  An additional screening visit may be scheduled for any 
follow-up as needed, but is not required.  
•!
Subject must fully understand the elements of the Informed Consent form, and sign and date the form 
prior to initiating protocol-specific procedures not covered in the JH200 screening protocol. 
•!
Subject must take and pass (with ≥70% understanding) a comprehension test. Study staff will review any 
questions that the subject may have and the subject will be able to retake the comprehension test if they 
do not pass the first time. 
•!
Assess inclusion and exclusion criteria. 
•!
Record demographics and medical history, including gender, date of birth, race, height, weight, BMI, and 
any allergies. 
•!
Complete physical examination including assessment of HEENT, heart, lungs, abdomen, skin, lymph 
nodes, neurological and musculoskeletal systems. 
•!
Take vital signs (heart rate, blood pressure, respiratory rate, and oral temperature).  
•!
Check health status. 
•!
Blood draw for hematology (complete blood count with differential), serum chemistries (sodium, 
potassium, creatinine, glucose, SGPT/ALT), IgA level, blood typing. 
•!
Urine for toxicology screen. 
•!
Functional Bowel Disorder survey. 
•!
Serum pregnancy test for women.  
Additionally, approximately -30 days prior to admission to the inpatient unit (allowable range: Day -30 to 
Day -5), subjects will have a follow-up medical history and brief physical exam to ensure ongoing eligibility. 
An educational brief may also be provided to the subjects at this visit. The following procedures will be 
performed: 
•!
Blood draw for serology (HIV, HCV and HbsAg) 
•!
Blood for exploratory endpoints 
•!
Confirm inclusion and exclusion criteria 
•!
Check health status 
If the initial screening visit is within the -30 day window, then all screening activities may be performed at 
the one visit. 
Attempts will be made to inform subjects of their screening laboratory results either in person or over the 
telephone prior to admission on day –3.  Subjects with clinically significant abnormalities (determined by PI) 
may be asked to have additional blood drawn.  If the result(s) is confirmed, subjects may be referred to their 
primary care physician.  A copy of the screening laboratory results may be provided to the subject at his/her 
request.!
6.2!
Inpatient Phase (Day –3 to Day 8) 
 
6.2.1! Admission (Study Day –3) 
The following procedures will occur on study day –3: 

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•!
Subjects will be admitted to the CIR   
•!
Inclusion and exclusion criteria will be reviewed to confirm continued eligibility 
•!
Vital signs (BP, HR and temperature) recorded 
•!
Day -3 complete physical exam  
•!
Medical history and concomitant medications since screening recorded 
•!
Serum and/or urine pregnancy tests (for female subjects) 
•!
Blood draw for hematology (complete blood count with differential), serum chemistries (sodium, 
potassium, creatinine, glucose, SGPT/ALT) 
•!
Blood, stool and saliva samples for immunology (may be collected on D-3 or D-2)  
•!
PVT demonstration 
6.2.2! Study Days –2 to 4 
On study day –2 subjects will be randomized as per section 5.2.  Dosing will occur three times a day 15 
minutes (range: 10 – 25 minutes) after each of three daily meals (breakfast, lunch and dinner) for a period of 7 
days (day -2 to day 4).  The time meals are completed will be recorded.  Day 0, the day of ETEC challenge, is 
an exception to this order of events (see section 6.2.3).  Subjects will receive test articles even if they do not 
eat. Vital signs (heart rate, blood pressure, and oral temperature) will be recorded at least three times daily. 
The study physician/nurse practitioner will monitor health status and adverse events by medical interview, 
and focused physical examinations. All stools will be collected, weighed and graded starting on Day -2 until 
discharge from the inpatient facility.  Following ETEC challenge, one to three stool samples will be cultured 
daily per the relevant SSP. Rectal swabs may be used when stool specimens cannot be produced. Blood, 
saliva, and stool samples will be collected according to Table 1!and the relevant SSP. 
6.2.3! ETEC Challenge (Day 0) 
The dose and fasting time were determined as a part of NMRC.2015.0007. !
On the day of ETEC challenge, subjects will be monitored as detailed above for days –2 to 4, with some 
modifications. Subjects will eat breakfast and then will receive test article/placebo in the morning 
approximately 90 minutes prior to challenge and will fast until the challenge.  Approximately 1 minute prior 
to challenge, subjects will ingest 120 ml of bicarbonate buffer (buffer formulation: 13.35 gram of sodium 
bicarbonate in 1000 mL of sterile water for irrigation). For challenge, subjects will drink a solution of the 
challenge inoculum suspended in the remaining 30 mL of bicarbonate buffer at the appropriate inoculum 
doses. A second 90-minute fast will commence from the time of challenge in which subjects can only take the 
second dose of test article/placebo, and sips of water. Fifteen minutes (range: 10 – 25 minutes) after 
challenge, they will receive the second dose of test article/ placebo. See Table 4 for tabular listing of Day 0 
schedule. Monitoring for post-challenge signs of adverse reactions will be conducted for at least 30 minutes, 
followed by taking vital signs.  No Test article/placebo dosing is scheduled after lunch, but routine dosing 
will commence again at dinner. Table 1 and Table 4 outline these events. The meal times will be documented 
on study day 0.   
Table 4. Order of events on day of challenge 
Event 
Volume 
(approximate) 
Breakfast 
- 
1st daily dose of test article/placebo (range 10-25 min) 
150 ml 
90 minute fast 
- 
Bicarbonate buffer 
120 ml 
1 minute interval (up to 2 minutes) 
- 

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Bicarbonate buffer + 1 x 1010 cfu B7A ETEC  
30 ml 
Interval of 15 minutes (range 10-25 min) 
- 
2nd daily dose of test article/placebo 
150 ml 
Fast at least 90 minutes from challenge 
- 
Lunch 
- 
Dinner 
- 
15 minutes after dinner complete (range 10-25 min) 
- 
3rd daily dose of test article/placebo 
150mL 
 
 
 
6.3!
Day 5-Discharge; Antibiotic Treatment  
All subjects will be treated with ciprofloxacin (500 mg by mouth twice daily for three days).  Alternate 
antibiotic treatment to which the strain is susceptible may also be considered as clinically appropriate. All 
antibiotic doses received prior to discharge will be directly observed by the investigator or designee. 
All subjects are scheduled for routine antibiotic treatment on Day 5 (approximately 120 hours after 
challenge), per the protocol time and events schedule. Early antibiotic treatment after challenge may 
commence when any of the following criteria are identified and a study physician considers it to be 
warranted:  
•!
Severe diarrhea (based on volume, 800 g in 24 hours)  
•!
Stool output consistent with moderate diarrhea for 48 hours  
•!
Mild or moderate diarrhea and 2 or more of the following symptoms: severe abdominal pain, severe 
abdominal cramps, severe nausea, severe headache, severe myalgias, any fever (≥ 38.0°C), or any 
vomiting 
•!
A study physician determines that early treatment is warranted for any other reason 
If, because of illness, a subject is unable to take oral antibiotics, intravenous antibiotics may be given (IV 
ciprofloxacin at an appropriate dose based on weight and clinical status).  Subjects meeting discharge criteria 
may be released with the remaining antibiotic treatment to be taken at home.  Subjects receiving early 
antibiotic therapy will NOT continue to receive test articles.  
On study day 5, blood specimens (for hematology and serum chemistry) will be obtained from all subjects. 
Urine samples may be obtained on any of the study days at the discretion of the PI to assess surreptitious 
antibiotic intake or protocol restricted drug intake.   
6.4!
Inpatient Discharge 
Discharge is routinely scheduled for day 8, when most subjects are expected to meet the discharge criteria of 
feeling well (with resolved or resolving symptoms of illness), having completed antibiotics, and having two 
consecutive stool cultures negative for ETEC.  A clinical check including vital signs and a physician/nurse 
practitioner assessment is required before discharge.  Blood, stool (except culture) and saliva specimens will 
be collected per Table 1 (from all subjects, inpatient and outpatient). 
6.5!
Outpatient Monitoring 
Day 28 (± 2 days) is the scheduled follow-up safety visit. Subjects will be questioned about their health status 
and Adverse Events (AEs) that have occurred since discharge. Any reported AEs and/or concomitant 
medications will be recorded on the appropriate eCRF.  Other procedures are vital signs, blood draw for 
serology and immunology. 

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Subjects will be told not to donate blood or blood products for one month following the completion of study 
participation and advised that the Red Cross will not allow blood donations for 1 year following participation 
in an investigational research study.!
A phone-check will be conducted approximately six months (+/- 1 mo) after challenge to track the occurrence 
of any medically significant new chronic illnesses or serious health event, and completion of functional bowel 
disorder survey.  
6.6!
Early Termination 
Subjects have the right to withdraw from the study at any time and for any reason without affecting the right 
to treatment by the investigator (for study-related conditions). The investigator also has the right to withdraw 
the subjects in the event of intercurrent illness, AEs, or for administrative/social reasons. 
An excessive number of withdrawals can affect the scientific validity of the study, therefore unnecessary 
withdrawal should be avoided. Should withdrawals occur, efforts will be made to ensure subject safety and 
continued monitoring as thoroughly as possible. In case of subject withdrawal, for whatever reason, a final 
trial evaluation must be completed stating the reasons. Withdrawals due to non-attendance must be followed-
up by the investigator to the extent possible to obtain the reason for non-attendance. 
Subjects withdrawing from the study after receiving BSIgG (or placebo) will be asked to return on study day 
5 for a brief physical exam and medical history, and blood draw for safety laboratory testing.  Subjects 
withdrawing after receiving the CS6 expressing B7A-ETEC challenge will receive antibiotics for outpatient 
treatment and will be educated on the importance of complying with treatment.  Attempts will be made to 
follow the subject for safety through study day 28. 
7.0!
STUDY INTERVENTION/INVESTIGATIONAL PRODUCT 
7.1!
Study Products 
7.1.1! Antigens for Bovine Immunization 
The antigens were used to vaccinate pre-selected cows for generation of the anti-CS6 and anti-B7A BSIgG 
products. 
7.1.1.1!
CS6 Antigen 
Recombinant CS6, given the lot designation 0840 was manufactured at the Walter Reed Army Institute of 
Research Pilot Bioproduction Facility under cGMP conditions in 26 Jan 2001 and released as a bulk material 
in 25 Jun 2001. Lot 0840 contains CS6 (derived from ETEC strain E8775), in a phosphate buffer, at a 
concentration of 2.56 mg/ml with an endotoxin content of 60 EU/ml. The manufacturing procedure for Lot 
0840 involved the following steps: Plasmid DNA, containing the CS6 operon (cssA, cssB, cssC, and cssD) 
from a tox- E8775 ETEC strain was partially digested with HindIII and ligated to pUC19, which had also 
been digested with HindIII. The ligated plasmid was transformed into Escherichia coli (E. coli) DH5α. The 
CS6 genes were then subsequently subcloned into a pUC19 vector containing a kanamycin resistance marker 
in place of the gene for ampicillin resistance. The resulting vector containing the CS6 operon and kanamycin 
resistance marker was transformed into DH5α and then moved into the E. coli expression strain HB101. This 
clone was given the designation M346 and was used to generate cGMP master and production cell banks.  

