A Phase 1 Double-Blinded, Placebo-Controlled, Dose Escalation Study to Evaluate the Safety and Immunogenicity of Double Mutant Heat-Labile Toxin LTR192G/L211A (dmLT) From Enterotoxigenic Escherichia Coli (ETEC) by Oral, Sublingual, or Intradermal Vaccination in Adults Residing in an Endemic Area
Phase: PHASE1
Conditions: Gastroenteritis Escherichia Coli, Immunisation
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

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A Phase 1 Double-Blinded, Placebo-Controlled, Dose Escalation Study to Evaluate the 
Safety and Immunogenicity of Double Mutant Heat-Labile Toxin LTR192G/L211A 
(dmLT) from Enterotoxigenic Escherichia coli (ETEC) by Oral, Sublingual, or 
Intradermal Vaccination in Adults Residing in an Endemic Area 
 
DMID Protocol Number: 14-0031 
 
DMID Funding Mechanism: VTEU Contract HHSN272201300022I 
Pharmaceutical Support Provided by: PATH 
IND Sponsor: DMID, NIAID 
 
Protocol Principal Investigator: Wilbur H. Chen, MD, MS 
VTEU Contract Principal Investigator: Karen Kotloff, MD 
Site Principal Investigator: Dr. Firdausi Qadri, PhD 
DMID Clinical Project Manager: Wendy Blocker, RN, MSN 
DMID Medical Monitor: Jorge Mejia-Galvis, MD 
DMID Medical Officer: Kay M. Tomashek, MD, MPH  
DMID Scientific Lead: Shahida Baqar, PhD.  
DMID Clinical Affairs Specialist: Hyung Koo, RN, BSN  
 
 
 
Version Number: 5.0 
 
06 November 2019 
 

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STATEMENT OF COMPLIANCE 
This trial will be conducted in accordance with Good Clinical Practices (GCP) as required by the 
following: 
• United States Code of Federal Regulations (CFR) applicable to clinical studies (45 CFR 
Part 46, 21 CFR Part 50, 21 CFR Part 54, 21 CFR Part 56, and 21 CFR Part 312); 
• International Conference on Harmonization (ICH) E6; 62 Federal Register 25691 (1997); 
• U.S. National Institutes of Health (NIH) Clinical Terms of Award, as applicable; 
• And the local laws and regulations of Bangladesh and International Centre for Diarrheal 
Disease Research, Bangladesh (icddr,b). 
Compliance with these standards provides public assurance that the rights, safety, and well-being 
of study participants are protected, consistent with the principles that have their origin in the 
Declaration of Helsinki. 
All key personnel (all individuals responsible for the design and conduct of this trial) have 
completed Human Subjects Protection Training. 
 
 

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SIGNATURE PAGE 
The signature below constitutes the approval of this protocol and the attachments, and provides 
the necessary assurances that this trial will be conducted according to all stipulations of the 
protocol, including all statements regarding confidentiality, and according to local (Bangladesh) 
legal and regulatory requirements and applicable U.S. federal regulations and ICH guidelines. 
 
Site Investigator: 
 
Signed: 
 
Date: 
 
 
Wilbur H. Chen, MD, MS 
Protocol Principal Investigator 
 
 

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TABLE OF CONTENTS  
Statement of Compliance .................................................................................................................2 
Signature Page .................................................................................................................................3 
List of Tables ...................................................................................................................................8 
List of Figures ..................................................................................................................................9 
List of Abbreviations .....................................................................................................................10 
Protocol Summary .........................................................................................................................13 
1 
Key Roles ..............................................................................................................................19 
2 
Background Information and Scientific Rationale ...............................................................22 
2.1 
Background Information ...........................................................................................22 
2.1.1 Background on the Study Sites .....................................................................30 
2.1.2 Clinical Experience with Oral dmLT ............................................................32 
2.1.3 Clinical Experience with Sublingual dmLT .................................................33 
2.1.4 Clinical Experience with Intradermal dmLT ................................................33 
2.1.5 Rationale .......................................................................................................34 
2.2 
Potential Risks and Benefits .....................................................................................35 
2.2.1 Potential Risks ..............................................................................................35 
2.2.2 Known Potential Benefits .............................................................................36 
2.2.3 Risk Benefit Ratio .........................................................................................36 
3 
Objectives .............................................................................................................................38 
3.1 
Study Objectives .......................................................................................................38 
3.2 
Study Outcome Measures .........................................................................................38 
3.2.1 Primary Outcome Measures ..........................................................................38 
3.2.2 Secondary Outcome Measures ......................................................................39 
3.2.3 Exploratory Outcome Measures ...................................................................39 
4 
Study Design .........................................................................................................................41 
4.1 
Dose Escalation Halting Criteria ..............................................................................42 
5 
Study Enrollment and Withdrawal........................................................................................44 
5.1 
Subject Inclusion Criteria .........................................................................................44 
5.2 
Subject Exclusion Criteria ........................................................................................44 
5.3 
Treatment Assignment Procedures ...........................................................................47 
5.3.1 Randomization Procedures ...........................................................................47 
5.3.2 Masking Procedures ......................................................................................47 
5.3.3 Reasons for Withdrawal and Discontinuation of Dosing .............................48 
5.3.4 Handling of Withdrawals ..............................................................................49 
5.3.5 Termination of Study ....................................................................................50 
6 
Study Intervention/Investigational Product ..........................................................................52 

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6.1 
Study Product Description ........................................................................................52 
6.1.1 Acquisition ....................................................................................................52 
6.1.2 Formulation, Packaging, and Labeling .........................................................53 
6.1.2.1 dmLT.................................................................................................53 
6.1.2.2 Sodium Bicarbonate, USP ................................................................53 
6.1.2.3 Water for Injection ............................................................................53 
6.1.2.4 Sterile Normal Saline ........................................................................53 
6.1.3 Product Storage and Stability........................................................................54 
6.1.3.1 dmLT.................................................................................................54 
6.1.3.2 Sodium Bicarbonate, USP ................................................................54 
6.1.3.3 Water for Injection (WFI) .................................................................54 
6.1.3.4 Sterile Normal Saline ........................................................................55 
6.2 
Dosage, Preparation and Administration of Study Intervention/Investigational 
Product ......................................................................................................................55 
6.2.1 Oral Administration of dmLT .......................................................................55 
6.2.2 Oral Administration of Placebo (Sodium Bicarbonate Buffer alone) ...........56 
6.2.3 Sublingual Administration of dmLT .............................................................56 
6.2.4 Sublingual Administration of Placebo (Normal Saline) ...............................57 
6.2.5 Intradermal Administration of dmLT ...........................................................57 
6.2.6 Intradermal Administration of Placebo (Normal Saline) ..............................57 
6.3 
Modification of Study Intervention/Investigational Product for a Participant .........57 
6.4 
Accountability Procedures for the Study Intervention/Investigational Product(s) ...57 
6.5 
Assessment of Subject Compliance with Study Intervention/Investigational 
Product ......................................................................................................................58 
6.6 
Concomitant Medications/Treatments ......................................................................58 
7 
Study Schedule......................................................................................................................60 
7.1 
Screening (Days -7 through -4).................................................................................60 
7.2 
Enrollment/Baseline ..................................................................................................61 
7.2.1 VISIT 1- First Vaccination (Day 1) ..............................................................61 
7.3 
Follow-up ..................................................................................................................62 
7.3.1 VISIT 2 (~1 week post-dose 1± 1 day) .........................................................62 
7.3.2 VISIT 3 - Second Vaccination (~14 days post dose 1 ±2 days) ...................63 
7.3.3 VISIT 4 (~1 week post-dose 2±1day) ...........................................................63 
7.3.4 VISIT 5 - Third Vaccination (~14 days post dose-2 ±2 days) ......................64 
7.3.5 VISIT 6 (~1 week post-dose 3±1 day) ..........................................................65 
7.3.6 VISIT 7 (~4 weeks post-dose 3 (window ±3 days) ......................................66 
7.3.7 VISIT 8 (~12weeks post-dose 3 (window ±1week) .....................................67 
7.4 
Final Study Visit .......................................................................................................67 

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7.4.1 VISIT 9 (~6 months post-dose 3, window ±2 weeks) ..................................67 
7.5 
Early Termination Visit ............................................................................................67 
7.6 
Unscheduled Visit .....................................................................................................68 
8 
Study Procedures/Evaluations ..............................................................................................69 
8.1 
Clinical Evaluations ..................................................................................................69 
8.2 
Laboratory Evaluations .............................................................................................71 
8.2.1 Clinical Laboratory Evaluations ...................................................................71 
8.2.2 Clinical Microbiology Evaluation.................................................................72 
8.2.3 Special Assays or Procedures .......................................................................72 
8.2.4 Specimen Preparation, Handling, and Shipping ...........................................73 
8.2.4.1 Instructions for Specimen Preparation, Handling, and Storage ........73 
8.2.4.2 Specimen Shipment ..........................................................................73 
9 
Assessment of Safety ............................................................................................................74 
9.1 
Specification of Safety Parameters ...........................................................................74 
9.2 
Methods and Timing for Assessing, Recording, and Analyzing Safety 
Parameters .................................................................................................................75 
9.2.1 Adverse Events .............................................................................................75 
9.2.2 Solicited Events ............................................................................................76 
9.2.3 Serious Adverse Events ................................................................................76 
9.2.4 Procedures to be followed in the Event of Abnormal Laboratory Test 
Values or Abnormal Clinical Findings .........................................................77 
9.3 
Reporting Procedures ................................................................................................78 
9.3.1 Serious Adverse Events ................................................................................78 
9.3.2 Regulatory Reporting for Studies Conducted Under DMID-Sponsored 
IND ...............................................................................................................79 
9.3.3 Reporting of Pregnancy ................................................................................79 
9.4 
Type and Duration of Follow-up of Subjects after Adverse Events .........................80 
9.5 
Halting Rules ............................................................................................................80 
9.6 
Safety Oversight........................................................................................................81 
9.6.1 DMID DSMB................................................................................................81 
9.6.2 DMID Independent Safety Monitor (ISM) ...................................................82 
9.6.3 Local DSMB .................................................................................................82 
9.6.4 Independent Protocol Safety Team (IPST) ...................................................83 
10 
Clinical Monitoring ...............................................................................................................84 
10.1 
Site Monitoring Plan .................................................................................................84 
11 
Statistical Considerations ......................................................................................................85 
11.1 
Study Hypotheses......................................................................................................85 
11.2 
Sample Size Considerations ......................................................................................85 

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11.3 
Planned Interim Analyses .........................................................................................85 
11.3.1 Safety Review ...............................................................................................85 
11.3.2 Immunogenicity Review ...............................................................................86 
11.4 
Final Analysis Plan ...................................................................................................86 
11.4.1 Analysis Populations .....................................................................................86 
11.4.2 Safety Data ....................................................................................................87 
11.4.3 Immunogenicity Data....................................................................................87 
12 
Source Documents and Access to Source Data/Documents .................................................89 
13 
Quality Control and Quality Assurance ................................................................................90 
14 
Ethics/Protection of Human Subjects ...................................................................................91 
14.1 
Ethical Standard ........................................................................................................91 
14.2 
Institutional Review Board .......................................................................................91 
14.3 
Informed Consent Process ........................................................................................92 
14.4 
Subject Confidentiality .............................................................................................94 
14.5 
Future Use of Stored Specimens ...............................................................................94 
15 
Data Handling and Record Keeping .....................................................................................96 
15.1 
Data Management Responsibilities...........................................................................96 
15.2 
Data Capture Methods ..............................................................................................96 
15.3 
Types of Data ............................................................................................................96 
15.4 
Timing/Reports .........................................................................................................97 
15.5 
Study Records Retention...........................................................................................97 
15.6 
Protocol Deviations ...................................................................................................97 
16 
Publication Policy .................................................................................................................98 
17 
Literature References ............................................................................................................99 
18 
Supplements/Appendices ....................................................................................................102 
Appendix A: Schedule of Events .................................................................................................103 
Appendix B: Acceptable Screening Lab Values ..........................................................................107 
Appendix C: Laboratory and Clinical Toxicity Grading Scales ..................................................108 
 
 

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LIST OF TABLES 
Table 1: Schematic of Study Design:............................................................................................ 17 
Table 2: Assessment of Dehydration and Treatment Guidance .................................................... 71 
Table 3: Assays to be performed at icddr,b .................................................................................. 73 
 
 

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LIST OF FIGURES 
Figure 1: SDS-PAGE gel of native LT, single mutant LT and double mutant LT ....................... 25 
Figure 2: Enzymatic activity of native LT, single mutant LT and double mutant LT .................. 26 
Figure 3: Comparative Enterotoxicity of native LT, single mutant LT and double mutant LT in 
mice. .............................................................................................................................................. 27 
Figure 4: Oral antigenicity and Adjuvanticity of dmLT (Lot 1575) ............................................. 28 
Figure 5: Magnitude of serum IgG and fecal IgA dmLT-specific responses to different routes of 
administration ............................................................................................................................... 29 