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The bulk lot of purified recombinant CS6, Lot 0840, was analyzed using a panel of assays designed to assess 
protein content, identity, sterility, and purity.  Protein content was quantified using the Lowry assay. A 
sterility test was conducted on Lot 0840 according to Code of Federal Regulations, Title 21, Section 610.12 
(21 CFR 610.12). Identity was ascertained by SDS PAGE and Western blot analysis, and purity assessed by 
densitometric analysis of the CS6 protein band(s) after SDS PAGE separation. Endotoxin contamination was 
determined by the limulus amebocyte lysate (LAL) assay and immunogenicity of the CS6 protein was 
assessed through the immunization of New Zealand white female rabbits, demonstrating a greater than tenfold 
increase in serum IgG titers over pre-immunization serum titers.!
7.1.1.2!
Whole-Cell Killed B7A ETEC  
Inactivated whole-cell B7A ETEC was prepared by performing a phenol incubation of B7A. CS6 expression 
levels were ascertained using a CS6-specific inhibition ELISA. To produce the final vaccine lot, B7A was 
grown in CFA broth for 8-20 hours at 37°C in 5 L fermenters.  Cells were harvested and inactivated with the 
optimal phenol concentration at 20°C with mild agitation for approximately 40 hours. The cells were washed 
and the OD600 adjusted to the desired final concentration. Vials containing a suitable amount of bacteria were 
prepared under aseptic conditions.  The final whole-cell B7A vaccine batch was analyzed for sterility, CS6 
content, pH, and visual appearance. !
7.1.2! BSIgG Products 
The IPs, anti-CS6 BSIgG, anti-whole cell killed B7A BSIgG, and nonhyperimmune BSIgG (placebo), were 
manufactured at SAB facilities. The anti-B7A BSIgG and anti-CS6 BSIgG are partially purified bovine 
polyclonal antibody products in development for use as a prophylactic for ETEC. These products were 
partially purified from bovine plasma collected from days 8, 11 and 14 post fourth (V4) through fifth (V5) 
vaccination with antigens/vaccine (CS6 and killed whole cell ETEC strain B7A) (see section 7.1.1). These 
products were manufactured into liquid form, with a total protein concentration of approximately >70 mg/mL 
formulated in phosphate buffered saline, and stored at -20°C ± 5ºC. 
7.1.2.1!
Hyperimmune Plasma Collection 
Plasma was pooled from three animals per vaccination group at approximately days 8, 11, and 14 post-
vaccination under sterile conditions by using an automated plasmapheresis system (Baxter Healthcare, 
Autopheresis C Model 200). For this system, two catheters (BD T Catheter 14 gauge 3.5”) were placed into 
the jugular of the donor. Whole blood was drawn through one catheter and was immediately stabilized with 
anticoagulant using an anticoagulant pump. The anticoagulant prevented the whole blood from clotting while 
the plasma is separated from the cells and platelets. The anticoagulant/whole blood was pumped through a 
spinning separation device which performed the plasma separation. The concentrated cells were returned to 
the donor using a cell pump through the second catheter. The plasma passed through the separation device 
and was collected into a bag (1-30L bioprocess film bags) on a scale. The total amount of plasma collected 
was up to 2.1% of the donor’s body weight. Dedicated rooms were used to collect and store plasma. 
7.1.2.2!
Negative Control Plasma (non-hyperimmune) 
Bovine plasma was collected in bioprocess bags from four non-immunized cattle using plasmapheresis 
method developed at SAB. Up to 2.1% of body weight of non-immune plasma per animal was collected and 
three collections were performed every three to four weeks. Collected plasma bags were checked by QC and 
stored frozen until their release for manufacturing. At the time of release, 1.42 kg of negative control antibody 
was produced. 

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7.1.3! Packaging of Final Product 
The liquid IP was packaged in multi-dose bottles.  Quality control (QC) tests were performed for the final 
liquid product.  

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Figure 2. Label for Anti-CS6 BSIgG Product 
!
Figure 3. Label for Anti-B7A BSIgG Product 
 
Figure 4. Label for Negative Control Plasma Product 
 

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7.1.4! Product Storage 
BSIgG products will be stored at SAB in a locked freezer. Temperatures are monitored routinely per Standard 
Operating Procedures (SOP). SAB will have dedicated freezers for each product for storage prior to clinical 
evaluation. 
7.1.5! Product Shipping 
Prior to the commencement of the study, the IP will be transferred directly to the Research Pharmacy, CIR.  
Any use of the IP will be done under the supervision of the Research Pharmacy, and the Research Pharmacist 
will maintain IP accountability log which tracks the status of all IP received.  Any bottles remaining at the end 
of the study will be returned to NMRC or destroyed per SSP. 
7.1.6! Dose Preparation 
Each dose of anti-CS6 BSIgG and anti-B7A whole cell killed BSIgG will be prepared per SSPs. A single 
unit-dose of both anti-CS6 BSIgG and anti-B7A whole cell BSIgG will contain approximately 1g of BIgG 
(pending the exact IgG content (>90% estimated)). The previous efficacy of 1g of Bovine IgG anti-CFA/I 
(AEMI Lot#43218) in preventing illness mediated by H10407 in the clinic [50]. As Bovine IgG anti- CS6 and 
anti-B7A are new products, the dose was fixed at 1g of IgG to normalize it with respect to Bovine IgG anti-
CFA/I.  More specifically, in these studies, the doses of bovine milk IgG product were protective against 
challenge with approximately 1 X 109 cfu of ETEC strain H10407. The anti-CS6 and anti B7A whole cell 
ETEC bovine serum IgG to be used in this trial are very similar in physical characteristics and potency (based 
on ELISA) to the anti-CFA/I and anti-CfaE bovine milk IgG used in the recent JHU study.  The non-
hyperimmune BSIgG will be prepared per the formulation SSP.  Section 6.2.2 describes how the test 
articles/placebo will be administered in relation to meals for Days -2 through 4. The test articles/placebo will 
be administered with bicarbonate buffer. 
7.2!
ETEC Challenge Strain 
A strain dose-finding study was recently completed with CS6- expressing ETEC strain B7A under BB IND 
16,517 (held by sponsor-investigator Dr. A.L. Bourgeois at CIR, JHBSPH).  Briefly, in a cohort of 28 subjects, 
we assessed the optimal dose (108, 109, and 1010 cfu) and fasting period (90 minutes and overnight) of the B7A 
challenge with B7A [identifying the optimal attack rate AR (seeking > 70.0%)]. The optimal dose and fasting 
time were determined from the outcome of NMRC.2015.0007. Subjects will be given approximately 1 x 1010 
cfu of ETEC B7A with a 90 minute fast in this study. 
7.2.1! Challenge Inoculum: The CS6-Expressing ETEC B7A 
The IP to be used for challenge is ETEC strain B7A (O148:H28- CS6+ LT+ST+).  It was manufactured at the 
WRAIR Pilot Bioproduction Facility (PBF) in 1997.  Each vial of the production cell bank contains 
approximately 9 x 108 cfu of live ETEC B7A in Luria Broth (LB) with 15% glycerol as cryopreservative.  
There is approximately 1 ml of the bacterial suspension per vial.  The lot number is 0481.  Vials are stored at -
80+10°C.  Bacteria are not given directly from the vials to subjects; they are inoculated into media and grown 
overnight. 
7.2.2! Packaging and Labeling 
The B7A challenge strain is stored as 1 ml aliquots in 2 ml cryostorage tubes held at –80°C ± 10°C under 
controlled conditions at the Pilot Bioproduction Facility, WRAIR. The cryovials are labeled as shown below: 

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The label is as follows:  
!
!
7.2.3! Product Characterization 
This Production Cell Bank (PCB) of B7A was used previously in a human challenge trial carried out by 
WRAIR investigators. Additionally, it was used in a challenge refinement investigation which optimized the 
dose and fasting period for its subsequent administration in this investigation (NMRC.2015.0007).  
Organisms prepared from the PCB will be used to challenge subjects participating in this trial.  This strain is 
susceptible to ciprofloxacin and other common antibiotics. 
7.2.4! Product Storage and Transfer 
The B7A vials are stored at ≤-80°C ± 10°C. The challenge strain will be transferred on dry ice from the 
WRAIR PBF to the CIR Enterics Research Laboratory at JHBSPH, logged in and stored at -80°C ± 10°C in a 
locked and temperature-monitored freezer.  Any use of these vials will be done under the supervision of the 
CIR Enterics Research Laboratory, JHSPH and tracked in an accountability log.  Any vials remaining at the 
end of the study will be disposed of (via autoclaving) or returned to NMRC/WRAIR for use in non-clinical 
research studies. 
7.2.5! Product Preparation 
Fresh, plate grown organisms will be used for challenge inocula, a standard approach for ETEC challenge 
studies. Approximately 48 hours before challenge, a vial of the cGMP Master Cell Bank will be thawed and 
streaked onto CF antigen agar (CFA without bile salts [CFA] agar) and Mac agar (to document purity of the 
cGMP PCB and E. coli verification). After 22-24 hours of incubation at 35-37°C, 10 colonies will be used to 
prepare a suspension in sterile saline (0.9%).  This suspension will be used to heavily inoculate approximately 
6 CFA agar plates for incubation at 35 -37°C. CFA agar plates will be harvested in sterile saline after 18 - 20 
hours and the resulting bacterial suspension further diluted in saline for optical density determination at 600 
nm. The optical density of the suspension will be adjusted to the appropriate concentration of bacterial cells 
depending on study group. The number of cfu in the inoculum will be determined by titrating and plating on 
agar plates before and after administration to subjects. The final inoculum will be examined by Gram stain for 
purity and for CS6 expression by agglutination in anti-B7A anti-serum. 
7.2.6! Product Administration 
A sodium bicarbonate (USP-grade) solution of 2 g/150 ml water will be prepared. Each subject will drink 120 
ml of this buffer one minute prior to ingesting the challenge inoculum, to neutralize gastric acidity. Within 2 
minutes, the subjects will drink the challenge inoculum dissolved in the remaining 30 ml of buffer. 

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The bicarbonate buffer is prepared from USP grade Sodium bicarbonate by dissolving 13.35 gram of sodium 
bicarbonate in 1000 mL of sterile water for irrigation.  When subjects ingest 150 mL of this buffer solution in 
conjunction with taking a dose of test article/placebo or receiving their ETEC challenge strain, they will 
receive a total dose of 2 grams of sodium bicarbonate 
7.3!
Accountability Procedures for the Investigational Products 
The investigator must ensure that the IP supplies are stored as specified in the protocol and in a secured area, 
with access limited to authorized study personnel. The investigator has the following responsibility for the 
products: maintaining inventory; maintaining accurate records of the receipt of IP, including date received, 
randomization code, manufacture or expiration date, amount received and disposition; holding the amount of 
product needed; and adequate storage and dispensing of the vaccines. A record will be maintained that 
includes the dispensation date, amount of IP dispensed, initials and identification number. The IP must be 
administered only at the specified institution.  Unused product will be shipped to: 
Steven Poole, PhD 
Naval Medical Research Center 
503 Robert Grant Avenue, Silver Spring, MD 20910-7500 
7.4!
Assessment of Subject Compliance with Investigational Products 
A member of the study team will witness the ingestion of the test article/placebo.   
8.0!
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 the sponsor.  
Study Safety Management: The research monitor and PI will review any safety concern. A data safety 
monitoring board (DSMB) is not required for this study.  
Research Monitor: The research monitor will function as an independent safety advocate for subjects per AR 
70-25 and Department of Defense (DoD) Instruction 3216.02. 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 IRBs, ORP HRPO, and USAMRMC Division of 
Regulated Activities and Compliance.  
The research monitor, in accordance with JHBSPH guidelines, will have the following responsibilities:  
•!
Evaluate ongoing safety data and make recommendations in order to ensure subjects safety as 
required  
•!
Be available for consultation by the clinical investigative team through the period of the clinical study 
in which there is an interaction with human subjects  
•!
Be available to review all SAEs and other unanticipated problems involving risk to subjects  
•!
Be available to discuss SAEs and significant safety issues  
•!
Provide clinical advice, in accordance with the study protocol, on the clinical management of 
subjects. This advice may include, but is not limited to  

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−!
Decisions on “borderline” laboratory values and eligibility for enrollment  
−!
Confirmation and discussion of treatment decisions for difficult clinical situations  
•!
Must document all clinical decisions including date, time and signature  
•!
Must communicate all decisions to the study PI and other study investigators, which must be stored 
with subject source documents  
All safety reports (i.e., serious adverse events, deviations, unanticipated problems involving risk and subject 
deaths) will be submitted to the JHSPH IRB and NMRC IRB.  
8.1!
Vital Signs 
Vital signs (temperature, blood pressure, heart rate) will be obtained throughout the inpatient period and at 
each study visit after discharge. Respiratory rates will be obtained on a case-by-case basis at the discretion of 
the study clinician. (See Table 5 for applicable AE coding.) 
Table 5. 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)  
38.0–38.4 
100.4–101.1 
38.5–38.9 
101.2–102.0 
>39.0 
>102.0 
 
Life threatening hyperthermia 
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. 
!
8.2!
Physical Examination 
A complete physical exam will be conducted during the screening visit and on Day -3 as part of the screening 
process; a targeted physical exam will be conducted prior to receipt of first IP, prior to challenge and daily 
during subject’s inpatient stay. Subsequent focused clinical assessments will occur at each study visit with 
specific attention to the identification of local, systemic or other adverse reactions. 
8.3!
Laboratory Assessments 
Venous blood samples will be collected for chemistry, hematology, and immunological parameters during the 
screening phase of this study and to provide a baseline sample.  Hematology and chemistry analyses will be 
performed by commercial laboratory (Quest, Incorporated in Baltimore City or by Johns Hopkins Medical 
Institutions). Additional specimens may be collected to confirm and evaluate any abnormal values.  
Additional blood for chemistry and hematology are planned for collection following experimental infection 
per the time and events schedule.  However, samples may be obtained as part of the clinical care of an 
individual subject.  The clinical toxicity grading scale that will be used as a guideline is based on the 

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Guidance for Industry: Toxicity Grading Scale for Healthy Adult and Adolescent Subjects enrolled in 
Preventive Vaccine Clinical Trials and the DAIDS Table for Grading and Severity of Adult and Pediatric 
Adverse Events. Final grading determination will be made by the PI based on normal lab values for the 
specific lab and clinical symptoms. Abnormal laboratory values based on hematology, and clinical chemistry 
(SGPT/ALT, glucose, creatinine, and electrolytes) after test article dosing will be considered an AE and 
severity determination by the investigator will be based on clinical symptoms and using the attached grading 
scale as a guideline. In the event of a clinically significant abnormal laboratory value, the test will be repeated 
and followed up, if clinically relevant.  Additional clinical laboratory evaluations may be performed at other 
times as required to follow up a serious or severe adverse event or as deemed necessary by the investigator. 
Slightly abnormal laboratory values that remain consistent from the time of screening throughout the study 
will not be recorded as AEs. 
Serologic evidence of chronic HIV-1, HCV, and HBV infections will be obtained during the screening 
process. Evidence of current infection will make a subject ineligible. Additional testing will not be performed 
as part of this study beyond second tier confirmatory tests on those with preliminary positive tests on ELISA 
after HIV and/or HCV serology. Targeted drug screenings are planned for this study at screening and at the 
discretion of the study clinician. 
A serum sample for pregnancy testing (female subjects) will be collected at the screening visit and on Day -3.  
A urine pregnancy test will be collected (female subjects) on Day -3 and at day 28. A positive pregnancy test 
prior to IP administration will result in disenrollment.  Any subjects who become pregnant during the study 
will 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. 
Table 6. Reference Ranges and Adverse Event Coding for Clinical Hematology Parameters 
Test 
Normal 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially Life 
Threatening 
(Grade 4) 
Hemoglobin (g/dL) 
(for screening purposes only) 
M: LLN = 11.0 
F: LLN = 10.5 
  
 
 
 
Hemoglobin, low 
 
M: 10.0 to 10.9 
F:9.5 to 10.4 
M: 9.0 to <10.0
F: 8.5 to <9.5 
M: 7.0 to <9.0 
F:6.5 to <8.5 
M: <7.0 
F: <6.5 
Eosinophils (cells/mm3) 
15-500 
551-1,500 
1,501-5,000 
> 5,000 
Hypereosinophili
c 
Leukocytes (white blood 
cells) (cells/mm3) 
2,500 to 10,800 
 
 
 
 
Leukopenia 
 
2,000 to 2,499 
1,500 to 1,999 1,000 to 1,499 
<1,000 
Leukocytosis 
 
10,801-15,000 
15,001- 
20,000 
20,001- 
25,000 
> 25,000 
Lymphocytes, low 
(cells/mm3) 
>650 
600 to <650 
500 to <600 
350 to <500 
<350 
Neutrophils, low (cells/mm3) 
>1,000 
800 to <1,000 
600to 799 
400to 599 
<400 
Platelets decreased 
(cells/mm3) 
≥125,000 
100,000 to 
124,999 
50,000 to 
<100,000 
25,000 to 
<50,000 
<25,000 
!

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Table 7. Reference Ranges and Adverse Event Coding for Blood Chemistry Parameters 
Test 
Normal 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially Life 
Threatening 
(Grade 4) 
BUN (elevation) 
7-25 
26-28 
29-31 
> 31 
Requires dialysis 
Creatinine (elevation) 
M: 0.7-1.4 
F: 0.5-1.1 
1.1 to 1.3 x ULN
 
> 1.3 to 1.8 x 
ULN OR 
Increase of > 
0.3 mg/dL 
above baseline 
> 1.8 to < 3.5 x 
ULN OR 
Increase of 1.5 
to < 2.0 x above 
baseline 
≥ 3.5 x ULN OR 
Increase of ≥ 2.0 x 
above baseline 
Glucose, Random (mg/dL)  
 
65 to115 
 
 
 
 
Hypoglycemia
 
55 to 64 
40 to <55 
30 to <40 
< 30 
Hyperglycemia
 
116 to 160 
>160 to 250 
>250 to 500 
> 500 
Potassium (mEq/L; mmol/L) 
3.4 to 5.6 
 
 
 
 
Hypokalemia
 
3.0 to < 3.4 
2.5 to <3.0 
2.0 to <2.5 
< 2.0 
Hyperkalemia
 
>5.6 to <6.0 
6.0 to <6.5 
6.5 to <7.0 
≥ 7.0 
SGPT/ALT (elevation) 
M:9 to 46 
F: 6 to 29 
1.25 to <2.5 x 
ULN 
2.5 to <5.0 x 
ULN 
5.0 to <10.0 x 
ULN 
≥ 10.0 x ULN 
Sodium (mEq/L; mmol/L) 
136 to145 
 
 
 
 
Hyponatremia
 
130 to <135 
125 to <130 
121 to <125 
≤ 120 
Hypernatremia
 
146 to <150 
150 to <154 
154 <160 
≥ 160 
 
8.4!
 
IND Safety Reporting 
The following terms, as defined by 21 CFR 312.32, apply to IND safety reporting. 
8.4.1! Adverse Event or Suspected Adverse Reaction 
Adverse event means any untoward medical occurrence associated with the use of a drug in humans, whether 
or not considered drug related. This includes an exacerbation or worsening of pre-existing conditions or 
events, intercurrent illnesses, injuries, or vaccine or drug interaction, or worsening of abnormal clinical 
laboratory values.  Anticipated day-to-day fluctuations of pre-existing conditions that do not represent a 
clinically significant exacerbation need not be considered AEs. Discrete episodes or worsening of chronic 
conditions occurring during a study period will be reported as AEs to assess changes in frequency or severity.  
Stable, pre-existing conditions and/or elective procedures are not AEs. 
AEs will be documented in terms of signs and symptoms observed by the investigator or designee or reported 
by the subjects at each study encounter, with a medical diagnosis stated. Pre-existing conditions or signs 
and/or symptoms (including any which are not recognized at study entry but are recognized during the study 
period) present in a subject prior to the start of the study will be recorded in the Medical History form within 