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LIST OF ABBREVIATIONS 
AE 
Adverse Event/Adverse Experience 
ALS 
Antibodies in Lymphocyte Supernatant 
ALT 
Alanine Aminotransferase, formerly called SGPT 
ANC 
Absolute Neutrophil Count 
ASC 
Antibody Secreting Cells 
AST 
Aspartate Aminotransferase, formerly called SGOT 
cAMP 
Cyclic Adenosine Monophosphate 
CFR 
Code of Federal Regulations 
cGMP 
Current Good Manufacturing Practice 
CI 
Confidence Interval 
CfaE 
CPM 
CFA/I minor pilin subunit 
Clinical Project Manager 
CFR 
Code of Federal Regulations 
CRF 
Case Report Form 
CT 
Cholera Toxin 
CTL 
Cytotoxic T Cell 
CVD 
Center for Vaccine Development-Global Health 
CyTOF 
Cytometry by Time-of-Flight, or Mass Cytometry 
DHHS 
Department of Health and Human Services 
DMID 
Division of Microbiology and Infectious Diseases, NIAID, NIH, 
DHHS 
dmLT 
Double-Mutant Heat-Labile Toxin 
DSMB 
Data and Safety Monitoring Board 
eCRF 
Electronic Case Report Form 
EHDB 
Enteric and Hepatic Disease Branch, DMID, NIAID, NIH, DHHS 
ELISA 
Enzyme-Linked Immunosorbent Assay 
ELISpot 
Enzyme-Linked ImmunoSpot Assay 
ETEC 
Enterotoxigenic Escherichia coli 
EVI 
Enteric Vaccine Initiative, of PATH 
FDA 
Food and Drug Administration, DHHS 
FWA 
Federal-wide Assurance 
GEMS 
Global Enteric Multicenter Study 
GCP 
Good Clinical Practice 
HBsAg 
Hepatitis B Virus Surface Antigen 
HCV 
Hepatitis C Virus 
HEENT 
Head, Eyes, Ears, Nose, Throat 

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Hg 
Hemoglobin 
HIPAA 
Health Insurance Portability and Accountability Act 
IB 
Investigator’s Brochure 
icddr,b 
International Centre for Diarrhoeal Disease Research, Bangladesh 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation 
ID 
Intradermal 
IEC 
Independent or Institutional Ethics Committee 
Ig 
Immunoglobulin 
IM 
Intramuscular 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
IVF 
Intravenous Fluids 
LT 
Labile Toxin, or E. coli Heat-Labile Enterotoxin 
MedDRA 
Medical Dictionary for Regulatory Activities 
mLT 
Single mutant Heat-Labile Toxin 
MM 
Medical Monitor 
MO 
Medical Officer 
MOP 
Manual of Procedures 
MSD  
moderate-to-severe diarrhea 
NIAID 
National Institute of Allergy and Infectious Diseases, NIH, DHHS 
NIH 
National Institutes of Health 
OCRA 
Office of Clinical Research Affairs, DMID, NIAID, NIH, DHHS 
OHRP 
Office for Human Research Protections, OASH, OS, DHHS 
ORA 
Office of Regulatory Affairs, DMID, NIAID, NIH, DHHS 
ORS 
Oral Rehydration Solution 
OVA 
Ovalbumin Antigen 
PATH 
Program for Appropriate Technology in Health, now simply “PATH” 
PBMC 
Peripheral Blood Mononuclear Cells 
PBS 
Phosphate Buffered Saline 
PHI 
Personal Health Information 
PI 
Principal Investigator 
PK 
Pharmacokinetics 
QA 
Quality Assurance 
QC 
Quality Control 
QMP 
Quality Management Plan 
SAE 
Serious Adverse Event/Serious Adverse Experience 

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SDCC 
Statistical and Data Coordinating Center 
SDS-PAGE 
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis 
SL 
Sublingual 
SMC 
Safety Monitoring Committee 
SOP 
Standard Operating Procedure 
ST 
Stable Toxin, or E. coli Heat-Stable Enterotoxin 
TCI 
Transcutaneous Immunization 
TD 
Traveler’s Diarrhea 
TT 
Tetanus Toxoid 
UMB 
University of Maryland, Baltimore 
US 
United States 
USAMRIID 
US Army Medical Research Institute of Infectious Diseases 
USP 
United States Pharmacopeia 
VTEU 
Vaccine and Treatment Evaluation Unit 
WBC 
White Blood Cell 
WFI 
Water For Injection 
WHO 
World Health Organization 
WRAIR 
Walter Reed Army Institute of Research 

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PROTOCOL SUMMARY 
Title: 
A Phase 1 Double-Blinded, Placebo-Controlled, Dose Escalation 
Study to Evaluate the Safety and Immunogenicity of Double 
Mutant Heat-Labile Toxin LTR192G/L211A (dmLT) from 
Enterotoxigenic Escherichia coli (ETEC) by Oral, Sublingual, or 
Intradermal Vaccination in Adults Residing in an Endemic Area 
Phase: 
1 
Population: 
Approximately 135 Healthy Adults, ages 18 to 45 years 
Number of Sites: 
Single site, Mirpur, Bangladesh 
Study Duration: 
Approximately 2.5 years, including 6 months of follow up for 
the last cohort 
Subject Participation 
Duration: 
Up to 9 months 
Description of Agent or 
Intervention: 
Attenuated, Recombinant Double Mutant Heat-Labile Toxin 
LTR192G/L211A (dmLT) from Enterotoxigenic Escherichia 
coli (ETEC) 
 
Study Objectives: 
 
 
Primary: 
• To assess the reactogenicity, safety and tolerability of 
dmLT when administered in three sequential doses, 
over a range of dosages by oral, sublingual, or 
intradermal routes  
Secondary: 
• To assess the long-term safety, from first vaccination 
through 6 months following the last dose of vaccine 
• To evaluate the serum anti-dmLT IgG and IgA 
response 
• To evaluate the IgG and IgA anti-dmLT Antibody 
Secreting Cell (ASC) response  

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• To evaluate the IgG and IgA anti-dmLT Antibodies 
in Lymphocyte Supernatant (ALS) response  
• To evaluate the total fecal IgA and fecal anti-dmLT 
IgA response  
• To evaluate the total salivary IgA and the saliva-
derived anti-dmLT IgA response  
Exploratory: 
• To measure the mucosal homing of IgA anti-dmLT 
ASC  
• To measure the serum toxin neutralizing antibody 
response  
• To measure the dmLT-specific IgG and IgA memory 
B cell response  
• To determine the dmLT-specific effector and memory 
T cell responses 
Study Outcome Measures: 
Primary Safety Endpoints: 
• The occurrence of solicited local site and systemic 
reactogenicity events from vaccination through 7 days 
after each dose of vaccine is administered 
 
• The occurrence of study withdrawal throughout the study 
 
• The occurrence of discontinuation of study vaccination 
during the study 
• The occurrence of unsolicited vaccine-related adverse 
events (AE), including laboratory AE, from first 
vaccination through 28 days after the last dose of vaccine 
is administered  
Secondary Safety Endpoints: 
• The occurrence of vaccine-related serious adverse events 
(SAE) from the first vaccination through 6 months after 
the last dose of vaccine is administered 
Secondary Immunogenicity Endpoints: 

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• At any time after vaccination, the proportion of 
participants with a ≥4-fold rise in dmLT-specific serum 
IgG and IgA titers over baseline measured by ELISA  
• At any time after vaccination the proportion of 
participants with >8 dmLT-specific IgA or IgG ASC / 
106 PBMC as measured by ELISpot  
• At any time after vaccination, the proportion of 
participants with ≥2-fold rise in ALS anti-dmLT-specific 
IgG and IgA titers over baseline measured by ELISA  
• At any time after vaccination, the proportion of 
participants with a ≥4-fold rise over baseline in dmLT-
specific fecal IgA titers measured by ELISA 
• At any time after vaccination, the proportion of 
participants with a ≥4-fold rise over baseline in dmLT-
specific salivary IgA titers measured by ELISA 
Exploratory Immunogenicity Endpoints: 
• Geometric mean titers of dmLT-specific serum IgG and 
IgA measured by ELISA at time points listed in 
Appendix A  
• Mean and median number of dmLT-specific IgG and IgA 
ASC measured by EliSpot at time points listed in 
Appendix A   
• Geometric mean titers of dmLT-specific IgG and IgA in 
ALS measured by ELISA at time points listed in 
Appendix A  
• Geometric mean titers of dmLT-specific fecal IgA 
measured by ELISA at time points listed in Appendix A 
• Geometric mean titers of dmLT-specific salivary IgA 
measured by ELISA at time points listed in Appendix A 
• The proportion of participants with dmLT-specific 
memory B cell response as measured by ELISpot at time 
points listed in Appendix A 
• At any time after vaccination, the proportion of 
participants with anti-dmLT IgG and IgA ASC in 
circulation expressing gut homing receptors (integrin 

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α4β7 in the absence or presence of CD62L) measured by 
EliSpot assay at time points listed in appendix A  
• At any time after vaccination, proportion of participants 
with ≥4-fold rise over baseline in toxin neutralization 
titers measured by Y-1 cell assay  
• At any time after vaccination, the proportion of 
participants with dmLT-specific effector and memory T 
cell responses as measured by CyTOF at time points 
listed in appendix A  
Description of Study Design: This is a phase 1 double-blinded, placebo-controlled, dose-
escalation study which will evaluate the safety and immune 
responses to a range of dosages of dmLT, when administered by 
the oral, sublingual, or intradermal route.  Two independent Data 
and Safety Monitoring Boards (DSMBs) will monitor this study.  
One, local DSMB will be convened by and advisory to the 
icddr,b Ethical Review Committee (ERC). The other will be a 
NIH DSMB that is convened and advisory to DMID.  See 
Section 9.6 Safety Oversight, for a description of the safety 
oversight to be conducted between the 2 independent review 
boards.  
Estimated Time to Complete 
Enrollment: 
Approximately 20 months 
 
 
 

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Table 1: Schematic of Study Design: 
Cohort 
Group 
Route 
dmLT 
dose (µg) 
 Target No. 
Participant 
Enrollment 
Minimum 
No. 
Participants 
Evaluable* 
Vaccination 
Days 
A 
A1 
A2 
Oral 
5 
placebo 
12 
3 
10 
1 
1, 15, 29 
ISM, MM, and the protocol PI will review Cohort A safety data collected through 7-days post vaccination of third 
dose of last participant for recommendation to proceed to the cohort B. 
B 
B1 
B2 
Oral 
25  
placebo 
12 
3 
10 
1 
1, 15, 29 
C 
C1 
C2 
Sublingual 
5 
placebo 
12 
3 
10 
1 
1, 15, 29 
ISM, MM, and the protocol PI will review Cohort C safety data collected 7-days post vaccination of third dose of 
last participant for recommendation to proceed to the cohort D. 
D 
D1 
D2 
Sublingual 
25 
placebo 
12 
3 
10 
1 
1, 15, 29 
E 
E1 
E2 
Intradermal 
0.3 
placebo 
12 
3 
10 
1 
1, 22, 43 
ISM, MM, and the protocol PI will review Cohort E safety data collected through 7-days post vaccination of third 
dose of last participant for recommendation to proceed to the cohort F. 
F 
F1 
F2 
Intradermal 
1.0 
placebo 
12 
3 
10 
1 
1, 22, 43 
Scheduled DMID DSMB meeting will be held to review cumulative safety data through 7 days after the third dose 
of vaccine and determine if study should proceed to Cohorts G, H and I.  
G 
G1 
G2 
Oral 
50 
placebo 
12 
3 
10 
1 
1, 15, 29 
H 
H1 
H2 
Sublingual 
50 
placebo 
12 
3 
10 
1 
1, 15, 29 
I 
I1 
I2 
Intradermal 
2.0 
placebo 
12 
3 
10 
1 
1, 22, 43 
Scheduled DMID DSMB meeting will be held to review the aggregate safety data through 28 days after the third 
dose of vaccine for all nine cohorts (A-I) 
A Final DMID DSMB meeting will be held to review the safety data following the completion of the study 
Note: the local DSMB will conduct a review of each cohort (A-I) by review of safety data through 7 days 
after the third dose of vaccine for each cohort. (see Section 9.6.3) The local DSMB will also be provided 
the final safety report, the same report that will be generated for the final DMID DSMB meeting.  
* Evaluable participants are defined as completing all 3 doses of vaccine and providing both 
safety information and protocol-required specimens through at least 7 days after the third dose of 

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vaccine. Participants receiving at least one dose of the investigational product must be followed 
for safety until the end of the study. If additional participants are required, to maintain blinding, 
additional participants are to be determined by Emmes; to keep the blind, at least one placebo 
will be included. 

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1 
KEY ROLES 
Individuals: 
 
DMID Scientific Lead: 
 
Shahida Baqar, PhD 
EHDB/DMID/NIAID/NIH/DHHS 
5601 Fishers Lane, Room 8F39 
Rockville, MD 20852 
Ph: 240-627-3458 
shahida.baqar@nih.gov 
Protocol Principal 
Investigator: 
 
Wilbur H. Chen, MD, MS 
Center for Vaccine Development-Global Health 
University of Maryland School of Medicine 
685 W. Baltimore Street, Suite 480 
Baltimore, MD 21201 
Ph: 410-706-1188 
wchen@som.umaryland.edu 
Site Principal Investigator: 
 
Firdausi Qadri, PhD 
Senior Director, Infectious Disease Division 
icddr,b 
68 Shaheed Tajuddin Ahmed Sarani, Mohakhali, Dhaka 1212, 
Bangladesh 
Ph: +8802 9827001-10, Ext-3465 
fqadri@icddrb.org 
VTEU Contract Principal 
Investigator: 
 
Karen L. Kotloff, MD 
Center for Vaccine Development-Global Health 
University of Maryland School of Medicine 
685 W. Baltimore Street, Suite 480 
Baltimore, MD 21201 
Ph: 410-706-6541 
kkotloff@som.umaryland.edu 
 

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Laboratory Principal 
Investigator: 
 
Marcelo B. Sztein, MD 
Center for Vaccine Development-Global Health 
University of Maryland School of Medicine 
685 W. Baltimore Street, Suite 480 
Baltimore, MD 21201 
Ph: 410-706-2345 
Cell: 443-538-1734 
msztein@som.umaryland.edu 
 
Applied Immunology Lab 
Principal Investigator: 
 
Marcela F. Pasetti, PhD 
Center for Vaccine Development-Global Health 
University of Maryland School of Medicine 
685 W. Baltimore Street, Suite 480 
Baltimore, MD 21201 
Ph: 410-706-2341 
Cell: 410-852-8957 
mpasetti@som.umaryland.edu 
 
DMID Medical Officer 
 
Kay M. Tomashek, MD, MPH, DTM 
OCRR/DMID/NIAID/NIH/DHHS 
5601 Fishers Lane, Room 7F39 
Rockville, MD 20852 
Ph: 240-669-2880 
kay.tomashek@nih.gov 
 
DMID Medical Monitor 
 
Jorge Mejia-Galvis, MD (C) 
OCRA/DMID/NIAID/NIH/DHHS 
5601 Fishers Lane, Room: 7E56 
Rockville, MD 20852 
Ph: 240-292-4713 
Jorge.mejia-galvis@nih.gov 
 
 

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Pharma/Biotech Support 
 
PATH  
Vaccine Development Global Program 
Enteric Vaccine Initiative 
455 Massachusetts Avenue, NW 
Washington, DC 20001, Suite 1000 
 
Senior Scientist: Lou Bourgeois, PhD, MPH 
Ph: 202-540-4413 
Fax: 202-457-1466 
lbourgeois@path.org 
 
Project Manager: Nicole Bauers, MS 
Ph: 202-540-4458 
nbauers@path.org 
 
Statistical and Data 
Coordinating Center 
 
The Emmes Corporation 
401 N. Washington St., Suite 700 
Rockville, MD 20850 
Group Email: enterics@emmes.com 
 
Institutions: 
 
Center for Vaccine Development-Global Health (CVD) 
University of Maryland School of Medicine 
685 W. Baltimore Street, 4th floor 
Baltimore, MD 21201, U.S.A. 
 