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the subject’s eCRF. AEs occurring after informed consent is obtained, but prior to test article receipt, will be 
documented in the Medical History form within the subject's eCRF as instructed by the Study Monitor.  
Hospitalization for elective surgery related to a pre-existing condition which did not increase in severity or 
frequency following initiation of the study, or for routine clinical procedures (including hospitalization for 
"social" reasons) that are not the result of an AE is not itself considered an AE, but must be recorded in the 
AE page of the eCRF. If hospitalization arises from a pre-existing condition, or was planned prior to the first 
test article dose, it will be recorded in the Medical History form of the eCRF. If planned after the first dose, it 
will only be recorded in the AE page of the eCRF. In both cases, it will be recorded as `Hospitalization (Not 
an AE)', and the relationship to test article receipts will be checked "No". Because hospitalization under these 
circumstances need not be considered an AE, it is therefore also not considered a SAE.  
A “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. The investigator is responsible for documentation of AEs according to the detailed 
guidelines set out below.  Subjects will be instructed to contact the investigator immediately should s/he 
manifest any signs or symptoms perceived as serious during the study period.  Approximately six months 
after study completion, subjects will be contacted by phone to document any intervening medically significant 
new chronic illnesses or serious health events.  These data will be documented in a telephone log and 
summarized in an annex to the final clinical study report. 
All AEs will be recorded on the appropriate AE form of the subject’s eCRF and recorded irrespective of 
severity or whether or not they are considered related to the test article or challenge inoculum. AEs will be 
tabulated separately for pre-and post-challenge data.  The assessment of the safety of the BSIgG and control 
products will be primarily limited to the 2 days prior to receipt of the ETEC challenge.  Following receipt of 
the challenge inoculum, gastrointestinal and systemic symptoms will likely attributable to B7A challenge 
strain unless temporally related to receipt of the BSIgG product/placebo or antibiotics. AEs occurring after 
receipt of the B7A challenge (day 0) will also be assessed as to their relationship with the challenge strain and 
the antibiotic (if treatment has started). 
8.4.1.1!
Solicited and Anticipated Adverse Events 
A solicited AE is a predetermined event, which may reflect safety concerns related to the IP. Previous clinical 
studies using much higher quantities of bovine colostrum products than planned for this study have been 
orally administered and well tolerated (e.g., 10 g/day anti-ETEC bovine milk IgG [93]; 30 g/day anti- C. 
parvum bovine milk IgG [103]; 30 g/day anti-S. flexneri bovine milk IgG [53]. 
This study includes a challenge with live CS6-expressing ETEC bacteria, and therefore all the symptoms of 
ETEC illness are expected.  The most common effects of ETEC infection are moderate to severe diarrhea 
(which may lead to dehydration and the need for oral or intravenous rehydration), and abdominal cramping.  
Fever, nausea with or without vomiting, chills, loss of appetite, headache, muscle aches and bloating may also 
occur. The following ETEC-associated AEs will be solicited daily during the challenge phase: 
1.! Abdominal cramping 
2.! Abdominal pain 
3.! Anorexia (poor appetite) 
4.! Arthralgias 
5.! Bloating 

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6.! Chills 
7.! Constipation 
8.! Excessive flatulence 
9.! Generalized myalgia 
10.!Headache 
11.!Lightheadedness 
12.!Malaise 
13.!Nausea 
14.!Urgency 
15.!Vomiting 
The following will be documented via clinical assessments during the inpatient challenge phase:  
1.! Diarrhea (via stool logs) 
2.! Hypovolemia 
3.! Fever (oral temperature > 100.4° F) 
8.4.1.2!
Serious Adverse Event 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 (abortion, stillbirth and any malformation/disease must be reported 
as an SAE). 
Although not considered SAEs, cancers will be reported in the same way as SAEs. Pertinent definitions 
include: 
•!
Life threatening - An AE is life threatening if the subject was at risk of death at the time of the event; 
it does not refer to an event that hypothetically might have caused death if it were more severe. 
•!
Disabling/incapacitating - An AE is incapacitating or disabling if it results in a substantial disruption 
of the subjects' ability to carry out normal life functions. This definition is not intended to include 
experiences of relatively minor medical significance such as headache, nausea, vomiting, diarrhea, 
influenza, injection site reactions and accidental trauma (e.g. sprained ankle). 
•!
Hospitalization: In general, hospitalization signifies that the subject has been detained (usually 
involving at least an overnight stay) at the hospital or emergency ward for treatment that would not 
have been appropriate in the physician's office or outpatient setting. Hospitalization for either elective 
surgery related to a pre-existing condition which did not increase in severity or frequency following 
initiation of the study or for routine clinical procedures (including hospitalization for "social" reasons) 
that are not the result of an adverse event need not be considered as AEs and are therefore not SAEs.  
•!
Routine Clinical Procedure: A procedure which takes place during the study and does not interfere 
with the test article administration or any of the ongoing protocol specific procedures. 
Note:  If anything untoward is reported during an elective procedure, that occurrence must be reported as an 
adverse event, either `serious' or non-serious according to the usual criteria. When in doubt as to whether 
‘hospitalization’ occurred or was necessary, the AE will be considered serious. Important medical events that 

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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 patient or 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. 
8.5!
Serious Adverse Events 
8.5.1! Unexpected Adverse Event or Unexpected Suspected Adverse Reaction 
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. 
8.5.2! Other Adverse Events 
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. 
8.6!
Relationship to Investigational Product (Assessment of Causality) 
The investigator or designee must assign a relationship of each AE to the receipt of the IP. The investigator or 
designee 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 IP, and identification of 
possible alternate etiologies including underlying disease, concurrent illness or concomitant medications. 
Every effort will be made to explain AEs and assess causal relationships, if any, to administration of the 
BSIgG test articles, B7A ETEC challenge, or antibiotic treatment.  AEs occurring on study days –2 to 0 will 
be assessed as to their relationship with the BSIgG test articles.  AEs occurring after receipt of the ETEC 
challenge (day 0) will be assessed as to their relationship with the BSIgG test articles, B7A challenge strain or 
the antibiotic, if applicable. The degree of certainty with which an AE can be attributed to these products (or 
alternative causes, e.g. natural history of the underlying diseases, concomitant therapy, etc.) will be 
determined by how well the event can be understood in terms of one or more of the following: 
•!
Reaction of similar nature having previously been observed with BIgG products, ETEC challenge strains, 
or antibiotic administration 
•!
Published literature accounts supporting causality 
•!
Temporal relationship with administration 

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The following guidelines should be used by investigators to assess the relationship of an AE to study product 
administration.  Only a physician or nurse practitioner can make this determination. The investigator will 
assess causality of all AEs as either ‘related’ or ‘unrelated’. Non-serious and serious adverse events will be 
evaluated as two distinct types of events given their different medical nature. If an event meets the criteria to 
be determined ‘serious’ it will be examined by the investigator to the extent possible to determine ALL 
contributing factors applicable to the event. Other possible contributors include: 
•!
Underlying disease 
•!
Other medication 
•!
Protocol required procedure 
•!
Other cause (specify) 
8.6.1! Causality 
Causality of all AEs will be assessed by the investigator using the following criteria: 
In the investigator’s opinion, is there a reasonable possibility that the AE may have been caused by the 
product under consideration? 
Definite 
The AE can only be explained by receipt of the product 
Probable 
AE occurs within a reasonable time after the administration of the product and cannot be 
reasonably explained by other factors (i.e., clinical condition, environmental / toxic factors 
or other treatments) 
Possible 
AE occurs within a reasonable time after the administration of the product but can also be 
reasonably explained by other factors (as mentioned above) 
Unrelated 
there is no suspicion that there is a relationship between the product and AE, there are 
other more likely causes, and administration of the product is not suspected to have 
contributed to the AE 
8.7!
Recording of Adverse Events 
8.7.1! Methods / Timing for Assessing, Recording and Analyzing Safety Endpoints 
All AEs either observed by the investigator or one of his/her clinical collaborators or reported by subjects 
spontaneously or in response to a direct question will be evaluated by a study investigator. The nature of each 
event, date of onset, outcome, severity and relationship to test article, challenge, and/or antibiotic will be 
established. Details of any symptomatic/corrective treatment will be recorded on the appropriate page of the 
eCRF. Subjects will be asked non-leading questions initially when soliciting AEs, followed by more direct 
questions as necessary.  AEs already documented in the eCRF, i.e., at a previous assessment, and designated 
as ‘ongoing’ will be reviewed at subsequent follow-up assessments. If resolved, documentation in the eCRF 
will be completed. If an AE changes significantly in frequency or intensity during a study period, a new 
record of the event will be started. 
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. Solicited AE's will be recorded as individual events. Unsolicited AE may be recorded as a diagnosis. 
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 IP, severity, 
and other possible etiologies. When an event has not resolved by the proscribed reporting period, it will be 
left open/without an end date on the AE eCRF and will be updated with end date or ongoing at visit.  

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The timeframe for the collection of AEs and SAEs begins at the time of IP administration through 28 days 
after receipt of the challenge strain. Additionally, subjects will be contacted by telephone approximately at 6 
months after challenge to assess for any new onset SAEs or AEs of special interest mandated by the FDA. 
8.7.2! Duration of Follow-up of Subjects after Adverse Events 
Investigators are required to follow 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. 
Investigators are not obligated to actively seek SAEs in former subjects; however, if a SAE, considered to be 
related to the IP is brought to the attention of the investigator at any time until closure of the study, the event 
will be reported. 
Investigators should follow-up adverse events at least until the final study visit. This may include repeat 
safety laboratory analysis. Outcome should be assessed as: 
•!
Resolved 
•!
Resolved with sequelae 
•!
Severity change (highest severity in a day will be recorded, if the severity on day 1 is mod, then 
mild and mod, it will be entered as moderate for the day only, then if on day 2 is mild, the 
moderate AE will stop and the AE will be reentered as mild) 
•!
Ongoing at day 28  
•!
Died 
•!
Lost to follow up 
All SAEs must be documented and followed until the event either resolves, subsides, stabilizes, disappears or 
is otherwise explained or the subject is lost to follow-up, but not longer than 6 months after the last receipt of 
test article. All follow-up activities have to be reported, if necessary on one or more consecutive SAE report 
forms, in a timely manner. All fields with additional or changed information must be completed and the report 
form will be forwarded to the study contact for reporting SAEs as soon as possible, but not more than 7 
calendar days after receipt of the new information. Clinically significant laboratory abnormalities will be 
followed up until they have returned to normal or until stable. Reports relative to the subsequent course of an 
AE noted for any subject must be submitted to the Sponsor. The outcome of SAEs will be assessed in the 
same manner as all AEs. 
8.7.3! Safety 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. 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 

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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 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 International Conference on Harmonization (ICH) guideline for Good Clinical Practice 
(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. 
During the challenge phase of the study, ETEC disease-specific adverse events will be graded in accordance 
to the table below. 
Table 8. Challenge Phase ETEC Infection Anticipated Adverse Event / Endpoint Assessments 
Adverse Event 
Severitya 
Parameter 
Diarrhea, based on highest output of 
loose/liquid stools in any 24-hour period. (A 
diarrhea episode ends when there is a 48 
hour window with no grade 3-5 stools.) 
1 
Mild: 2-3 loose /liquid stools totaling ≤400g 
2  
Moderate: 4 to 5 loose/liquid stools or 401-800 g of 
loose/liquid stool  
3 
Severe: 6 or more loose/liquid stools totaling >800g 
4 
Life threatening  
Body temperature (t) 
1 
>100.4oF and <101.1oF (38.0-38.4oC) 
2 
>101.1oF and <102.0oF (38.5-38.9oC) 
3 
>102.0oF (39.0oC) 
4 
Life threatening hyperthermia 
Vomiting 
1 
One episode within a 24-hour period 
2 
Two episodes within a 24-hour period 
3 
More than two episodes with a 24-hour period 
4 
Life threatening consequence of emesis 
Other solicited and non-solicited adverse 
events 
1 
Discomfort noted, but no disruption of normal daily 
activities; slightly bothersome; relieved with or without 
symptomatic treatment. 
2 
Discomfort sufficient to reduce or affect normal daily 
activity to some degree; bothersome; interferes with 
activities, only partially relieved with symptomatic 
treatment. 
3 
Discomfort sufficient to reduce or affect normal daily 
activity considerably; prevents regular activities; not 
relieved with symptomatic treatment. 
4 
Life threatening 
a1=mild; 2=moderate; 3=severe; 4=life threatening. 
 
8.8!
Reporting Adverse Events 
The PI will report all AEs to the sponsor and the local IRB in the appropriate safety, annual, and/or final 
reports. The NMRC staff in conjunction with the clinical site will draft annual and final clinical study reports 
and provide files to the sponsor for review and submission to the FDA. 

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8.8.1! Reporting Serious and Unexpected Adverse Events 
All SAEs must be reported immediately by the investigator without filtration, whether or not regarded as 
possibly attributable to the test articles, placebo, or antibiotic.  SAE reports will be provided to the Sponsor, 
medical monitor, JHSPH IRB, and NMRC IRB.  The investigator must report SAEs within one calendar day 
of becoming aware of the event by telephone, fax or e-mail (if appropriate) to the study contact for reporting 
SAEs as described in the protocol. This initial notification will include minimal, but sufficient information to 
permit identification of the reporter, the subject, the test articles, AEs, and date of onset. The investigator will 
not wait for additional information to fully document the event before notifying. The first notification will be 
confirmed by an acknowledgement letter. The report is then to be followed by submission of a completed 
SAE Report Form provided by the sponsor as soon as possible but not more than 3 calendar days past the 
initial report, detailing relevant aspects of the AE in question. All investigator actions and event outcomes 
must also be reported immediately. SAE Report Forms are to be used for documentation of these various 
aspects regarding the event.   Hospital records and autopsy reports will be obtained if applicable. 
The 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 medical monitor will comment on the event outcomes, and in the case of a SAE or death, 
comment on the relationship to participation in the study.  The medical monitor will indicate concurrence or 
nonconcurrence with the details of the report provided by the investigator.  Reports for events determined by 
either the investigator or medical monitor to be possibly, probably, or definitely related to participation and 
reports of events resulting in death will be promptly forwarded to all germane IRBs (JHSPH IRB, NMRC 
IRB). 
Unanticipated problems involving risk to subjects or others, SAEs related to participation in the study and all 
subject deaths will be promptly reported by phone (301-619-2165), by email, or by facsimile (301-619-7803) 
to the NMRC IRB.  A complete written report will follow the initial notification. 
8.8.1.1!
Reporting to the Sponsor 
All SAEs and unexpected AEs must be reported promptly (within 72 hours) to the sponsor as per 
21 CFR 312.64, whether or not the event is considered related to study product. Further, the investigator 
should comply with relevant study site SOPs on reporting SAEs. 
The minimum information that the investigator will provide to the sponsor is specified in Table 10.  

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Table 9. Study Contacts for Reporting Serious Adverse Events 
Sponsor 
 
A. Louis Bourgeois, Ph.D., M.P.H. 
Center for Immunization Research 
Department of International Health 
Johns Hopkins Bloomberg School of Public Health 
624 N. Broadway, HH, Rm 205 
Baltimore, MD 20215 
Institutional Review Board  
JHSPH IRB Office 
615 N. Wolfe Street 
Suite E1100 
Baltimore, Maryland 21205 
Phone: 410-955-3193 
Toll-Free: 1-888-262-3242 
Fax: 410-502-0584 
Email: JHSPH.irboffice@jhu.edu!
Collaborating Institutional Review 
Board 
Naval Medical Research Center (NMRC) IRB 
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 
Alexandra Singer, MD 
Malaria Department 
Naval Medical Research Center 
503 Robert Grant Avenue 
Silver Spring, MD 20910 
Telephone: 301-295-0007 
Fax: 301-295-8025 
E-mail: Alexandra.l.singer.mil@mail.mil 
 
Table 10. SAE Information to Be Reported to the Sponsor 
Notification Method 
Information to be Provided 
Email or Telephone 
(within 72 hours) 
IND number, sponsor study number, name of the IP, and investigator name and contact 
number 
 
Subject identification number 
 
SAE, onset date, date of IP administration, severity, relationship, and subject’s current status 
AND 
 
Email or Fax 
Cover sheet or letter 
 
Adverse event case report form 
 
Serious adverse event report form 
 
Concomitant medication case report form or a list of concomitant medications 
 
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 # _____, 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 will 
report unexpected SAEs associated with the use of the challenge strain to the FDA as specified at 21 CFR 
312.32 (c).  

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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 and research monitor.  
Reporting to the sponsor 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 and the research monitor. 
8.8.1.2!
Reporting to the IRB 
Unanticipated problems involving risk to subjects or others, serious adverse events related to participation in 
the study and all subject deaths should be promptly reported by phone, email, or fax to the local JHSPH IRB 
and NMRC IRB. A written report will follow the initial notification. 
Investigators are required to forward safety information provided by the sponsor’s representative to the IRB. 
All SAEs will be reported to the JHSPH IRB according to IRB guidelines. 
JHSPH IRB Guidelines: IRB Phone 410-955-3193; Fax 410-502-0584. Investigators are required to promptly 
report adverse events that fit the following criteria using the Problem/Event Report Form: 
Event (including adverse event reports, injuries, side effects, breaches of confidentiality, or other 
problems) that occurs any time during or after the research study, which in the opinion of the 
principal investigator: 
1.! Involved harm to one or more participants or others, or placed one or more 
participants or others at increased risk of harm; 
2.! Is unexpected (an event is “unexpected” when it is not described with specificity in 
the protocol and informed consent document; or if described with specificity, it 
occurs beyond the expected frequency and/or severity identified); and 
3.! Is related to the research procedures (an event is “related to the research procedures” 
if in the opinion of the principal investigator, it was more likely than not to be caused 
by the research procedures.) 
 
Table 11. IRB Contact Information 
IRB 
Telephone 
Fax 
Address 
NMRC 
301-319-7276 
301-319-7277 
500 Robert Grant Ave 
Silver Spring, MD 20910 
JHSPH IRB 
410-955-3193; 
1-888-262-3242  
410-502-0584 
jhsph.irboffice@jhu.edu 
!
8.8.2! Immediately Reportable Events 
8.8.2.1!
Pregnancy 
Each pregnancy must be reported immediately (within 72 hours of identification) by email or fax to the 
sponsor and the IRB. The investigator must report any pregnancy on study subjects to the Research Monitor 
within 14 calendar days of learning of this occurrence. 
Subjects who become pregnant after Day 0 through 3 months after the last study visit will be followed to 
term, and the following information will be gathered for outcome: date of delivery and health status of the 

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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 IP may have interfered with the effectiveness of contraception or there was a serious 
complication in the pregnancy including a spontaneous abortion or an elective termination for medical 
rationale. 
A pregnancy outcome other than abortion, stillbirth, and any malformation/disease as well as follow-up of the 
infant must be reported by the Investigator within 14 days of learning of its occurrence using local site 
procedures. 
8.8.2.2!
AE-related Withdrawal of Consent 
Any AE-related withdrawal of consent during the study must be reported immediately (within 24 hours of 
identification) by email or fax to the sponsor and the IRB. 
8.8.2.3!
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 Agencies including legal or medical actions and any instances of serious or continuing 
noncompliance with the regulations or requirements will be reported immediately to IRB and the sponsor. 
8.8.3! IND Reporting 
8.8.3.1!
Annual Reports 
The NMRC lead investigator will be responsible for the preparation of a detailed annual synopsis of clinical 
activity, including adverse events, for submission to the sponsor. Each annual report will summarize IND 
activity for 1 year beginning approximately 3 months before the IND FDA anniversary date. The sponsor will 
notify the NMRC lead investigator of the due date with sufficient time for the NMRC lead investigator to 
assemble the required information. 
8.8.3.2!
Final Clinical Study Report 
A final study report will be prepared in accordance with “Guidance for Industry: Submission of Abbreviated 
Reports and Synopses in Support of Marketing Applications” and ICH E3 Guideline “Structure and Content 
of Clinical Study Reports” and provided to the sponsor for review and approval. The sponsor representative 
will use this report to prepare the final clinical study report for submission to the FDA.  The investigative 
team will report all AEs to the sponsor and the local IRB in the appropriate safety, annual, and/or final 
reports.  
8.9!
 
Safety Criteria for Stopping Doses 
The PI, along with the research monitor, may determine if certain events warrant discontinuation of challenges 
and/or IP administration for all subjects in a cohort. If any of the additional following events occur, 
administration of the IP will be discontinued for all subjects in that cohort, and the PI and the research 
monitor will undertake a thorough review of the events: 
•!
The occurrence of one or more serious adverse events (SAEs) determined to be related to the IP. 
•!
One serious or unexpected AE evaluated by the PI, research monitor and sponsor determined to be an 

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unacceptable risk to the health and safety of other subjects. 
•!
Systemic allergic reaction, including but not limited to generalized urticaria, generalized petechiae, or 
erythema multiforme, occurring in two or more subjects in a group.  Bronchospasm or anaphylaxis 
occurring in any subject.  
Based on prior experience with ETEC challenge studies, it is expected that some subjects will have severe 
AEs (such as severe diarrhea). 
AEs which will prompt stopping the BSIgG test article administrations for an individual subject include: 
•!
SAEs unrelated to the test articles (event will be discussed with the medical monitor so as to 
determine if the event precludes further participation and vaccination.) 
•!
The investigator deems that stopping test article administration is in the best interest of the subject 
Additional reasons for subject withdrawal include: 
•!
The subject does not wish to continue with the study 
•!
The subject is lost to follow-up 
Further challenge, in accordance with the protocol, may be resumed with the concurrence of the research 
monitor, sponsor, PI, and the FDA.  
8.10!
Treatment of Adverse Events 
Treatment of an AE is the responsibility of the investigator according to the best treatment currently available. 
The applied measures will be recorded in the eCRF of the subject. 
8.11!
 