International Centre for Diarrhoeal Disease Research, 
Bangladesh (icddr,b) 
Dhaka, Bangladesh 
68 Shaheed Tajuddin Ahmed Sarani, Mohakhali, Dhaka 1212, 
Bangladesh 
 
 
 

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2 
BACKGROUND INFORMATION AND SCIENTIFIC 
RATIONALE 
2.1 
Background Information 
Enterotoxigenic Escherichia coli (ETEC), one of six recognized diarrheagenic E. coli, is a 
mucosal non-invasive bacterial enteropathogen that causes acute watery diarrhea.  The Center for 
Vaccine Development (CVD)’s recent Global Enteric Multicenter Study (GEMS) confirmed 
ETEC as one of the top 5 attributable pathogens responsible for causing moderate-to-severe 
diarrhea (MSD) among infants and young children of developing countries.1  ETEC is also the 
most common cause of diarrhea for persons from industrialized countries who visit developing 
countries. ETEC is estimated to cause approximately 400 million diarrheal episodes and 380,000 
deaths globally each year (WHO 2009).  Nonetheless, despite the public health importance, there 
is no licensed vaccine to prevent ETEC.  
Heat-labile Toxin or LT 
ETEC strains produce a highly immunogenic heat-labile toxin (LT) and/or a small (18-19 amino 
acid polypeptide), poorly immunogenic heat-stable toxin (ST).  LT has been more extensively 
studied as a potential vaccine antigen. The LT protein, which structurally and functionally acts 
like cholera toxin (CT), is a multimeric (1 A-subunit: 5 B-subunits) enzyme that catalyzes ADP 
ribosylation of GTP binding proteins within polarized intestinal epithelial cells. The A-subunit is 
the toxic enzymatic moiety while the B-subunits facilitate toxin uptake by binding GM1 and 
other galactose-containing cell-surface components.  The result of A-subunit intoxication is up-
regulation of intracellular cAMP leading to hypersecretion of water and electrolytes in the small 
intestine, resulting in diarrhea and potentially severe dehydration.   LT is also believed to serve 
as an accessory colonization factor for ETEC strains, in that, LT expressing strains colonize 
better and are more virulent in animal models.2-4 In GEMS, ST only and ST plus LT producing 
ETEC were associated with MSD but LT-only ETEC strains were not.   
As a principal virulence factor of ETEC, LT has been studied as a potential vaccine antigen5 and 
has been shown to confer short term protection in animal models and in limited human field 
trials.6-8 An ETEC vaccine candidate based on native LT delivered by transcutaneous 
immunization (TCI) with a dermal patch was found to be immunogenic, and demonstrated some 
efficacy in Phase 2b and Phase 3 field efficacy trials in travelers to Guatemala and Mexico.8,9 In 
addition, recent field studies of cholera vaccines which are capable of inducing cross-reactive 
anti-LT toxin immunity also indicate that an anti-LT based vaccine can be protective at least in 
travelers against ETEC and that anti-LT vaccines may provide protection against a broader array 
of ETEC pathotypes than originally anticipated.10-12 

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LT or its derivatives have also been extensively studied as a robust immunostimulating adjuvant 
to vaccines.13,14 Thus, the LT of ETEC is a unique protein with the potential to be both a stand-
alone vaccine as well as a mucosal adjuvant for other co-administered vaccine antigens. The 
adjuvanting property of this protein may be particularly relevant for use in ETEC or other enteric 
vaccine candidates or combination vaccines since enteric vaccines have historically been poorly 
immunogenic when given to infants and young children in developing countries.15  Consequently 
the inclusion of a non-toxic mutant of the LT protein in future enteric vaccines formulations 
would not only help protect vaccinees against ETEC but might serve to substantially improved 
the antibody and cellular responses to other ETEC, Shigella, Cholera, Rotavirus or Typhoid 
antigens administered along with it.5,14 
However, native LT has considerable enterotoxicity for humans, thereby limiting its use as an 
adjuvant.  For example, in a study conducted at the CVD and at Lausanne University Hospital, 
oral doses of 5 and 10 µg of native LT, administered with a recombinant Helicobacter pylori 
urease protein, elicited diarrhea among 67% (16 of 24).16In a subsequent study of lower native 
LT doses, administered with a H. pylori urease vaccine, mild diarrhea (1-4 loose stools) occurred 
in 50% (6 of 12) of volunteers exposed to 2.5 µg of native LT.17 
Single-mutant LT (LTR192G) or mLT 
To render LT safe for human use, site-directed mutagenesis was performed to eliminate the 
ADP-ribosylating enzymatic activity of the A subunit while maintaining the immunogenic B 
subunit.  A single mutant (mLT) LTR192G was constructed by substituting glycine for arginine 
at position 192 within the disulfide subtended region of the A subunit, separating the A1 from 
A2.18The US Army Medical Research Institute of Infectious Diseases (USAMRIID) conducted a 
randomized, placebo-controlled, dose-escalation study in 36 healthy adult subjects who received 
a single oral dose of mLT (5, 25, 50, or 100 µg) or placebo. An oral dose of mLT at 100 µg 
elicited severe diarrhea (>1L within 24 h) among 2 of 12 recipients.19  When mLT was orally 
administered at the CVD, in the context of an adjuvant to an inactivated whole-cell H. pylori 
vaccine, 5 of 34 (16%) subjects who received 25 µg of mLT developed diarrhea (3-17 stools 
over 1-3 days).  One of 3 subjects given 25 µg of mLT alone also developed diarrhea (33%).20 
Mild-to-moderate diarrhea was also observed with a Campylobacter whole-cell vaccine 
adjuvanted with mLT.  Nine of 39 subjects (23%) developed mild-to-moderate diarrhea within 
24-36 hours after receiving their first dose of the vaccine plus 25 µg mLT adjuvant 
combination.21  Whereas, in a study of a microencapsulated E. coli surface antigen C6 (meCS6), 
60 subjects received meCS6 with or without 2 µg of mLT (significantly lower dose than in 
previous studies) and there was no difference in the rates of diarrhea and limited adjuvanticity 
observed.22 

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In a phase 1 study comparing the safety and immunogenicity of fimbriae CFA/I minor pilin 
subunit E (CfaE) tip-adhesion protein with 100 ng of mLT  via transcutaneous (TCI) and 
intradermal (ID) routes, the ID route was shown to be superior to TCI in inducing both serum 
and mucosal responses to the mLT adjuvanted CfaE candidate vaccine. Tenderness and pain was 
more commonly reported with ID compared to TCI vaccination but was generally mild and brief. 
There were no clear systemic adverse events attributable to the ID administration of vaccine. 
Vaccine site reactions (rash) and pruritus were common but the severity was generally mild and 
resolved in all subjects. Six months after receiving the first vaccine dose, 5 subjects (10.2%) 
reported a persistent vaccine site reaction. Additionally, persistent skin color changes were 
reported by 22 subjects 44.9%) that included hypopigmentation (2 subjects; 4.1%) and 
hyperpigmentation (20 subjects; 40.8%).  The frequency of skin pigmentation changes decreased 
over the following six months with only 9 subjects (18.4%) reporting persistent hyper-(3 
subjects; 4.1%) or hypopigmentation (6 subjects; 12.2%) 1 year following the first vaccination. 
(Unpublished data from S. Savarino, NMRC; NCT01644565) 
Based on the tolerable safety profile and superior immunogenicity results (unpublished data), it 
was determined that the ID administered 25 µg of CfaE with mLT would be utilized in a Phase 
2b challenge study as a proof-of-concept for parenteral administration of an ETEC antigen 
(unpublished data; NCT01922856). The vaccination and challenge phases have completed and 
similar to the phase 1 trial, among the 56 vaccine recipients, there were no vaccine-attributable 
systemic adverse events while vaccine site reactions (rash) and pruritus were common.  Subjects 
are currently undergoing long-term safety follow-up at the 6- and 12-month post vaccination 
time points. 
Derivation and Characterization of double-mutant LT (R192G/L211A) or dmLT 
A second-generation derivative, double-mutant LT (R192G/L211A) or dmLT, was constructed 
by a further substitution of leucine for alanine at amino acid position 211.23This substitution was 
initially believed to be a putative pepsin-sensitive proteolytic cleavage site, but there is no 
evidence that LT is cleaved by pepsin either in vitro or in vivo (data not shown), so the 
biochemical basis of attenuation is unclear. 
dmLT has been characterized in four ways: a) sodium dodecyl sulfate polyacrylamide gel 
electrophoresis (SDS-PAGE, Figure 1), b) in vitro cAMP production (Figure 2), c) enterotoxicity 
in mice (Figure 3), and d) immunogenic and adjuvant capacities in mice (Figure 4A and B).  
These structural, biological, and immunogenic analyses confirmed that this material is in fact 
dmLT; that native LT-like molecules retaining the trypsin sensitivity and cAMP inducing 
activity of the wild-type holotoxin are not present; that dmLT lacks enterotoxicity at the doses 
tested in mice, and that the mutant retains the ability to induce immune response to itself as well 
as adjuvant the immune response to a model protein antigen, like tetanus toxoid (TT).  SDS-

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PAGE was used to confirm that, unlike the native LT holotoxin, the A subunit of dmLT was not 
susceptible to trypsin cleavage.  In vitro cAMP induction and in vivo patent mouse assays were 
used to determine the extent to which dmLT may have reduced enzymatic or enterotoxic activity 
compared to the wild-type toxin, while oral immunization of mice was used to demonstrate that 
the mutant retains its ability to serve as both an antigen and adjuvant. 
Figure 1: SDS-PAGE gel of native LT, single mutant LT and double mutant LT 
 
 
 
 
 
 
 
 
SDS-PAGE gel of 10 µg of native LT (lanes 2 and 3), LTR192G (lanes 4 and 5), or 
LT(R192G/L211A) Lot No. 1575 with (lane 7) or without (lane 6) treatment with 200 ng of 
trypsin for 60 min at 37oC. This assay demonstrates trypsin cleavage (activation) of native LT 
(lane 3) and confirms the absence of native LT-like molecules in the LTR192G and LT 
(R192G/L211A; Lot No 1575) preparations. A comparison of lanes 6 and 7 confirms the 
resistance of Lot No. 1575 to trypsin digestion (lane 7) and confirms the absence of any native 
LT in Lot No. 1575 (comparing lane 3 with lane 7). SDS-PAGE analysis of trypsin treated and 
untreated preparations: lane 1, molecular weight markers; lane 2, native LT; lane 3, native LT 
treated with trypsin; lane 4, LTR192G; lane 5, LTR192G treated with trypsin; lane 6, 
LT(R192G/L211A); lane 7, LT(R192G/L211A) treated with trypsin. (Unpublished data, from 
current Investigator’s Brochure). 
 
 

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Figure 2: Enzymatic activity of native LT, single mutant LT and double mutant LT 
 
 
 
 
 
 
 
 
 
The enzymatic activity of LT, LT(R192G) and LT(R192G/L211A) based on cAMP induction 
(accumulation) as measured in cultured Caco-2 cells. Dilutions of trypsin-treated native LT were 
added to one set of wells to achieve a final concentration of 0.001 µg of native LT per well. One 
microgram of trypsin-treated LTR192G or LT(R192G/L211A) was added to another set of wells.  
At 1, 2, 3, and 4 hours after toxin addition, cells were washed twice with cold phosphate buffered 
saline (PBS).  Intracellular cAMP was extracted by adding 0.4 mL of 0.1 N HCl to each well and 
incubated at room temperature for 20 minutes.  cAMP was detected using an ELISA-based low 
pH cAMP kit (Figure 2).  The inability of 1 µg of LT(R192G/L211A) to elicit as much 
intracellular cAMP as 0.001 µg of native LT indicates that this mutant enterotoxin is attenuated 
in enzymatic activity in comparison to native LT (Unpublished data from J.D. Clements, 
Tulane). 
 

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Figure 3: Comparative Enterotoxicity of native LT, single mutant LT and double mutant 
LT in mice.  
 