Study Termination Criteria 
The PI, research monitor, NMRC IRB, JHSPH IRB, Sponsor, or FDA may stop or suspend the use of this 
product at any time. 
8.12!
Six Month Follow-up Safety Surveillance 
Data will begin to be entered into the study database beginning on or after the inpatient period for a cohort, 
verified, and subsequently locked.  However, approximately 6 months after study completion, subjects will be 
contacted by phone to track the occurrence of any medically significant new chronic illnesses or serious 
health events and functional bowel disorder survey. If a subject cannot be contacted after three attempts, a 
registered letter will be mailed asking them to contact a study investigator. These data will be documented in 
a telephone log and summarized in an annex to the final clinical study report.!
9.0!
CLINICAL MONITORING 
Monitoring will be conducted according to an approved monitoring plan.  Local monitoring will commence 
prior to beginning, at initiation, during the study, and at closeout. 
The study monitor shall be available for consultation with the investigator. The study monitor or other 
authorized representatives of the Sponsor may inspect all documents and records maintained by the 
investigator, including, but not limited to, medical records (office, clinic or hospital) and pharmacy records 
for the subject in this study. The clinical study site will permit access to such records. The investigator will 
obtain, as part of informed consent, permission for authorized representatives of the Sponsor, or regulatory 
authorities, to review, in confidence, any records identifying individuals in this clinical study.  

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The investigator will notify the Sponsor within 24 hours following contact by a regulatory agency. The 
investigator and study coordinator will be available to respond to reasonable requests and audit queries made 
by authorized representatives of regulatory agencies. The investigator will provide the Sponsor with copies of 
all correspondence that may affect the review of the current study or his/her qualification as an investigator in 
clinical studies conducted by the Sponsor. The Sponsor will provide any needed assistance in responding to 
regulatory audits or correspondence. The investigator will permit independent auditors (employees of the 
Sponsor or an external company designated by the Sponsor) to verify source data validation of the regularly 
monitored clinical trial. The auditors will compare the entries in the eCRFs with the source data, and evaluate 
the study site for its adherence to the clinical study protocol and GCP guidelines and applicable regulatory 
requirements.  
The study team and data management group will arrange visits prior to beginning, at initiation, during the 
study, and at closeout by the study monitor or designee. 
10.0!
STATISTICAL CONSIDERATIONS 
10.1!
Introduction 
Safety, efficacy, clinical outcomes, and immunogenicity data will be entered into the eCRFs using standard 
software for data management.  Data will be edited with standard strategies for range and consistency checks.  
AEs for all subjects, regardless of the number of doses they have received, will be included in the safety 
analysis.  The null hypotheses for this study are that the diarrhea rates will be the same in groups receiving the 
placebo and the 1) anti-CS6 BSIgG, 2) anti-B7A whole cell BSIgG. 
10.2!
Sample Size Considerations 
The hypothesis being considered is that (1) anti-CS6 BSIgG confers ≥ 60% protective efficacy against 
diarrhea upon challenge with B7A (in comparison to the placebo group); and (2) anti-B7A whole cell killed 
BSIgG confers ≥ 60% protective efficacy against diarrhea upon challenge with B7A (in comparison to the 
placebo group).  An attack rate of 87.5% of diarrheal illness has been found with B7A at an inoculum of 1 X 
1010 cfu.  For the current study, assuming a one-sided alpha = 0.05 and an attack rate of 80% in the placebo 
group, the power to detect a preliminary efficacy of 60% in the immunoprophylaxis groups is 90% (Figure 3) 
when groups each contain 20 subjects. 
Figure 5. Power Curve for Sample Size Calculation 
 
Sample Size per Group 
10 
12 
14 
16 
18 
20 
22 
24 
Power 
50 
60 
70 
80 
90 
100 

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10.3!
Analysis 
10.3.1! Safety 
Assessment of BSIgG product safety is limited to the two days prior to receipt of the ETEC challenge dose 
(day –2 and –1).  Unless AEs are temporally related to receipt of BSIgG products, most will likely be 
attributed to the ETEC inoculum.  During each day of the inpatient period, subjects will be monitored for 
loose stools (not meeting the diarrhea definition), diarrhea, nausea, vomiting, abdominal cramps, fever, 
headache, abdominal tenderness, abdominal distention and an abnormal abdominal exam.  Additionally, 
subjects will have vital signs taken at least 3 times per day (in cases of moderate to severe diarrhea postural 
BP and pulse will be taken as necessary for clinical management according to the judgement of the 
physician/nurse practitioner). 
The sample size is designed to indicate trends but not to show statistically significant differences between 
groups. All subjects who receive BSIgG products or placebo, irrespective of number of doses or receipt of the 
ETEC challenge will be included in the safety analyses.  AEs will be summarized and compared between 
study groups for the periods prior to and after ETEC challenge.  Summaries of the number and proportion of 
subjects who report a given coded term will be reported. Safety data, including AEs, vital signs, and 
laboratory tests will be listed by study subject. 
10.3.2! Protective Efficacy of anti-CS6 and anti-B7A Whole Cell Killed BSIgG Products 
The primary endpoint for determination of efficacy is moderate to severe diarrhea occurring during previously 
defined post-challenge period.  However, subjects will be monitored for additional GI and non-GI complaints 
daily.  Side effects, coded as ‘possible, probable or definite relationship’, as defined in the protocol will be 
listed (group, time of onset, duration, and severity). During each day of the inpatient period, subjects will be 
monitored for loose stools (Grade 3-5 not meeting the diarrhea definition), diarrhea, nausea, vomiting, 
abdominal pain or cramps, fever, headache, abdominal tenderness, abdominal distention or otherwise 
abnormal abdominal exam, along with solicited symptoms noted above. Vital signs will be taken 3 times a 
day or more, particularly if the subject meets the study definition for severe diarrhea. All AEs will be 
summarized and compared between dose groups. Safety data, including AEs, stool information, specified 
vital signs, and laboratory tests, will be listed by study subject. The planned statistical evaluation is based on 
the proportion of subjects meeting prospectively defined clinical, microbiological and immunological 
endpoints. The attack rate will be calculated for all study groups, using the standard definition of: (# with 
endpoint / # receiving inoculum) x 100%. Summary tables will also be created to detail quantitative and 
temporal features of the illness such as diarrhea stool frequency and volume, maximum temperature observed, 
and time to illness and infection. Continuous variables will be analyzed using nonparametric statistics. In 
addition, tables will be prepared to list each commonly observed adverse event, the number of subjects 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 and life-threatening). 
Data will be analyzed to determine the incidence of diarrheal illness as outlined above among subjects in the 
placebo vs. each of the passive BSIgG prophylactic groups (with no alpha adjustment for multiple 
comparisons’).  Each rate and PE estimate will be calculated with 95% confidence intervals. Preliminary 
vaccine PE (%) will be calculated by the formula below: 
PE% = diarrhea incidence (placebo) – diarrhea incidence (prophylactic)   X 100                                                         
diarrhea incidence (placebo) 

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Additional efficacy analyses will evaluate protection from colonization, moderate and severe diarrhea, 
moderate and severe abdominal cramps, moderate and severe nausea, as well as evaluating time to diarrhea 
onset, diarrhea duration (controlling for early antibiotic treatment), need for intravenous re-hydration and 
early antibiotic treatment. 
Initial efficacy analyses will be based on an intent to treat and will include all subjects who receive each of 
the BSIgG products/placebo and the B7A challenge.  A secondary, per protocol analysis will limit the number 
of subjects evaluated.  Subjects who miss more than one dose of BSIgG (or placebo) in the 24 hours prior to 
receipt of the challenge inoculum will be excluded from this secondary analysis.  Subjects who miss more 
than one dose of BSIgG in the 72 hours following receipt of the challenge inoculum and who do not meet the 
primary outcome before missing their second dose will also be excluded.  A similar analysis will be 
performed for the secondary outcomes of moderate and severe diarrhea.  Analysis of subjects who miss doses 
not included in this time period will only be descriptive in nature. 
10.3.3! Immunogenicity 
Immunologic outcomes following ETEC challenge will be reported and compared between the three study 
groups. Analysis will include both ordinal (responder rates) and continuous (geometric mean titers) outcomes.  
Immunological outcomes will also be summarized in a tabular format and graphed to demonstrate kinetics of 
response. Qualitative (responder rates) and quantitative assessments (log transformed values) will be 
analyzed. Median increases (fold rises) of antibody concentrations and seroconversion rates may be calculated 
along with their 95% confidence intervals. Geometric mean titers may also be determined and presented with 
their 95% confidence intervals. All statistical tests will be interpreted in a two-tailed fashion using an alpha = 
0.05 with no alpha adjustments for multiple comparisons. 
11.0!
DATA MANAGEMENT 
The investigator will maintain complete and accurate documentation for the study, including records of 
medical treatments external to the research received during the study, records detailing the progress of the 
study for subjects, laboratory reports, source documents, signed informed consent forms, drug disposition 
records, correspondence with the IRB, the study monitor and the sponsor, AE reports, and information 
regarding subject discontinuation and completion of the study. All required data will be clearly and accurately 
recorded by authorized study personnel in the eCRFs. Only designated study site personnel will record or 
change data in an eCRF. The investigator will be responsible for procuring data and for quality of data 
recorded in the eCRFs.  Complete source documentation (study visits, laboratory reports, etc) is kept for each 
subject in his/her individual study chart.  All laboratory specimens, reports, study data collection, and 
administrative forms will be identified by coded number only to maintain participant confidentiality.  eCRFs 
using coded identifiers will be used to record data for subjects enrolled in the study.  Forms, lists, logbooks, 
appointment books, and any other listings that link participant ID numbers to other identifying information 
will be stored in a separate, locked file in an area with limited access. All information regarding study 
subjects is kept in password-protected computer files or in locked file cabinets that can be accessed only by 
authorized study personnel.  Samples are identified by coded subject number only.  Chart information and 
information from study records is not released without written permission of the volunteer. 
The source documents will be retained at the site. All study related documents will be kept in locked cabinets 
in locked rooms with limited access.  Information in the electronic database is password-protected and access 
is available only to authorized research team members.  Additionally, each authorized research team member 
is assigned a level of security clearance (also password-protected) with mandatory password changes every 90 
days) for the purpose of limiting access to certain areas or functions of the database.  Any information printed 
from this database is stored in locked files until its use is complete and then shredded. 

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For this study, an electronic data capture (EDC) database system will be used for the collection of the study 
data. The EDC database system will be designed based on the protocol requirements, the approved eCRF 
layouts and specifications, and in accordance with 21 CFR 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 into the eCRF 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, the PI, and the data 
management group. 
12.0!
RECORD AND SPECIMEN ARCHIVAL 
All records pertaining to this protocol will be stored in a locked filing cabinet at JHU or at an offsite, locked 
storage facility per regulations for a minimum of 5 years.  Access to these records will be limited to 
researchers in the Enteric Disease Department at NMRC and the JHU CIR as well as those responsible for 
regulatory monitoring of data to include representatives of the DoD and JHU. A copy of study records will be 
made available to the Sponsor. The investigator will obtain permission from the sponsor in writing before 
destroying any study records and the sponsor will notify the investigator in writing when records can be 
destroyed. Relevant IRBs will be notified in writing prior to destruction of any research records.  Specimens 
will be stored indefinitely in the JHU or the ETEC laboratory at NMRC. 
13.0!
OBLIGATIONS AND ROLES OF THE SPONSOR, INVESTIGATOR AND STUDY 
PERSONNEL 
This study will be conducted using GCP and in accordance with all federal regulations regarding the 
protection of human participants in research including The Nuremberg Code, The Belmont Report, US 21 
CFR Part 50 – Protection of Human Subjects, 32 CFR 219 (The Common Rule) and all regulations pertinent 
to the Department of Defense. 
The investigators agree to conduct the research in strict accordance with this protocol, the ICH Guideline for 
GCP (CPMP/ICH/135/95), as well as in conformity with any federal, provincial or local regulations regarding 
the conduct of clinical studies. The sponsor and investigator must comply with all applicable regulations. In 
addition, the investigator must follow local and institutional requirements including, but not limited to, IP, 
clinical research, informed consent and IRB regulations. The Sponsor will provide notification to the 
investigator of protocol and amendment approvals by regulatory authorities when applicable. Except where 
the investigator's signature is specifically required, it is understood that the term "investigator" as used in this 
protocol and on source documents refers to the investigator or appropriate study personnel that the 
investigator designates to perform a certain duty. The investigator is ultimately responsible for the conduct of 
all aspects of the study. Sub-investigators or other appropriate study personnel are eligible to sign for the 
investigator on designated source documents. 
14.0!
QUALITY CONTROL AND ASSURANCE 
14.1!
QA/QC monitoring 
During the study, the investigator will maintain complete and accurate documentation for the study, including 
records detailing the progress of the study for each subject, laboratory reports, eCRFs, signed informed 
consent forms for each study subject, drug disposition records, correspondence with the IRB, the study 
monitor and the sponsor, adverse event reports and information regarding subject discontinuation and 

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completion of the study. All required study data will be clearly and accurately recorded by authorized study 
personnel in the eCRFs. Only designated study site personnel shall record or change data in an eCRF. During 
the study, the investigator will be responsible for the procurement of data and for quality of data recorded in 
the eCRFs. The study monitor will ensure accuracy of the eCRFs. 
14.2!
Protocol Deviation Management 
All amendments to the protocol, consent form and/or questionnaires, including a change of PI, will be 
submitted to the JHSPH IRB and NMRC IRB for review and approval prior to implementation. Other-than-
minimal-risk changes and all unanticipated major problems involving human subjects or others will be 
reported promptly to the IRBs, and no such changes will be made to the research without IRB approval unless 
necessary to eliminate apparent immediate hazards to human subjects. Minor minimal-risk deviations 
necessitated during the course of the trial will be made on site as needed, and documented for subsequent 
review within a reasonable time period. Deviations from the protocol that potentially impact on subject safety 
will be promptly reported to the Research Monitor, IRBs, and the Sponsor. Other deviations will be reported 
at the time of continuing review. 
15.0!
HUMAN SUBJECTS PROTECTIONS CONSIDERATIONS 
15.1!
Risks / Benefit 
15.1.1! Risks 
The BSIgG products are expected to be safe with possible mild to moderate discomfort likely related to 
consumption of the sodium bicarbonate buffer, such as bad taste, bloating, nausea, gas, etc.  As with any 
investigational drug or biologic, there is a possibility of severe allergic reaction. 
Naturally acquired illness caused by ETEC ranges from mild-to-severe watery diarrhea. Nausea, vomiting, 
abdominal cramping, headache, abdominal gurgling or gas, anorexia, fever, muscle and/or joint aches, and 
malaise, may occur. For most adults the illness is not life threatening but often leads to mild to moderate 
dehydration and significant inconvenience associated with loss of sleep and activity. Study facilities will have 
personnel and resources capable to manage diarrheal illness and potential complications.  Side effects to the 
antibiotic (ciprofloxacin) used to treat the ETEC infection are possible. 
Therapeutic antibiotics for use in this study are licensed approved medications that have been used 
extensively and shown to be very safe with only rare side effects. The most commonly reported side effects 
for ciprofloxacin are gastrointestinal symptoms (nausea, vomiting, and diarrhea) in as many as 5 persons in 
100. Other reported symptoms in less than 1 person in a 100 include rash, dizziness, and headache. Rarely, 
allergic reactions to these medications have been observed. Ciprofloxacin is not recommended for use in 
pregnancy due to concerns of joint damage to the unborn child (based on studies in young animals). 
Pregnancy is exclusionary for study participation and is documented through testing prior to study 
interventions and provided discussion on methods to prevent pregnancy during study. Fluoroquinolones, 
including ciprofloxacin, are associated with an increased risk of tendonitis and tendon rupture in all ages. The 
risk of developing fluoroquinolone-associated tendonitis and tendon rupture is further increased in older 
patients usually over 60 years of age, in patients taking corticosteroid drugs, and in patients with kidney heart 
or lung transplants, all of whom are excluded from this study. Clostridium difficile associated diarrhea 
(CDAD/pseudomembranous colitis) has been reported with use of nearly all antibacterial agents.  
Good nursing practices are performed during blood draws, which minimizes the risk to the subject. Hand-
washing and sanitary disposal of feces (including pretreatment with bleach) are the main elements of personal 
hygiene and will minimize the spread by person-to-person infection; hand washing will be emphasized to the 

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subjects and subjects will be instructed not to share food or beverages. Subjects and staff will be trained in 
proper techniques of hand washing. Subjects will be instructed as to the importance of completing the 3-day 
course of antibiotics and this instruction will be documented. Risk of secondary transmission is highly 
unlikely due to antibiotic treatment and because subjects are required to submit two confirmed, consecutive 
negative stool samples prior to discharge.  
There is a minimal risk of pain, hematoma or infection at the site of venipuncture. The maximum amount of 
blood drawn from a subject in total, and daily, will fall within applicable regulations. 
There may be physical, psychological and social risks if subjects test positive for hepatitis B, hepatitis C 
and/or HIV. Subjects testing positive will be counseled and referred for treatment. 
Recent studies also suggest an increased risk of post-infectious irritable bowel syndrome (PI-IBS) following 
bacterial enteritis, and infection with ETEC has been found to be associated with these sequelae [35, 115-
117].  PI-IBS, a functional bowel disorder characterized by unexplained abdominal discomfort or pain 
associated with changes in normal bowel patterns, has been described in a recent systematic review to occur 
6-7 times more frequently after an acute enteric infection compared to similar matched controls without such 
a history [118]. 
Medical records associated with this protocol are subject to provisions of the Privacy Act of 1974, 5 U.S.C., 
Section 552A, and AR 340-21. All data and medical information obtained about subjects will be considered 
privileged and held in confidence. Subjects will not be identified by name in any published 
report/presentation of the results. Complete confidentiality cannot be promised to subjects who are military 
personnel, because appropriate medical command authorities may require reporting information bearing on 
the health of their personnel. Representatives of the Sponsor, NMRC IRB, JHSPH IRB, or FDA may inspect 
the records of this research as part of their responsibility to oversee research and ensure protection of subjects. 
Study results and data may be published in scientific/medical journals; the identity of individual subjects will 
not be disclosed. 
15.1.2! Risk Mitigation Strategies 
Subjects will be questioned and examined daily for evidence of infection and diarrhea complications. Vital 
signs will be recorded at least three times per day. Based on prior studies, infected subjects tend to develop 
illness with incubation periods of approximately 1-3 days. Therapeutic benefit seems to be optimal if 
treatment is given within the first three days of symptom onset. The risk of diarrhea complications will be 
minimized by a conservative approach to timing of antibiotic administration well within an interval that has 
been shown to be efficacious as well as daily clinical monitoring. Stool output will be closely monitored. The 
plan will be to treat all subjects no later than day 5 post-dosing.  
Aggressive fluid management will be undertaken to ensure the most common complication, dehydration, does 
not occur. The procedures to institute early oral and/or intravenous rehydration therapy are detailed above. In 
addition to rehydration therapy, prospectively defined criteria and procedures to institute early antibiotic 
therapy are also fully described above. In order to ensure clinical resolution and limit the potential for 
secondary spread upon discharge, predefined discharge criteria have been established. Subjects will be 
discharged from the inpatient phase of the study when clinical symptoms are resolved or resolving AND two 
consecutive stool cultures are negative for ETEC.  
Systemic or severe gastrointestinal complications rarely occur with ETEC infection. The following clinical 
findings necessitate immediate consideration and management of complicated enteritis: 
•!
Physical examination compatible with an acute abdomen  

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•!
Severe GI bleeding (any evidence of GI blood loss other than hemoccult positivity only, with 
evidence of hemodynamic instability, decrease in hemoglobin, hypovolemia) 
•!
Sepsis (high fever: temp. >102ºF (39ºC), rigors, hemodynamic instability).  
Any of these findings require prompt clinical management and discussion with the independent Research 
Monitor.  
The ETEC strain has the potential for risk to both the environment and to the research personnel; however, the 
risk to the environment in regards to potential transmission outside of the CIR facility is low. There is a 
minimal risk of acquiring ETEC infection associated with subject inoculum administration, patient care 
activities on the ward, or processing ETEC-infected stool. The risk to the environment will be reduced by 
ensuring that all human waste products from inpatients are disinfected with bleach prior to disposal, ensuring 
all subjects comply with discharge criteria (two consecutive negative stool cultures for ETEC), emphasizing 
importance of handwashing for subjects and staff, ensuring proper disposal/cleaning of linen, and cohorting 
subjects in the CIR while shedding ETEC.  Additionally, subjects will not be discharged until they are no 
longer shedding the challenge strain as per procedures outlined in the protocol. 
Subjects with prior history of abnormal bowel patterns who might be at higher risk of post-infectious sequelae 
are excluded. Predefined criteria to assure early treatment as appropriate also may further reduce risk of post-
infectious sequelae and is likely to reduce the risk associated with PI-IBS given the positive association 
between diarrheal illness duration and PI-IBS risk [119] . 
There is no risk associated with collecting stool samples; however slight discomfort is possible when using 
rectal swabs. A breach of confidentiality in which private health information is made public is possible.  
There may be physical, psychological and social risks if subjects test positive for hepatitis B, hepatitis C 
and/or HIV.  Subjects testing positive will be counseled and referred for treatment.  Medical records 
associated with this protocol are subject to provisions of the Privacy Act of 1974, 5 U.S.C., Section 552A, and 
AR 340-21.  All data and medical information obtained about subjects will be considered privileged and held 
in confidence.  Subjects will not be identified by name in any published report/presentation of the results.  
The sponsor and the FDA may inspect the records of this research as part of their responsibility to oversee 
research and ensure protection of subjects.  Study results and data may be published in scientific/medical 
journals; the identity of individual subjects will not be disclosed. 
15.1.3! Benefits 
There is no benefit that can be guaranteed to subjects for participating in this research study. However, there 
is potential societal benefit of the development of a product to prevent ETEC. 
15.2!
Subject Compensation 
Compensation for participation will occur as detailed below. Compensation will be provided only for 
completed study procedures designated for compensatory payment. If a Subject is eligible to participate in the 
investigational protocol after screening, and s/he completes all study visits, procedures and follows all the 
rules s/he will receive the following compensation: 
If enrolled in the study, the Subject will be compensated for participation time and travel in this trial as 
follows:  
•!
$80 total for screening (only if enrolled in the study or presents as an alternate) 
•!
$2,400 for the inpatient period (as long as all study requirements are met) 