The comparative enterotoxicity of native LT and LTR192G/L211A (Lot No. 1575) in the Patent 
Mouse Model. For the patent mouse assay, four female BALB/c mice per group were inoculated 
with 0.5 mL of buffer; 5, 25, and 125 µg native LT; or 50, 125, and 250 µg LT(R192G/L211A) 
Lot No. 1575. Inoculations were made intragastrically with a blunt-tip feeding needle. Following 
inoculation, animals remained in their cages without food but with water ad libitum for 3 hours 
and were then sacrificed. The entire intestine from duodenum to anus from each mouse was 
removed carefully to retain any accumulated fluid; residual mesentery was eliminated before 
weighing. The carcass was weighed separately and a gut:carcass ratio (Y axis Figure 3) was 
calculated for each animal. Mean gut:carcass ratio for 50 µg of LT(R192G/L211A) should be 
less than that obtained with 5 µg of native LT.  As shown in Figure 4, the reduced enterotoxicity 
of LT(R192G/L211A) was clearly evident since 250 µg of Lot No. 1575 elicited no more fluid 
than the buffer control and the gut:carcass ratio was less than 5 µg of native LT at any of the 
dilutions tested (Unpublished data, from Investigator’s Brochure). 
 
 

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Figure 4: Oral antigenicity and Adjuvanticity of dmLT (Lot 1575) 
 
 
Animals that were orally immunized with LT(R192G/L211A) developed a significant serum 
anti-LT IgG response (Figure 4A) confirming the oral antigenicity of LT(R192G/L211A). The 
adjuvanticity of LT (R192G/L211A) was monitored biologically in an oral adjuvanticity assay 
using TT (Statens Serum Institute, Denmark) as a test antigen. Groups of 10 BALB/c mice were 
immunized orally with a blunt-tip feeding needle, once per week for 3 weeks, with either 100 µg 
of TT alone or with the same dose of TT admixed with 25 µg of LT(R192G/L211A). One week 
after the third immunization, serum anti-TT was determined by ELISA, and the adjuvant activity 
of Lot No. 1575 was highly significant in this assay. As seen in Figure 4B, LT(R192G/L211A) 
significantly increased the serum anti-TT IgG response in orally immunized animals compared to 
the response in animals receiving TT alone.  This assay confirms the oral adjuvanticity of 
LT(R192G/L211A). (Unpublished data, from current Investigator’s Brochure) 
 
Sublingual and Intradermal dmLT 
Recent animal studies have demonstrated that the sublingual (SL) route of administration elicits 
serum (IgG) and local intestinal fecal (IgA) antibodies to vaccine antigens that are comparable or 
better than those induced by the intradermal (ID) and oral (per os [PO]) routes.  These 
observations may be particularly relevant for enteric vaccines.  As shown in the Figure 5, the 
magnitude of fecal anti-dmLT IgA response was better in those animals receiving dmLT by the 
SL versus ID route, which further suggests that the SL route may be better for inducing local 
intestinal anti-dmLT antibodies than the ID or oral route of delivery (Unpublished data from J.D. 
Clements, Tulane). 
 
 

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Figure 5: Magnitude of serum IgG and fecal IgA dmLT-specific responses to different 
routes of administration 
 
 
 
 
 
 
 
 
 
 
 
 
 
Other animal studies have shown that the SL and ID routes are not only well tolerated, but 
induce mucosal immune responses on a broad range of mucosal surfaces, including the 
respiratory, gastrointestinal, and urogenital tracts.14  Chemotactic responses of IgG and IgA 
antibody secreting cells (ASCs) after SL administration of 2 µg of CT showed that CCL28 (a β-
chemokine that possesses2 or more adjacent cysteines) plays a critical role in the selective 
homing of IgA ASCs into genital tissues as well as other mucosal sites.24  SL administration of a 
combination CT and live influenza virus at vaccine doses that would be lethal by the nasal route 
were well tolerated (no significant histopathology noted) and highly immunogenic in mice.25  
Administration of non-replicating antigens (ovalbumin antigen [OVA]),25Bacillus subtilis-
expressing TT fragment C,26-28 inactivated whole cell Pneumococcal vaccine,29 Helicobacter 
pylori,20 and live influenza virus,24 some in the presence of CT or mutant LT proteins (mLT and 
dmLT), has been shown to induce broad-based systemic and mucosal immune responses in the 
targeted mucosal area. In 3 of the 5 above mentioned vaccinations via the SL route, subsequent 
challenge with the respective wild type strains of human papilloma virus (HPV),30Helicobacter 
pylori,31 and Streptococcus pneumoniae29 protection was conferred and  strong antigen-specific 
serum IgG and IgA responses in the targeted mucosal tissues were observed.  
Accumulating preclinical data continues to indicate that the SL route is a very robust and 
encouraging new option for effective mucosal immunization; however, the utility (safety and 
efficacy) of this route for immunizing humans has yet to be established.32 As a result, studies 
exploring the SL route are pivotal for the mucosal vaccine development field as another option 
for effective active mucosal immunization with protein antigens like dmLT. The significant 

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attenuation of dmLT is expected to further eliminate the possibility of facial palsy, which has 
been observed with native LT when delivered by the intranasal route.33 
Buoyed by these positive results, multiple enteric vaccine studies are being planned for the use of 
dmLT as an adjuvant (private communication with PATH-Enteric Vaccine Initiative). 
Significant questions remain to be addressed in future studies.  First, how will dmLT perform 
(through the assessment of safety and immunogenicity) in an endemic country, when compared 
to studies performed in an industrialized country?  Second, are there significant differences in the 
responses by the route of administration (oral, SL, or ID)? 
2.1.1 
Background on the Study Sites 
Center for Vaccine Development-Global Health (CVD) - Baltimore, Maryland, U.S.A. 
The CVD has been an NIAID Vaccine and Treatment Evaluation Unit (VTEU) for ~40 years and 
has a formidable track record of research on vaccines needed primarily by developing countries 
and travelers, including vaccines against bacterial enteric infections, such as ETEC. The CVD’s 
Clinical Research Unit has conducted many clinical trials, including multiple trials of ETEC 
vaccine candidates including, LT, mLT, and dmLT.  The Unit is supported by the CVD’s 
Regulatory Affairs & Quality Management Office, with both domestic and international 
regulatory affairs specialists, and a quality management coordinator. CVD Research Labs are 
fully equipped for immunology, molecular biology, and bacteriology, including safe handling of 
vaccines and bacteria. 
The CVD Immunology Group is composed of 3 integrated components that work together using 
state-of-the-art equipment to measure a broad array of immunologic responses: a Cellular 
Immunology Section, a Flow Cytometry Core, and an Applied Immunology Unit.   
The CVD’s Immunology Group performs the protocol-required assays for DMID 09-0066 (oral 
dmLT), DMID 12-0023 (sublingual dmLT), and DMID 13-0013 (intradermal dmLT).  Thus, 
training and sharing of the methods and reagents by the staff of the Immunology Group will be 
central to ensuring that the assays at icddr,b are qualified for the present study. 
The CVD’s Flow Cytometry Core houses: a Beckman-Coulter (former Dako-Cytomation) 
MoFlo flow cytometer/cell sorter system equipped with 3 lasers (i.e., up to 12 simultaneous 
parameters), a custom Becton-Dickinson LSR-II Flow cytometer analyzer equipped with 4 lasers 
(i.e., up to 16 simultaneous parameters), and a DVS Sciences CyTOF Mass Cytometer, which 
uses mass spectrometry to allow the simultaneous detection of at least 35 metal-labeled 
antibodies.  The CyTOF Mass Cytometer will be used for the T cell assays in the present study. 
 

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International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) - Dhaka, 
Bangladesh   
The icddr,b is a renowned international research institute addressing fundamental global health 
needs; a leader in endemic diarrheal disease research, including ETEC. For the present study, the 
icddr,b will operate under a subcontract with the CVD.  The icddr,b routinely is engaged in 
research, training, and extension activities and also provides significant clinical services to 
thousands of impoverished patients. The centre has both national and international staff, 
including public-health scientists, epidemiologists, demographers, social and behavioral 
scientists, laboratory scientists, clinicians, nutritionists, Information Technology (IT) 
professionals, and research experts in emerging and re-emerging infectious diseases and vaccine 
sciences. 
The icddr,b main campus is located in Dhaka (the main hospital, clinical labs and all extensive 
research laboratories are at this site). The centre also has a variety of urban and rural field sites, 
all of which have been extensively involved in vaccine research, clinical trials and observational 
studies. There are three major field sites available: Matlab, Mirpur, and Bandarban. 
Mirpur Field Site: The Mirpur research site is located in the Dhaka Metropolitan area, 
approximately 7 km from the main icddr,b Dhaka hospital, and has been used for field studies 
since 1987. Mirpur is densely populated with approximately 3.5 million individuals. This 
research clinic has enjoyed a long-standing relationship with this stable community. The Mirpur 
research clinic is composed of five floors, with each floor consisting of 4 rooms; approximately 
7000 square feet of research space.  This dedicated clinic building is situated within the same 
neighborhood as the study population. The facility contains a participant waiting area, several 
examination rooms, a staff work and file room, a specimen processing area, archive room and a 
meeting room. The specimen processing area contains a refrigerator and a centrifuge with 24 
hour generator back-up. The building has 24 hours security coverage and internet service. All 
clinical and laboratory specimens are sent to the clinical and research laboratories of the main 
icddr,b campus daily maintaining cold chain requirements. Numerous natural history studies as 
well as GCP based vaccine trials have been conducted or planned in this site and include oral 
cholera, oral ETEC vaccine(s) and typhoid vaccine studies.  Socio-demographic information is 
also available from this area. A geographic information system (GIS) was used for mapping of 
this area and identifying households and other information of these households have been 
completed using PDA (Personal digital assistance) devices and android TABs.  
The Mucosal Immunology & Vaccinology Laboratory at the icddr,b is a BSL2 level facility with 
internal and external quality assurance procedures that are carried out for the laboratory assays 
and their results. All standard operating procedures (SOPs) for studies are updated frequently for 
meeting study requirements. The laboratory is equipped with six biohazard safety hoods for 

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processing of biological samples and for maintaining sterile conditions for specimen processing. 
For the fractionation of samples, refrigerated table top centrifuges, high speed centrifuges 
(Beckman) and ultracentrifuges (Beckman L7-80 and a Beckman L5-65B) are also available. 
Incubators with carbon-dioxide gas attachment are present for the study of B cell and T cell 
responses. There is a cryostat for sectioning of frozen sections and a microtome for paraffin 
sections. Four ELISA readers including two kinetic ones are linked to a computer for 
determining antibody responses in study samples. Facilities for ELISPOT and “Antibody in 
Lymphocyte Supernatant” (ALS) assays are available. A cytotoxic T cell (CTL) automatic 
counter and stereomicroscopes, for enumeration of ASCs, are available. There are facilities for 
carrying out extraction of lymphocytes from gut biopsies.  In addition, facilities for carrying out 
intestinal lavages and fecal extracts are used for assessing mucosal antibody responses. There are 
six -80°C low temperature freezers, two liquid nitrogen freezers, and a number of additional 
freezers (-20°C) and refrigerators used for storage of specimens from vaccine trial and studies of 
infectious diseases. The Flow Cytometry Core facility that is coordinated by and used by this lab 
have a Becton Dickinson Facs Caliburs and FACSAria™ III cell sorter. The FACSAria™ III is 
currently configured with 3 lasers and is capable of detecting 9 fluorochromes (this capacity is 
expandable if needed) as well as a 96-well plate adapter for single cell sorting. 
2.1.2 
Clinical Experience with Oral dmLT 
The National Institute of Allergy and Infectious Disease (NIAID) sponsored a Phase 1 dose-
escalation study conducted at the CVD and University of Cincinnati in 36 healthy adult 
volunteers who received a single oral dose of 5, 25, 50, or 100 µg dmLT (DMID Protocol 09-
0066, under IND 14399; NCT01147445). The immune responses were measured by dmLT-
specific serum IgA and IgG, fecal IgA, ASC, and ALS assays.  No participants experienced 
diarrhea in any dosage group and the vaccine was well tolerated.  Although the immune 
responses were limited in those receiving 5 µg or 25 µg of dmLT, the immune response was 
greater in those receiving 50 µg or 100 µg of dmLT, with a possible plateauing of immune 
responses at 50 µg34. 
A Phase 1 study of an inactivated oral multivalent ETEC vaccine (ETVAX, expressing CFA/I, 
CS3, CS5, CS6, and LTB) with dmLT as an adjuvant was conducted in 129 healthy Swedish 
adults. The vaccine with dmLT was safe and well tolerated; dmLT appeared to further enhance 
mucosal immune responses to CF antigens which were present in low amounts in the vaccine.35 
A Phase 1/2 study of oral administration of an inactivated ETEC vaccine (ETVAX) with and 
without dmLT as an adjuvant was initiated October 2015 and is ongoing at the icddr,b (VAC 
014/OEV-122; NCT02531802). 
A Phase 2 study of the oral ETEC vaccine (ETVAX) administered with dmLT is currently being 
conducted among healthy adults in Bangladesh by icddr,b. (NCT02531802) investigators.  