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•!
$80 for outpatient study visit: Days 28 
•!
$60 for the follow up telephone contact: Day 180 
•!
$400 bonus upon completion of inpatient phase and outpatient visits 
The payment schedule is: 
•!
$2,480 at the completion of the inpatient period (approx. Day 8) 
•!
$480 on Day 28 
•!
$60 after completion of the telephone contact follow up, Day 180 
Maximum compensation is $3,020 for participation. 
If a subject is not eligible for discharge on day 8 because of illness or not having 2 consecutive negative stool 
culture results s/he will receive $200 per additional inpatient day. Subjects will not be paid for missed 
outpatient visits, and may forfeit some or all of their bonus as a result of missed visits or non-compliance. 
For active duty military subjects, compensation for this study depends on duty status. 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. 
•!
$50 total for screening (only if enrolled in the study or presents as an alternate) 
•!
$250 for the inpatient period (as long as all study requirements are met) 
•!
$50 for outpatient study visit: Day 28 
•!
$0 for the follow up telephone contact: Day 180 
•!
$0 bonus upon completion of inpatient phase and outpatient visits 
 
Maximum compensation is $350 for participation of an active duty service member. 
 
15.3!
Research-Related Injury 
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 military-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. 
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 a DoD hospital or clinic. It cannot be determined in 
advance which DoD hospital or clinic will provide care. If the subject receives care for research-related 
injuries outside of a DoD hospital or clinic, the subject or the subject’s insurance will be responsible for 
medical expenses. 

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During the challenge phase, subjects who require medical treatment beyond what can be provided safely at 
the CIR will be transferred to the Johns Hopkins Hospital or Johns Hopkins Bayview Medical Center for care. 
If a subject is injured during the study, the study doctor will help the subjects find medical care. Medical care 
at Johns Hopkins is open to all subjects as it is to all sick or injured people. Neither Johns Hopkins 
Bloomberg School of Public Health nor the John Hopkins Hospitals have any plan to provide compensation to 
the subjects if they experience injury or other bad effects which are not the fault of the study doctors. Subjects 
will only be treated for injuries that are directly caused by the research study. In the event this occurs, the 
sponsor agrees to reimburse the Hospital for all reasonable expenses incurred by the Hospital in providing 
medical treatment and/or hospitalization reasonably necessary to address any injury to a Subject that, in the 
reasonable judgment of Hospital and Sponsor, occurs directly as a result of the administration of the IMPs or 
performance of study procedures in accordance with the Protocol, but only to the extent such expenses are 
not:  
•!
the result of a foreseeable side effect as indicated in the Protocol  
•!
reimbursed by (or submitted for reimbursement to) the Subject’s insurance or any governmental 
program or other third-party payer providing medical or hospital coverage; provided, however, that 
this provision shall not obligate Hospital to submit such costs to the prospective Subject’s insurance 
or any governmental program or other third-party payer coverage  
•!
attributable to a failure of Hospital, or any of the Investigator Personnel, including PI, to adhere to the 
terms of the Protocol, Sponsor’s written instructions or Applicable Law  
•!
attributable to the negligence, recklessness or willful misconduct or omission of Hospital or any of its 
Investigator Personnel, including PI  
•!
attributable to a pre-existing abnormal medical condition or underlying disease of the Subject or 
treatment that would have been provided to the Subject in the ordinary course notwithstanding 
participation in the study, or  
•!
attributable to the failure of the Subject to follow the reasonable instructions of Investigator Personnel 
or Subject’s physician. 
Transportation to and from military hospitals or clinics will not be provided. No reimbursement is available if 
the subject incurs medical expenses to treat research-related injuries from outside or private providers. 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. 
15.4!
Compensation for Investigators 
There is no financial compensation for investigators in this study. All investigators will be required to 
complete a form for the disclosure of significant financial interest. 

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15.5!
Fair and Equitable Selection of Subjects 
Subjects will not be discriminated against on the basis of race, sex, or religion. Due to the early stage of 
development of this IP, we have excluded individuals under 18 and women who are pregnant or nursing and 
we have excluded individuals who are over the age of 50 due to the frequency of exclusionary medical 
conditions. Any individual who is unable to consent due to any reason will not be included in this study. 
15.6!
Informed Consent 
The informed consent process and document(s) will be reviewed and approved by the NMRC IRB and the 
JHSPH IRB prior to initiation of the study. The consent document(s) will contain 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.  Subjects will receive an oral presentation of the study in language (i.e., using 
lay terms as appropriate) they can understand. Subjects will be given the written, IRB-approved informed 
consent, allowed ample time to read the consent, allowed to ask questions about the study, have the questions 
answered, and given time to decide if he/she would like to participate in the study. To document subjects’ 
understanding of informed consent, immediately before the consent is signed, the person obtaining consent 
will administer a brief quiz or comprehension test.  A subject must achieve ≥70% correct to be eligible for 
inclusion in the study. Incorrect answers will be discussed with subjects to reinforce the consent. Subjects 
who fail the comprehension test on the first attempt will be given one additional opportunity, either on the 
same day or another day, to take the test after reviewing the quiz, re-reading the consent, and listening to the 
study brief again.  A final acceptable test score is ≥70% answered correctly.  Subjects failing the 
comprehension test on the second attempt are not eligible for study enrollment. No coercion or influence is 
allowed in obtaining subjects’ consent.  Before subjects participate in the study, consent forms will be signed 
and dated by subjects as well as by the PI or designee.  Subjects will receive copies of the signed consent 
prior to participation.  As part of the consent process, subjects will also be asked to read and sign a Medical 
Records/Lab Results Release, with an opportunity to ask questions, if relevant. Subjects will also be asked to 
sign a separate information form for HIV-1 testing. The consent document indicates that by signature, the 
subject, or where appropriate, legal guardian, permits access to relevant 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, 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 CIR at a later time to sign it.  The subject should 
understand that the study product is investigational 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. 
•!
The individual is free to ask any questions that will allow him/her to understand the nature of the 
protocol. 

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A description of this clinical trial will be available on http://www.ClinicalTrials.gov, as required by US law.  
All non-exempt research involving human subjects shall, at a minimum, meet the requirement of 32 CFR 
219.116(a)(6) in the Code of Federal Regulations. 
15.7!
Recruitment 
Newspaper ads and study fliers posted on the JHU campus and community bulletin boards will be used to 
recruit prospective subjects.  Additionally, subjects in previous studies that have expressed interest in 
participating in future trials will be contacted about the proposed study.  All study-related advertisements will 
be reviewed and approved by the JHSPH IRB, NMRC IRB and HRPO-ORP, if applicable.  Subjects 
responding to the advertisements by a phone call to the center will be screened for eligibility based on a 
standard screening questionnaire administered by the CIR recruiter.  Some elements of the 
inclusion/exclusion criteria will be discussed with the subject at that time and a preliminary determination 
will be made regarding the individual’s eligibility for study participation. Active duty military members will 
not specifically be recruited for this study. 
16.! PRIVACY AND CONFIDENTIALITY 
16.1.! Storage of Data and Samples 
All original records involving this protocol will be stored securely at CIR or a locked, offsite storage facility 
for at least 5 years. Copies of databases will be stored securely at NMRC (and made available to the Sponsor). 
All samples will be stored under appropriate conditions in laboratories in the Enteric Disease Department at 
NMRC and/or the JHU laboratories. 
16.2.! Provisions Protecting Privacy and Confidentiality 
Individual subject medical information obtained as a result of this study is considered confidential and 
disclosure to third parties, other than those cited below, is prohibited. Subject confidentiality will be further 
ensured by utilizing subject identification code numbers and subject initials. Neither NMRC nor the JHSPH 
are HIPAA-covered entities. 
Confidentiality agreements may be developed with other clinical trials groups (e.g. at the University of 
Maryland Vaccine Research Center or Walter Reed Clinical Trials Center), and the investigative team may 
check verbally with these sites to see if subjects have participated in studies that would preclude their 
participation in this study. No written list will be exchanged with these sites. 
16.3.! Safeguards for Vulnerable Subjects 
This study will not include individuals less than 18, incarcerated or unable to meet the requirements to sign 
the informed consent form. Military personnel will not be specifically recruited for this study. All active duty 
military subjects will need to have written permission from their superior to participate in this study. 
17.! PROTOCOL REVIEW PROCESS 
The protocol will undergo scientific and ethical review at the two primary collaborating institutions: CIR and 
NMRC. In addition to these reviews, the JHU Biosafety Committee and Pharmacy and Therapeutics 
Committee will review the protocol. The protocol will also require FDA review as part of the IND 
application. The IND sponsor will be Dr. Louis Bourgeois. Continuing review will be undertaken in 
accordance with existing regulations. 

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The investigator may deviate from the protocol without prior approval when the change is necessary to 
eliminate an apparent immediate hazard to the subject. In that event, the investigator will notify the sponsor 
promptly by phone, will notify the CIR (JHSPH IRB) and NMRC IRB, and will confirm notification to the 
sponsor in writing within 5 working days after the change is implemented. All protocol deviations, including 
minor deviations not impacting subject safety, will be noted in the continuing review reports, the annual 
report to the Sponsor, and in the final study report. Any modification to the protocol, consent form and/or 
questionnaires, including changing the PI, must be submitted to both IRBs for review and approval prior to 
implementation of the modification. Any deviation to the protocol that may have an effect on the safety or 
rights of the subject or the integrity of the study must be reported to the NMRC ORA, JHSPH IRB and 
USAMRMC HRPO-ORP, if applicable, as soon as the deviation is identified. 
18.! 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. 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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