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A Phase 1/2b study of a live, attenuated oral ETEC vaccine (ACE527) adjuvanted with dmLT 
was conducted in the U.S. (VAC 006; NCT01739231).  The trial results have not yet been 
published, but the data have been presented at scientific conferences. Immunization with 
ACE527 administered with or without dmLT was safe and immunogenic. The addition of dmLT 
to the vaccine formulation did not significantly change its safety or immunogenicity profile.  
When a total of 57 participants were challenged with ~107 cfu ETEC strain H10407, participants 
in the ACE527+dmLT vaccine group were highly protected; only 3/13 subjects developing 
severe diarrhea (PE=65.9%, P=0.003 one-sided Barnard’s test). Meanwhile, 7/13 (53.8%) 
subjects receiving ACE527 alone developed severe diarrhea (PE=20.5%; P=0.205). 
ACE527+dmLT was also 58.5% efficacious in protecting against diarrhea of any severity 
(P=0.016) and substantially reduced H10407 shedding post-challenge. There were no significant 
adverse events (AEs) associated with orally administered dmLT as adjuvant.36 
2.1.3 
Clinical Experience with Sublingual dmLT 
NIAID is currently sponsoring a Phase 1 dose-escalation study in  64 healthy adult volunteers 
who are to receive three sequential SL doses of 1, 5, 25, or 50 µg dmLT; a final cohort will 
compare sublingual vs. oral administration at the dose level showing optimal immunogenicity 
without any safety concerns (DMID Protocol 12-0023, under IND 14399; NCT02052934).  
Delivery of dmLT to Cohorts 1-4 were completed and no safety concerns were identified with up 
to the 3 doses of 50 µg dmLT delivered orally. The CVD’s Applied Immunology Laboratory has 
performed all the immunologic assays for this study. The study is completed; there have been no 
noted safety concerns, currently a clinical study report (CSR) is being finalized .  There are no 
other human trials, to our knowledge, that have been completed using SL route of administration 
of dmLT as a vaccine or adjuvant. 
2.1.4 
Clinical Experience with Intradermal dmLT 
NIAID is sponsoring a Phase 1 dose-escalation study of three sequential ID doses of 0.1, 0.3, 1.0, 
or 2.0 µg dmLT, spaced by 21 days (DMID Protocol 13-0013, under IND 16836 
NCT02531685). The CVD’s Applied Immunology Laboratory is performing all the 
immunologic assays for this study.  DMID 13-0013 trial started in April of 2016 at the CCHMC, 
and it is the first in human trial with dmLT administered via the ID route. Currently, three doses 
of 0.1 µg, 0.3 µg and1.0 µg have been delivered to subjects; no safety signals were seen nor any 
halting rules were met. 
The first generation mLT (LTR192G), which is more toxic in animal (IB V1.0 16 May 2017) 
than the double-mutant being tested here, was evaluated in a phase 1 trial of ID administered 
CfaE with or without 100 ng LTR192G. In this study, local reaction at the vaccine injection site 
were the most common vaccine-related adverse events and were generally mild in severity. 

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Hypo/hyperpigmentation were observed for 6 months in 45% of the participants and up to 1 year 
in 18% of the study participants.  Preliminary immunogenicity results have revealed robust anti-
LTB and anti-CfaE serologic and ASC responses (IgG/IgA), as well as high hemagglutination 
inhibition titers after ID immunization37.   
 
2.1.5 
Rationale 
ETEC remains a major cause of childhood diarrhea in low-middle income (i.e., developing) 
countries and is endemic to areas with poor infrastructure for sanitation and without access to 
clean water.  The use of dmLT is proposed as an oral, sublingual, or intradermal traveler’s 
diarrhea (TD) vaccine for persons from industrialized (i.e., developed) countries that visit high-
risk developing (non-industrialized) countries.  It is envisioned that dmLT may also be used as a 
component of candidate ETEC vaccines under development for use in infants and young children 
living in ETEC endemic areas. The vaccine should provide its best protection against ETEC 
diarrhea associated with infection by strains expressing the LT toxin alone or in combination 
with ST toxin (LT/ST).  Native LT given by the transcutaneous immunization (TCI) route has 
been shown to protect travelers against TD due to LT producing ETEC strains in two field trials; 
8,15 however, broader protection against ETEC might be achievable if the vaccine is given by the 
mucosal route (oral or SL), or by routes that may induce both systemic and mucosal immunity 
such as ID. Non-native LT vaccine such as dmLT may have beneficial effects beyond the 
anticipated protection against ETEC expressing LT or LT/ST. Two cellular ETEC vaccines that 
have recently completed clinical trials included study arms in which dmLT was added as a 
vaccine component to improve the anti-LT toxin responses induced by the vaccine, as well as 
potentially improve mucosal responses to other co-administered ETEC surface antigens. In the 
case of ETVAX vaccine, the anti-LT toxin and anti ETEC colonization factor antigen immune 
responses were significantly improved with the addition of the lower dose of dmLT adjuvant.26 
Furthermore, adding dmLT to the live attenuated ETEC vaccine formulation offered significant 
protection against ETEC challenge 6-7 months after the primary immunization series that was 
otherwise not conferred by vaccine alone.38 
Compared to oral immunization, SL or ID immunization may increase dose-sparing and 
potentially eliminate the need for buffering to neutralize gastric acid, therefore providing greater 
protein antigen stability. Both the SL and ID routes may also help bypass gut enteropathy, which 
serves as a major barrier to oral vaccination. The increased dose sparing would help reduce the 
cost of the final vaccine product.   
The optimal protection to be afforded against enteric diarrheal pathogens is believed to be 
through the elicitation of robust mucosal immune responses. The addition of LT may serve as 
both a protective antigen and/or a potent immunostimulating adjuvant to vaccine candidates.  

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The aforementioned DMID-sponsored studies on dmLT have been or are being conducted in the 
U.S. (an industrialized country) and there are unanswered questions regarding the immune 
responses through different routes of administration in a non-industrialized country. This study is 
designed to assess the safety and immune responses of a range of doses of dmLT when 
administered by the oral, SL, or ID route, in volunteers residing in an endemic country for ETEC 
disease.  The proposed indication for dmLT is to protect against ETEC diarrhea associated with 
infection by strains expressing the LT toxin alone or in combination with the heat-stable toxin 
(ST) as a component of an oral, sublingual, or intradermal diarrhea vaccine for those residing in 
high risk endemic regions as well as international travelers to these high-risk regions.  
2.2 
Potential Risks and Benefits 
2.2.1 
Potential Risks 
The study product, dmLT, is a non-infectious product; the risk of direct transmission from study 
participants to the community or research members is negligible. The study product has been 
given to humans as a stand-alone single component antigen and as an adjuvant with other 
candidate vaccines. There have been few adverse events reported. Any candidate vaccine has the 
risk of allergic reaction; these reactions are rare and unpredictable. 
Risk of AEs when dmLT is administered by the oral route. The enterotoxicity observed with 
native LT has, in theory, been eliminated with the two mutations that have been introduced. 
There is the possibility of diarrhea with dmLT, if there is residual enterotoxicity. Other possible 
adverse effects of residual enterotoxicity might include abdominal cramping or discomfort, gas 
or bloating, nausea, vomiting and/or decreased appetite.   
Risk of AEs when dmLT is administered by the SL route. Sublingual administration of dmLT 
is not expected to allow for direct contact with the nasal passages and thus there is no 
expectation for Bell’s palsy. Whereas, intranasal delivery of native LT (and a K63 mutant) with 
influenza and other experimental vaccines has been shown to be associated with transient 
peripheral facial nerve palsies (Bell’s palsy) in a small but significant number of volunteers.33,39  
These events were attributed to retrograde axonal transport of the toxin by binding neuronal 
ganglioside and/or an inflammatory immune response.40,41 Other possible adverse events are 
irritation of the tongue or oral cavity. As with oral dmLT, there may be a risk for mild 
enterotoxicity, including abdominal cramping or discomfort, gas or bloating, nausea, vomiting 
and/or decreased appetite.   
Risk of AEs when dmLT is administered by the ID route. dmLT administered via the ID 
route is currently being tested in humans under the DMID 13-0013 protocol. ID administration 
of 100 ng or 300 ng of dmLT to two separate cohorts of the 10 subjects each is completed. To 

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date no safety concerns were noted, and a 3rd cohort to receive 1 µg is planned to start in July 
2017.  Nonetheless, ID vaccination may be associated with injection site edema, erythema, pain, 
pruritus, induration, plaques, hypopigmentation, hyperpigmentation, and vesicles in a dose-
dependent manner. 
Risk with venipuncture and blood collection. Drawing blood may cause transient discomfort 
and fainting. Fainting is usually transient and managed by having the participant lie down. 
Bruising at the blood draw site may occur, but can be prevented or lessened by applying pressure 
to the venipuncture site for several minutes.  Breaking the skin during venipuncture or ID 
vaccination may cause transient discomfort, fainting, or infection.  The use of sterile technique 
and materials will minimize the risk of infection.   
Risk to breach of confidentiality. Participants will be asked to provide personal health 
information (PHI). All attempts will be made to keep this PHI confidential, within the limits of 
the local laws and regulations. There is the chance that unauthorized persons will gain access to 
PHI. To minimize this risk, all records will be maintained in locked file cabinets or locked 
rooms, when not in use.  When in use, research records will be under the direct supervision of 
study team members.  Electronic files will be password protected. Only persons who are 
involved in the conduct, oversight, monitoring, or auditing of this study will be allowed access to 
the PHI that is collected.   
There may be other unknown risks, discomforts, or side effects from dmLT.  Any significant 
adverse events which are deemed possibly related to dmLT will be disclosed, as appropriate. 
2.2.2 
Known Potential Benefits 
The receipt of dmLT could provide partial protection against an infection with ETEC.  Because 
this study is placebo-controlled and allocation is random and double-blinded, all participants 
should assume no definitive direct benefit.  Nonetheless, due to the global public health 
importance of ETEC infections and vaccines which could be adjuvanted with dmLT, participants 
provide a greater societal benefit for helping the study team members gain important knowledge 
on dmLT. 
2.2.3 
Risk Benefit Ratio 
The burden of ETEC infections in Bangladesh is high, it is a country with endemic ETEC, where 
ETEC infections are common. There is the possibility of some protection with participation in 
this study, but some participants will receive placebo and the vaccine may not work.  Previous 
studies of the vaccine have not demonstrated excessive toxicity or reactogenicity.  The 
assessment of the safety and tolerability of the vaccine is the primary objective.  There is also a 
potential for a societal benefit, in that an ETEC vaccine may be helpful for decreasing the burden 

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associated with this common infection.  Therefore, the risks of the study are balanced by 
benefits. 
 

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3 
OBJECTIVES 
3.1 
Study Objectives 
 
Primary: 
• To assess the reactogenicity, safety and tolerability of dmLT when administered in 
three sequential doses, over a range of dosages by oral, sublingual, or intradermal 
routes  
Secondary: 
• To assess the long-term safety, from first vaccination through 6 months following the 
last dose of vaccine 
• To evaluate the serum anti-dmLT IgG and IgA response 
• To evaluate the IgG and IgA anti-dmLT Antibody Secreting Cell (ASC) response  
• To evaluate the IgG and IgA anti-dmLT Antibodies in Lymphocyte Supernatant 
(ALS) response  
• To evaluate the total fecal IgA and fecal anti-dmLT IgA response  
• To evaluate the total salivary IgA and the saliva-derived anti-dmLT IgA response  
Exploratory: 
• To measure the mucosal homing of IgA anti-dmLT ASC  
• To measure the serum toxin neutralizing antibody response  
• To measure the dmLT-specific IgG and IgA memory B cell response  
• To determine the dmLT-specific effector and memory T cell responses 
3.2 
Study Outcome Measures 
3.2.1 
Primary Outcome Measures 
The Primary Safety Endpoints to be used to evaluate reactogenicity, safety and tolerability are 
as follows: 
• The occurrence of solicited local site and systemic reactogenicity events from vaccination 
through 7 days after each dose of vaccine is administered 
• The occurrence of study withdrawals throughout the study  
• The occurrence of discontinuation of study vaccination during the study 

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• The occurrence of unsolicited vaccine-related adverse events (AE), including laboratory 
AE, from first vaccination through 28 days after the last dose of vaccine is administered  
3.2.2 
Secondary Outcome Measures 
The Secondary Safety Endpoints to be used to evaluate the safety of this study are: 
• The occurrence of vaccine-related serious adverse events (SAE) from the first 
vaccination through 6 months after the last dose of vaccine is administered 
 
The Secondary Immunogenicity Endpoints to be used to evaluate the immune response to 
vaccination will consist of the following: 
• At any time after vaccination, the proportion of participants with a ≥4-fold rise in dmLT-
specific serum IgG and IgA titers over baseline measured by ELISA  
• At any time after vaccination the proportion of participants with >8 dmLT-specific IgA 
or IgG ASC / 106 PBMC as measured by ELISpot  
• At any time after vaccination, the proportion of participants with ≥2-fold rise in ALS 
anti-dmLT-specific IgG and IgA titers over baseline measured by ELISA  
• At any time after vaccination, the proportion of participants with a ≥4-fold rise over 
baseline in dmLT-specific fecal IgA titers measured by ELISA 
• At any time after vaccination, the proportion of participants with a ≥4-fold rise over 
baseline in dmLT-specific salivary IgA titers measured by ELISA 
3.2.3 
Exploratory Outcome Measures 
The Exploratory Endpoints to be used to evaluate the immune response to vaccination will 
consist of the following: 
• Geometric mean titers of dmLT-specific IgG and IgA in ALS measured by ELISA at 
time points listed in Appendix A  
• Geometric mean titers of dmLT-specific serum IgG and IgA measured by ELISA at time 
points listed in Appendix A  
• Mean and median number of dmLT-specific IgG and IgA ASC measured by EliSpot at 
time points listed in Appendix A   
• Geometric mean titers of dmLT-specific fecal IgA measured by ELISA at time points 
listed in Appendix A 
• Geometric mean titers of dmLT-specific salivary IgA measured by ELISA at time points 
listed in Appendix A 

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• The proportion of participants with dmLT-specific memory B cell response as measured 
by ELISpot at time points listed in Appendix A 
• At any time after vaccination, the proportion of participants with anti-dmLT IgG and IgA 
ASC in circulation expressing gut homing receptors (integrin α4β7 in the absence or 
presence of CD62L) measured by EliSpot assay at time points listed in appendix A  
• At any time after vaccination, proportion of participants with ≥4-fold rise over baseline in 
toxin neutralization titers measured by Y-1 cell assay  
• At any time after vaccination, the proportion of participants with dmLT-specific effector 
and memory T cell responses as measured by CyTOF at time points listed in appendix A  

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4 
STUDY DESIGN 
This is an outpatient Phase 1 double-blinded, placebo-controlled, dose-escalation trial in 
approximately 135 (plus any additional participants if needed to have minimal evaluable 
participants / cohort as described in Table 1) apparently healthy adult volunteers, age 18-45 
years, who meet all the eligibility criteria and reside in Bangladesh, an endemic country for 
ETEC infection.  The study population will be recruited from Mirpur, a densely populated urban 
slum community of Dhaka, Bangladesh, known as having a high rate of diarrheal, respiratory 
and enteric disease.  
This clinical trial is designed to assess the safety, reactogenicity, tolerability, and 
immunogenicity of a range of dosages of dmLT administered by three different routes: oral, SL, 
or ID.  The schematic of study design is presented in Table 1. The oral and SL routes of 
administration will evaluate dosages of 5, 25, and 50 µg of dmLT; the ID route of administration 
will evaluate dosages of 0.3, 1.0, and 2.0 µg of dmLT.  Participants will receive a total of three 
sequential doses of dmLT; the oral and SL routes will be at days 1, 15, and 29 and the ID route 
will be at days 1, 22, and 43.  After each dose of study product, participants will be observed for 
30-90 minutes, depending on the route of administration. 
Safety, reactogenicity, and tolerability of the study products will be evaluated for each 
participant. Solicited local and systemic reactogenicity will be collected for 7 days after each 
vaccine administration. Memory Aids and digital thermometers will be given to participants to 
record solicited local and systemic reactogenicity including elevated body temperature (i.e., 
fever) at home, and they will be reviewed with the participants.  Unsolicited AEs will be 
collected from the time of each study vaccination through 28 days after the last vaccination. 
Safety laboratories will be collected on Visit 7 or about 28 days after the last vaccination. 
Serious adverse events (SAEs) will be collected from the time of the first study vaccination 
through 6 months after the last study vaccination. Tolerability of the study products will be 
assessed by recording the occurrence of and reason for study withdrawals and discontinuation of 
study vaccination during the study.  
The evaluation of immunogenicity will include dmLT-specific antibody responses in serum, 
fecal, salivary and ALS samples, in addition levels of ASC in circulation will be determined.  
Memory B cells specific for dmLT will also be evaluated.  An in-depth evaluation of effector 
and memory T cells response, including homing potential, cytokine/chemokine production 
profile, degranulation capacity, and activation potential, will also be performed.  In order to 
maximize our ability to compare immune responses between US and Bangladesh studies, the 
timing of the sample collections for the immunological assays needs to be comparable. The 
timing of the specimen collection for the immunological assays were selected based on previous 

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DMID-funded dmLT studies conducted in the U.S. (i.e., DMID protocols 09-0066 and 12-0023) 
and the ongoing study 13-0013.  
Additional participants may be enrolled to ensure there are at least 11 evaluable participants (10 
vaccinees and 1 placebo) in each of the cohorts (cohorts A through I).  Evaluable participants are 
defined as completing all 3 doses of vaccine and providing both safety and protocol-required 
specimens through at least 7 days after the third dose of vaccine. If additional participants are 
required, to maintain blinding, treatment assignments of additional participants are to be 
determined by Emmes. 
The duration of the study for each participant will be up to 9 months (inclusive of the screening 
period through 6 months after the third dose of vaccine, plus a 2-week window on the last visit), 
depending on the route of dmLT administered.  Details on the study procedures, evaluations, and 
study schedule are in Sections 7, 8 and Appendix A, respectively. 
4.1 
Dose Escalation Halting Criteria 
Prior to dose escalation to the next cohort within a route of administration group (see Table 1), 
safety data through 7 days post vaccination of the third dose will be reviewed. Specifically, the 
Statistical and Data Coordinating Center (SDCC) will provide the cumulative safety data and 
notify the protocol PI, medical monitor (MM), and DMID Independent Safety Monitor (ISM), 
that the following criteria have not been met based on a review of the safety data collected in the 
seven days after the third vaccination of the last participant in the current cohort. Only one 
cohort will be enrolled at a time, and enrollment will proceed from Cohort A to B; Cohort C to 
D, and Cohort E to F if the halting criteria described in Section 9.5 are NOT met. 
In addition, there will be no progression to the next cohort within a route of administration group 
if the following dose escalation halting criteria is met: 
• The site PI, protocol PI, MM, DMID ISM or local DSMB identify a safety concern that 
would prompt a review by the DMID DSMB. 
If the above dose escalation halting criteria are NOT met, the dose escalation may proceed to the 
next successive route of administration cohort (see section 9.6.3). 
If any of the above dose escalation halting criteria are met, then escalation to the next successive 
route of administration cohort will not proceed and the data will be reviewed by the DMID 
DSMB (see section 9.6.1).  The enrollment for the next cohort may begin when all three of the 
below requirements are met: 
• DMID DSMB recommendation to proceed,  
• DMID concurs with the DMID DSMB recommendation to proceed,   

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• Local DSMB recommendation to proceed.  
Upon completion of Cohorts A through F, a scheduled DMID DSMB meeting will be convened 
to review all the safety data (clinical labs and reported events) collected in the seven days after 
the third vaccination in each cohort. Progression to Cohorts G, H and I will occur when all three 
of the below requirements are met: 
• DMID DSMB recommendation to proceed  
• DMID concurs with the DMID DSMB recommendation to proceed 
• Local DSMB recommendation to proceed  
As the IND sponsor, DMID retains the final decision-making authority to stop the study at any 
point. 
 

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5 
STUDY ENROLLMENT AND WITHDRAWAL 
5.1 
Subject Inclusion Criteria 
Study participants are eligible for this study if they fulfill the inclusion criteria below: 
1. Male or female age 18-45 years old, inclusive. 
2. Provides written informed consent before initiation of any study procedures. 
3. Healthy as judged by the site investigators and determined by medical history, 
medication history, and physical examination. 
4. Capable of understanding, consenting, and complying with all the study visits and 
procedures. 
5. Body Mass Index of no less than 18.5 
6. Agrees not to participate in another clinical trial during the study period. 
7. Agrees to complete all study visits and procedures. 
8. Agrees not to donate blood to a blood bank for 12 months after receiving the last vaccine. 
5.2 
Subject Exclusion Criteria 
Participants will be ineligible for any of the following conditions or reasons: 
1. Women who are pregnant or lactating or have a positive urine pregnancy test at screening 
or on the day of vaccinations. 
Note: all women presenting for screening will have urine pregnancy testing.  “Females of 
childbearing potential must agree to use an efficacious hormonal or barrier method of birth 
control from screening and  through 28 days post last dose of vaccine. Abstinence is also 
acceptable.”  
2. Presence or history of a chronic medical condition* that would, in the opinion of the 
investigator, render vaccination unsafe or interfere with the evaluation of the vaccine. 
* Note: this may include, but is not limited to: significant renal disease, unstable or 
progressive neurological disorders, diabetes, heart disease, asthma, lung disease, liver 
disease, organ transplant recipients and cancer. 
3. Presence of a significant dermatologic condition*, or tattoo(s), scarring or significant skin 
damage at the vaccination site that would impede evaluation of local reactogenicity. 
* Note: this may include severe eczema, psoriasis or history of keloid formation. Participants 
with history of squamous cell or basal cell skin cancer that has been surgically excised and 

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considered cured may be enrolled in the study if the skin cancer site is healed and is not at 
proposed vaccine administration site. 
4. Any developmental abnormality of the palate. 
5. Participants diagnosed with autoimmune disorders, chronic inflammatory disorders or 
neurological disorders with a potential autoimmune correlation. 
6. Use of long-term (≥2 weeks) oral steroids, intranasal or topical prednisone (or equivalent), 
parenteral steroids, or high-dose inhaled steroids (>800 μg/day of beclomethasone 
dipropionate or equivalent) within the preceding 6 months. 
7. Has major psychiatric illness* during last 12 months that in the investigator’s opinion 
would preclude participation. 
* Note: Participants taking antipsychotic or antimanic drugs should not be enrolled. These 
include: aripiprazole, clozapine, ziprasidone, haloperidol, molindone, lamotrigine, 
gabapentin, topiramate, loxapine, thioridazine, thiothixene, pimozide, fluphenazine, 
risperidone, mesoridazine, quetiapine, trifluoperazine, chlorprothixene, chlorpromazine, 
perphenazine, olanzapine, carbamazepine, divalproex sodium, lithium carbonate, or lithium 
citrate. Participants taking a single antidepressant drug and are stable without de-
compensating symptoms in the preceding 3 months can be enrolled in the study. 
8. Use of prescription or over-the-counter (OTC) anti-inflammatory medications* 48 hours 
prior to receiving the investigational product.  
* Note: This includes naproxen, aspirin, ibuprofen, and other non-steroidal anti-
inflammatory drugs. 
9. Gastrointestinal symptoms* in the past 24 hours or abdominal pain lasting for more than 2 
weeks in the past 6 months. 
*Note: this may include, but is not limited to: abdominal pain or cramps, loss of appetite, 
nausea, general ill-feeling or vomiting. 
10. Moderate or severe diarrheal illness* during the 6 weeks prior to enrollment. 
*Note: Moderate or severe diarrheal illness is defined by the passage of ≥ 4 unformed or 
loose stools (mix of liquid and solid components) in a 24 hour period 
11. History of chronic gastrointestinal illness*. 
*Note: this includes severe dyspepsia or gastroesophageal reflux disease, constipation, 
irritable bowel syndrome (IBS), hemorrhoids, diverticular disease, colitis, colon polyps, 
colon cancer, and inflammatory bowel disease. Mild or moderate heartburn or epigastric 
pain occurring no more than three times per week is permitted. 

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12. Regular use (weekly or more often) of laxatives, anti-diarrheal, anti-constipation, or 
antacid therapy. 
13. History of major gastrointestinal surgery, excluding uncomplicated appendectomy or 
cholecystectomy. 
14. History of systemic antimicrobial treatment (i.e., topical treatments are not an exclusion) 
during the week prior to any administration of dmLT. 
15. Acute febrile illness (body temperature ≥38°C) during the week prior to enrollment. 
16. Abnormal screening laboratories*. 
*Note: screening labs include white blood cell count (WBC), absolute neutrophil count 
(ANC), hemoglobin (Hg), platelet count, serum creatinine, serum albumin, alanine 
aminotransferase (ALT, also known as SGPT), and serologic testing for Hepatitis B virus 
surface antigen (HBsAg) and Hepatitis C virus (HCV) antibody.  See Appendix B for 
acceptable values. Abnormal vital signs. See Appendix C for abnormal values. 
17. Isolation of specific bacteria* from screening stool cultures. 
*Note: bacteria include ETEC, Vibrio cholerae, and Shigella spp. Salmonella and 
Campylobacter will not be evaluated as part this criterion. 
18. Received an inactivated licensed vaccine within 2 weeks of enrollment or live licensed 
vaccine within 4 weeks of enrollment. 
19. Received a cholera (licensed or experimental) vaccine, E. coli vaccine, or Shigella vaccine 
in the last 3 years. 
20. History of receiving immune globulin or other blood product within the 3 months before 
enrollment in this study. 
21. Currently enrolled in another study, involving an experimental agent. Participants 
involved in observational studies or surveys remain eligible. 
22. Any condition that would, in the opinion of the Site Investigator, place the participant at 
an unacceptable risk of injury or render the participant unable to meet the requirements of 
the protocol.  
23. Known allergies to study compound or components of the study vaccine. 
24. Donating blood in the 8 weeks prior to study entry. 

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5.3 
Treatment Assignment Procedures 
5.3.1 
Randomization Procedures 
No exemptions are granted on Subject Inclusion/Exclusion Criteria in DMID-sponsored studies. 
Questions about eligibility should be directed toward the DMID Medical Officer.  Per 
International Conference on Harmonisation (ICH) guideline E6: Good Clinical Practice (GCP), 
screening records will be maintained to document the reason why an individual was screened, 
but failed trial entry criteria.  Screening information will be recorded in the Statistical and Data 
Coordinating Center’s (SDCC’s) Advantage eClinicalSM (Electronic Data Capture System). 
Once consented and upon entry of demographic data and confirmation of eligibility for this trial, 
the participant will be enrolled. Enrollment of participants will be done online, using the 
enrollment module of Advantage eClinicalSM.  Participants within each cohort will be randomly 
assigned to receive study product or placebo, as shown in Table 1.  The list of randomized 
treatment assignments will be prepared by statisticians at the SDCC and included in the 
enrollment module for this trial.  Advantage eClinicalSM will assign each participant a treatment 
code from the list after the demographic and eligibility data have been entered into the system.  
A designated individual at the site will be provided with a treatment key, which links the 
treatment code to the actual treatment assignment, which will be maintained in a secure location.  
Instructions for use of the enrollment module are included in the Advantage User’s Guide. 
Manual back-up randomization procedures and instructions are provided in the MOP for use if 
the site temporarily loses access to the Internet or the online enrollment system is unavailable. 
The enrollment and randomization for all cohorts will be done as a dose-escalation, with the 
lower dosage cohort enrolled before the higher dosage cohort within a given route of 
administration (Table 1).   
5.3.2 
Masking Procedures 
This is a double-blind study. While participants will not be blinded to cohort, they will be 
blinded to group assignment (i.e., placebo vs. vaccine) within a cohort. 
Participants, investigators, and study personnel performing any study-related assessments 
following study product administration, and laboratory personnel performing immunologic 
assessments will be blinded to the administration of vaccine or placebo, within each cohort.  
The randomization scheme will be generated by the SDCC and provided to the assigned 
unblinded study personnel (e.g., pharmacist preparing study products and unblinded study 
product administrators).   

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The unblinded study product administrator is a study personnel licensed, registered, or certified 
to administer vaccines, but will not be involved in study-related assessments or have participant 
contact for data collection following study injection.  
Both DSMBs may receive data in aggregate and blinded by treatment arm, or may be unblinded 
to individual participant treatment assignments, as needed, to adequately assess safety issues. 
There will be one designated physician within the Independent Protocol Safety Team (IPST) that 
will be assigned to communicate with CROMS Pharmacovigilance for purposes of reporting 
SAEs and to request unblinding (see Section 9.6.4 and MOP for additional details). 
5.3.3 
Reasons for Withdrawal and Discontinuation of Dosing 
A participant may voluntarily withdraw their consent for study participation at any time and for 
any reason, without penalty. 
A participant may be withdrawn from study participation for any of the following reasons: 
• Medical disease or condition, or any new clinical findings for which continued 
participation, in the opinion of the investigator, would compromise the safety or welfare 
of the volunteer, or would interfere with the volunteer’s successful completion of the 
study, or would interfere with the evaluation of responses. 
• Participant no longer meets eligibility criteria. 
• As deemed necessary by the investigator for noncompliance or other reasons. 
• Participant withdrawal of consent. 
• Participant lost to follow-up. 
• Participant dies. 
• Termination of this study. 
• New information becomes available that makes further participation unsafe. 
The second or third dose of study product will not be administered for any of the following 
reasons: 
• Medical condition for which continued participation, in the opinion of the investigator, 
would pose a risk to the participant or would likely confound interpretation of the results. 
• Presence of signs or symptoms that could confound or confuse the assessment of 
reactogenicity.  Study vaccination should be postponed/deferred until signs, symptoms, 
or acute illness have resolved and if within acceptable protocol-specified window for that 
visit.  If outside this window, the DMID Medical Officer must first approve the 
vaccination and the documentation of approval should be filed in the research record. 
• Any unresolved or continuing solicited or unsolicited Grade 3 AE deemed to be vaccine 
related until the time of next dose.  An unresolved or continuing Grade 1 or 2 AE is 

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permissible unless, in the opinion of the investigator, it would render study vaccination 
unsafe or interfere with the evaluation of responses. 
• Solicited or unsolicited Grade 3 AE that occurs without alternative etiology through 7 
days following the previous vaccinations. 
• Solicited AEs, the reactogenicity events occurring through 7 days of each vaccine: 
o Systemic reactions to include: feverishness (chills/shivering/sweating), fatigue 
(tiredness, malaise, general unwell feeling), myalgia (body ache, muscular pain) 
headache, diarrhea*, nausea*, vomiting* or abdominal discomfort*. Systemic 
reactions will also include the measurement of a daily oral temperature. 
o Local reactions include: 
 For oral: irritation of oral cavity or tongue, diarrhea, nausea, vomiting, 
abdominal discomfort  
 For SL: irritation of oral cavity or tongue, or facial nerve disturbance, 
diarrhea, nausea, vomiting, abdominal discomfort 
 For ID: injection site pain, redness, swelling, bruising, itching, hypo/hyper 
pigmentation and induration, vesicles or hardened mass 
* For the oral and SL routes of administration, diarrhea, nausea, vomiting, and 
abdominal discomfort will be evaluated as local reactions. 
• Participant no longer meets eligibility criteria (e.g., new onset illness or condition that 
meets exclusion criteria)  
• As deemed necessary by the investigator for noncompliance or other reasons. 
• Participant refusal of further study vaccinations. 
• Participant withdrawal of consent. 
• Participant lost to follow-up. 
• Termination of this study. 
• New information becomes available that makes further participation unsafe. 
5.3.4 
Handling of Withdrawals 
The primary reason for withdrawal from this study will be recorded on the Study Status data 
collection form. Participants will be encouraged to complete the Early Termination Visit. The 
Early Termination Visit procedures are listed in Section 7.5.  
The investigator will make at least three documented attempts to contact any participant who 
does not return for scheduled follow-up.  Although the participant is not obliged to give reasons 
for withdrawing early, the investigator should make a reasonable effort to ascertain the reason(s) 
while fully respecting the participant’s rights.  An investigator may also withdraw a participant 
from the study.  If a participant withdraws early, an early withdrawal assessment should be 

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obtained via a clinic visit or via telephone contact if this is the only available means. Attempts 
will be made to follow all AEs through resolution, as applicable. Any withdrawals, terminations, 
or losses to follow up will be recorded on the appropriate source document. 
Participants who have received the first vaccination may choose to discontinue receipt of study 
vaccine for any reason but may choose to remain in the study (i.e., not withdraw from study). In 
addition, a participant may be discontinued from receipt of the second or third vaccination. 
However, a discontinuation from vaccination will not result in automatic withdrawal from the 
study. Participants meeting criteria for discontinuation of study vaccination must not receive any 
further study vaccine, but should continue to be monitored for safety and immunogenicity if this 
does not result in a safety risk for the participant and the participant agrees to continue with 
safety and immunogenicity monitoring.  Participants that voluntarily discontinue additional 
doses of vaccine should also be monitored for safety and immunogenicity, if the participant 
consents. 
Participants who provide informed consent and are randomized but do not receive study product 
may be replaced.  
For the oral cohorts, participants that vomit or spit out the first dose of study product within 90 
minutes of taking the study product will be replaced.  Any participant with a failure in 
administering or receiving a first dose of study product will be replaced.   
Participants who withdraw, or are withdrawn or terminated from the study, or are lost to follow-
up after signing the informed consent form, randomization, and receipt of study vaccine will not 
be replaced. 
Additional participants may be enrolled to ensure there are at least 11 evaluable participants in 
each cohort (i.e., 10 vaccinees and 1 placebo per cohort).  Evaluable participants are defined as at 
least completing 3 doses of vaccine and providing both safety and protocol-required specimens 
through at least 7 days after the third dose of vaccine.  
If replacement or additional participants are required and because the study will remain double-
blinded, these participants are to be determined by Emmes. The procedures for replacement of 
study participants can be found in the Manual of Procedures (MOP). Any withdrawals, 
terminations, or losses to follow up will be recorded on the appropriate source document. 
 
5.3.5 
Termination of Study 
Although the study Sponsor has every intention of completing this study, the Sponsor reserves 
the right to terminate the study at any time for clinical or administrative reasons. Reasons for 

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termination include, but are not limited to study closure due to DSMB review and 
recommendation and at the discretion of DMID (Sponsor). 
 

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6 
STUDY INTERVENTION/INVESTIGATIONAL 
PRODUCT 
6.1 
Study Product Description 
LT(R192G/L211A), or dmLT, is a derivative of wild-type ETEC heat-labile enterotoxin that has 
been genetically modified by replacing the arginine at amino acid position 192 with glycine and 
the leucine at amino acid position 211 with alanine. These two amino acid substitutions take 
place in proteolytic cleavage sites which are critical for activation of the secreted toxin 
molecules. The protein has been designated LT(R192G/L211A) and has been extensively 
evaluated in pre-clinical animal studies for its ability to induce anti-dmLT antibody responses, as 
well as its capacity to adjuvant the immune responses for co-administered antigens. This 
Investigational Product requires special handling as a BSL-2 agent. 
6.1.1 
Acquisition 
The dmLT vaccine will be provided by PATH Vaccine Solutions (PVS) and upon DMID 
authorization, will be transferred to the following address: 
DMID Clinical Materials Services (CMS) 
Fisher Bio Services 
20439 Seneca Meadows Parkway 
Germantown, MD 20876, U.S.A. 
Tel: (240) 477-1350 
Fax: (240) 477-1360 
Email: DMID.CMS@thermofisher.com 
 
 
Sodium bicarbonate, water for injection (WFI), sterile empty borosilicate glass vials for vaccine 
dilution and sterile normal saline for use in the study will be provided by the DMID CMS.   
 
All study products will be shipped to the clinical research site upon request and approval from 
DMID. 

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6.1.2 
Formulation, Packaging, and Labeling 
6.1.2.1 
dmLT 
The bulk LT (R192G/L211A), or dmLT, was produced to current Good Manufacturing Practice 
(cGMP) specifications by IDT Biologika.  The product, will be formulated as a freeze-dried 
(lyophilized), white to off-white cake, containing ~500 µg of dmLT in a sodium phosphate 
buffer supplemented with 5% lactose to stabilizer.  The product will be provided in a 2 mL 
injection vial, 2 R, made of clear borosilicate glass, and sealed by a 13 mm dark grey rubber 
freeze-drying stopper and a crimped 13 mm aluminum cap, color: silver/red.  Vial labels will 
include the cautionary statement: “Caution: New drug -Limited by Federal Law to 
investigational use”. 
6.1.2.2 
Sodium Bicarbonate, USP 
The sodium bicarbonate (NaHCO3) is a white, crystalline powder that has a molecular weight of 
84.01 gram/Mol and meets United States Pharmacopeia (USP) standards. This product comes in 
a 500 g plastic container and is manufactured under GMP guidelines. This product is used for the 
preparation and delivery of orally administered dmLT and for the placebo for the oral route of 
administration only. A label with the statement “Caution: New drug -Limited by Federal Law to 
investigational use” will be placed on the immediate package. 
6.1.2.3 
Water for Injection 
The USP grade sterile water for injection (WFI) is nonpyrogenic and contains no bacteriostatic 
agent, or added buffer.  It is supplied as single-dose container and should be used to reconstitute 
the lyophilized vaccine (dmLT) vial and to prepare the sodium bicarbonate buffer solution used 
for delivery of the orally administered dmLT. A label with the statement “Caution: New drug -
Limited by Federal Law to investigational use” will be placed on the immediate package. 
6.1.2.4 
Sterile Normal Saline 
The USP grade 0.9% Sodium Chloride or normal saline is a sterile, nonpyrogenic, isotonic 
solution; each mL contains sodium chloride 9 mg.  It contains no bacteriostatic agent, 
antimicrobial agent, preservatives, or added buffer and is supplied only in single-dose containers.  
The solution may contain hydrochloric acid and/or sodium hydroxide for pH adjustment (pH 5.3, 
range 4.5-7.0). This product should be used to dilute the vaccine to the desired concentration and 
as the placebo for the sublingual and intradermal routes of administration only. A label with the 
statement “Caution: New drug -Limited by Federal Law to investigational use” will be placed on 
the immediate package. 

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6.1.3 
Product Storage and Stability 
The temperature of the storage unit must be manually recorded daily (excluding non-business 
days and holidays, as applicable) and continuously monitored and recorded during the course of 
this trial per the participating site standard operating procedures (SOPs), and documentation will 
be maintained. If the temperature fluctuates outside of the required range, the affected study 
product(s) must be quarantined at the correct storage temperature and labeled as ‘Do Not Use’ 
(until further notice). The participating site’s research pharmacist must alert the site principal 
investigator and study coordinator, if the temperature fluctuates outside of the required range. In 
the event the temperature fluctuates outside of the required range, including accidental deep-
freezing or disruption of the cold chain, the affected study product(s) must not be administered. 
The site principal investigator or responsible person should immediately contact the DMID 
Product Support Team at DMIDProductSupportTeam@niaid.nih.gov for further instructions before 
any additional study vaccinations are administered. Based on the information collected, DMID 
and/or the manufacturer will determine whether the affected study product(s) can be used. If it 
cannot be used, the site will receive specific instructions on how to return the affected study 
product(s) to the DMID CMS or destroy it on site. Additional instructions for quarantine and 
DMID contact information are provided in the protocol-specific MOP. 
6.1.3.1 
dmLT 
The recommended storage temperature as marked on the label is ≤-10°C (-10°C or colder), 
which is also indicated in the Investigator’s Brochure.  Once rehydrated with sterile water for 
injection, as described in the pharmacy manual and preparation worksheets, dmLT should be 
held at 2-8˚C. 
6.1.3.2 
Sodium Bicarbonate, USP 
The USP-grade sodium bicarbonate is preserved in well-closed containers. The Material Safety 
Data Sheet indicates that the product is stable under ordinary conditions of use and storage. The 
sodium bicarbonate is to be stored between 15°C and 30°C (59°F and 86°F). 
6.1.3.3 
Water for Injection (WFI) 
The sterile water for injection must be stored at 20°C to 25°C (excursions between 15°C to 30°C 
are permitted according to USP, Controlled Room Temperature). Before use in dmLT 
reconstitution, WFI will be stored between 2°C and 8°C overnight (at least 18 hours) and 
alternatively may be stored at 2°C and 8°C for up to 18 months, not to exceed the product 
manufacturer expiry date. 

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6.1.3.4 
Sterile Normal Saline 
Sterile normal saline (0.9% Sodium Chloride, USP) must be stored at 20°C to 25°C (excursions 
between 15°C to 30°C are permitted according to USP, Controlled Room Temperature). Before 
use in dmLT dilutions, normal saline will be stored between 2°C and 8°C overnight (at least 18 
hours) and alternatively may be stored at 2°C and 8°C for up to 18 months, not to exceed the 
product manufacturer expiry date. 
6.2 
Dosage, Preparation and Administration of Study 
Intervention/Investigational Product 
Reconstitution of lyophilized dmLT. The vial containing the lyophilized dmLT product is to be 
reconstituted by the addition of 0.5 mL of cold WFIUSP Pharmaceutical grade,  according to the 
directions in the vaccine preparation worksheet.  The reconstituted vial, now containing dmLT at 
1 mg/mL (or 1000 µg per mL), should be clear in appearance and the vial can be further diluted 
to specification. Once reconstituted (1 mg / mL), dmLT can be stored on wet ice or refrigerated 
for up to 6 hours.   As per the DMID Guidelines for Clinical Study Product Management, written 
authorization from CPM/DMID for destruction of study products will be obtained before 
disposing reconstituted products (See MOP for further details). 
Dilution of reconstituted dmLT. The desired dilutions of dmLT are then made, using cold 
sterile normal saline, according to the dosage to be administered and the route of administration. 
The dilution worksheets and details of storage, once dmLT vaccine has been diluted, are 
provided in the study-specific Manual of Procedures. 
Preparation of Sodium Bicarbonate Buffer Solution (for Oral Route of Administration 
only). The sodium bicarbonate buffer solution should be prepared by the Investigational 
Pharmacist (or trained designee) and maintained at room temperature until used.  Two grams (2 
g) of sodium bicarbonate powder will be weighed and placed into an appropriate container (e.g., 
150 mL bottle or larger).  One hundred fifty mL (150 mL) of sterile WFI will then be added to 
the container and the solution mixed until crystals are completely dissolved. 
Preparation of Sterile Normal Saline Placebo (for SL and ID Route of Administration 
only).  The placebo will be prepared by withdrawing sufficient volume of sterile normal saline to 
administer a 0.1mL dose.  Apply a new needle prior to administration for the ID route. 
The vaccine or placebo by either route will be administered by a member of the research team 
who is licensed to administer medications or vaccines. 
6.2.1 
Oral Administration of dmLT 

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Participants will have nothing to eat or drink for 90 minutes before and after dosing.  For oral 
routes of administration, the sodium bicarbonate buffer solution (2 g sodium bicarbonate in 150 
mL of sterile WFI) is split into a 120 mL and 30 mL aliquot (e.g., into two disposable drinking 
cups).  The designated amount of dmLT is transferred from the working dilution, as specified, 
into 30 mL of sodium bicarbonate buffer solution.  Once the dmLT is added to the sodium 
bicarbonate buffer solution it must be administered within 15 minutes to the study participant.  
For oral vaccination, participants first ingest 120 mL of sodium bicarbonate buffer and then 
within 1-5 minutes ingest the designated dosage of dmLT, suspended in 30 mL sodium 
bicarbonate buffer solution. For the oral cohorts, participants that vomit or spit out study product 
within 90 minutes of taking the study product will be replaced. 
6.2.2 
Oral Administration of Placebo (Sodium Bicarbonate Buffer alone) 
Participants will have nothing to eat or drink for 90 minutes before and after dosing.  As with 
active vaccination, a sodium bicarbonate buffer solution will be prepared (2 g sodium 
bicarbonate in 150 mL of sterile WFI) and maintained at room temperature until use.  To 
maintain the blind, participants first ingest 120 mL of the sodium bicarbonate buffer and then 
within 1-5 minutes ingest the remaining 30 mL of sodium bicarbonate buffer solution. For the 
oral cohorts, participants that vomit or spit out study product within 90 minutes of taking the 
study product will be replaced. 
6.2.3 
Sublingual Administration of dmLT 
For SL routes of administration, participants will have nothing to eat or drink for 30 minutes 
before and after dosing.  Participants will be asked to gargle and rinse their mouths for 10±5 
minutes with bottled water or treated tap water that is supplied by study staff.  After this is 
completed, a sterile gauze will be placed under the participant’s tongue for ~1 minute ±15 
seconds. Afterwards, the gauze will be removed, and the participant will receive the sublingual 
vaccine immediately.  The designated amount of vaccine will be given in a 100 µL (0.1 mL) 
volume, diluted with normal saline, and be delivered underneath the tongue using a calibrated 
tuberculin or insulin syringe (or equivalent).  After delivery of vaccine, participants will be 
instructed to tilt their head forward, chin to chest, for at least 1 minute.  Afterwards, participants 
will be instructed to bring their head to normal position and swallow.  Participants will be 
reminded not to eat, drink, or rinse the mouth for 30 minutes following dosing. For the SL 
cohorts, participants that vomit or spit out study product within 30 minutes of taking the study 
product will be replaced. 

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6.2.4 
Sublingual Administration of Placebo (Normal Saline) 
For SL routes of administration, participants will have nothing to eat or drink for 30 minutes 
before and after dosing.  To maintain the blind, participants will undergo the same procedure as 
described above in Section 6.2.3 for the active SL vaccination, except 100 µL (0.1mL) of normal 
saline will be delivered under the tongue instead of active study product. For the SL cohorts, 
participants that vomit or spit out study product within 30 minutes of taking the study product 
will be replaced 
6.2.5 
Intradermal Administration of dmLT 
For intradermal routes of administration, the arms of the participant will be examined for the 
most appropriate injection site, considering the avoidance of scars, tattoos, marks, etc. which 
may obscure or make the evaluation of local site reactogenicity difficult. Subsequent doses of 
vaccine, may be administered in the same arm or the arm may be switched as preferred by the 
participant. The designated amount of vaccine will be delivered in a single 100 µL (0.1 mL) 
volume by the ID route, using a tuberculin syringe (or equivalent) with a single-use sterile 25 
gauge (or smaller) needle. 
6.2.6 
Intradermal Administration of Placebo (Normal Saline) 
To maintain the blind, participants will have the same procedure as described above in Section 
6.2.5 for the active ID vaccination, except 100 µL (0.1 mL) of normal sterile saline will be 
administered by the ID route instead of active study product.  
6.3 
Modification of Study Intervention/Investigational Product for a 
Participant 
Not applicable 
6.4 
Accountability Procedures for the Study Intervention/Investigational 
Product(s) 
The site principal investigator is responsible for ensuring study product distribution and 
disposition and has ultimate responsibility for study product accountability. The Investigator may 
delegate to the Site Research Pharmacist responsibility for study product accountability.  Study 
product accountability records should include date received, date prepared, date administered, 
time of preparation, quantity administered, and the subject identification number to whom the 
study product was administered. The designated research pharmacist(s) will be responsible for 
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pharmacy records must be available for inspection by the DMID monitor and is subject to 
inspection by a regulatory agency (e.g. FDA) at any time. Aliquots of dmLT dilutions in saline 
prepared for each cohort dosing level should be saved and stored at -80ºC±15°C  for later 
shipment to PATH’s analytical laboratory subcontractor for dmLT dose verification (see MOP 
for details on shipping instructions). Temporary storage on dry ice for up to 24 hours is allowed.  
The dose verification frozen aliquots may be stored as a reagent (i.e., does not need to be stored 
under the conditions of an investigational product).  Used and unused study product (dmLT 
vials) will be retained and monitored until written authorization from the Study Sponsor 
(CPM/DMID) for destruction of study products is obtained.  Final disposition of the unused 
study products will be determined by DMID and communicated to the participating sites by the 
DMID Clinical Project Manager. 
 
6.5 
Assessment of Subject Compliance with Study 
Intervention/Investigational Product 
Participants will be vaccinated and observed for the respective time following dosing by a 
blinded member of the study team who is licensed to administer medications or vaccines. 
Observation times are approximately 90 minutes following oral dosing and 30 minutes following 
SL and ID dosing. 
There will be no fasting requirement for the 30 minute observation period following ID dosing. 
6.6 
Concomitant Medications/Treatments 
Participants are permitted to take birth control pills and vitamins throughout the course of the 
study; herbal supplements are not permitted.  In addition, participants will be instructed that 
prescription or OTC anti-inflammatory medications will not be permitted in the 48 hours prior to 
receiving the investigational product. This includes medications that contain naproxen, aspirin, 
ibuprofen, and other non-steroidal anti-inflammatory drugs. All concomitant medications (e.g., 
vitamins, antacids, analgesics, prescription medications) taken 28 days prior to vaccination will 
be recorded in the concomitant medication data collection form.  Any medications taken during 
the 7 days following a dose of vaccine are to be recorded on the participant’s memory aid and/or 
reported to a research team member upon follow up clinic visit.  All analgesics and prescription 
medications from 8 to 28 days post-vaccination will be recorded in the concomitant medication 
data collection form.    
If the investigator learns that the participant has taken a prohibited medication (see 
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product, the investigator will contact the DMID CPM and MO for instructions regarding the 
participant’s continuation in the study. 
 

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7 
STUDY SCHEDULE 
7.1 
Screening (Days -7 through -4) 
Recruitment: Participants for this study will be recruited from the Mirpur field site. Study staff 
will visit nearby households of the Mirpur field clinic and have preliminary discussions with 
adults regarding the study, study procedures, and eligibility. During this process, study staff will 
identify potential participants for the study and invite them to come to the Mirpur field clinic at a 
scheduled time for a screening visit.  
Research staff will obtain written consent per the standard informed consent process before 
conducting protocol-specific screening activities. 
After signing the ICF, the following screening procedures will be performed: 
• 
Confirm signed informed consent form.  
• 
Collect demographic information, medical history, and concomitant medication history 
• 
Record oral temperature, pulse, and blood pressure 
• 
Perform an abbreviated physical examination, to include: head, eyes, ears, nose, throat 
(HEENT); lymph nodes; skin; pulmonary; cardiovascular; abdominal; neurological; and 
musculoskeletal systems  
• 
Review eligibility criteria 
• 
Collect approximately 2 mL of venous blood, for screening labs: complete blood counts 
(CBC) with differential for WBC, Hg, ANC, platelets count, creatinine, albumin, ALT, 
serum bilirubin, HBsAg, and HCV antibody 
• 
Collect 13 mL of venous blood for cryopreservation of PBMC, for effector T cell assays 
• 
Collect screening stool for culture and future use.  
o The study staff will provide a stool container to the potential participant and give 
instructions to collect stool.  Participants will bring the stool sample back to the field 
clinic, within 8 hours of collection, for the completion of the screening stool for 
culture.  
o 1-2 gm of stool will be frozen at -80°C±15°C for future use microbiome studies 
Prospective participants will be carefully screened to ensure that they are in excellent physical 
and mental health, and that they meet the eligibility criteria Screening tests will be conducted 4-7 
days before Day 1 (the day of first vaccination with dmLT).  All women will have a urine 
pregnancy test performed during screening and a urine pregnancy test on the vaccination days.  
To be eligible the result of the urine pregnancy test must be negative. During screening, 
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of administration cohort so that they are able to anticipate the required duration of fasting 
necessary prior to vaccination. 
7.2 
Enrollment/Baseline 
7.2.1 
VISIT 1- First Vaccination (Day 1) 
• 
Confirm ongoing consent and review eligibility criteria prior to performing any study 
procedures, to include updating the medical history and concomitant medications 
• 
Record oral temperature, pulse, and blood pressure 
• 
Targeted physical examination, if indicated based on review of medical history 
• 
Urine pregnancy test will be obtained from all women must be negative prior to dosing.  
Provide instructions to women participants to avoid pregnancy through the 28 days from 
receipt of the last dose of vaccine. 
• 
Collect approximately 15 mL of venous blood: 
‒ 3 mL for serum ELISA and neutralization assays 
‒ 2 mL for ASC assay 
‒ 2 mL for ALS assay 
‒ 8 mL for cryopreservation of PBMC, for memory B cell assays 
‒ Collect plasma from PBMC tubes 
• 
Collect stool (approximately 4 gm) and saliva (approximately 2 mL) for IgA assay. 
• 
1-2 gm of stool freeze at -80°C±15°C for future use microbiome studies 
• 
Perform Pre-vaccination reactogenicity assessment. Note: the elements of the 
reactogenicity assessment will vary based on the route of administration of vaccine. The 
pre-vaccination assessment will serve as the baseline for the comparison of post-
vaccination reactogenicity. 
• 
Administer vaccine, per the randomization code and observing the required pre-
vaccination fasting for oral and rinsing for SL dosing 
• 
Provide memory aid and other 7-day solicited AE and reactogenicity instructions 
• 
Provide digital thermometer for recording of daily oral temperature for seven days 
following administration of study product. Participants will be instructed on the use of the 
digital thermometer. 
• 
Perform post-vaccination reactogenicity assessment. Participants will be examined at the 
end of a 30 minute observation period following each vaccination for the SL and ID 
cohorts, and at the end of a 90 minute observation period for oral cohorts. 

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o Reactogenicity Assessments: Will include brief history for assessment of AE/SAEs 
just prior to and following vaccination (Days 1, 15, and 29 for oral/SL; Days 1, 22, 
and 43 for ID), which includes an assessment of systemic and local reactions 
according to section 9.1.   
• 
Provide stool sample collection container and instructions on how to collect stool sample 
• 
Confirm date and time for Visit 2 
7.3 
Follow-up 
7.3.1 
VISIT 2 (~1 week post-dose 1± 1 day) 
• 
Confirm ongoing consent prior to performing any study procedures. Conduct eligibility 
review. Review any AEs or SAEs that have occurred since the last visit as well as update 
the medical history and concomitant medications as necessary. Assess for injection site 
necrosis within 7 days of vaccination.    
o Assess for urticaria, laryngospasm, bronchospasm, or anaphylaxis determined to be 
related to vaccine and within 7 days of vaccination. 
o Assess for participants experience facial nerve neuropathy, confirmed by an 
investigator and determined to be related to vaccine. 
• 
Record oral temperature, pulse, and blood pressure 
• 
Targeted physical examination, if indicated based on review of medical history 
• 
Collect approximately 8 mL of venous blood: 
‒ 2 mL for serum ELISA and neutralization assays 
‒ 2 mL for ASC assay 
‒ 2 mL for ALS assay 
‒ 2 mL for ASC with homing 
‒ Collect plasma from PBMC tubes 
• 
Collect stool (approximately 4 gm) and saliva (approximately 2 mL) for IgA assay. 
• 
1-2 gm of stool frozen at -80°C±15°C for future use microbiome studies 
• 
Collect memory aid/reactogenicity information  
• 
Provide stool sample collection container and instructions on how to collect stool sample 
• 
Provide instructions to women participants to avoid pregnancy through the 28 days from 
receipt of the last dose of vaccine. 
• 
Confirm date and time for Visit 3 

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7.3.2 
VISIT 3 - Second Vaccination (~14 days post dose 1 ±2 days) 
• 
This visit is ~14 days after the first dose for oral and SL cohorts and ~21 days after the 
first dose for the ID cohorts (window ±2 days) 
• 
Confirm ongoing consent and review eligibility criteria with the participant prior to 
performing any study procedures, to include updating the medical history and 
concomitant medications 
• 
Record oral temperature, pulse, and blood pressure 
• 
Targeted physical examination, if indicated based on review of medical history 
• 
Collect approximately 6 mL of venous blood: 
‒ 2 mL for serum ELISA and neutralization assays 
‒ 2 mL for ASC assay 
‒ 2 mL for ALS assay 
• 
Collect stool (approximately 4 gm) and saliva (approximately 2 mL) 
• 
1-2 gm of stool frozen at -80°C±15°C for future use microbiome studies 
• 
Urine pregnancy test for all women will be performed and must be negative prior to 
dosing. Provide instructions to women participants to avoid pregnancy through the 28 
days from receipt of the last dose of vaccine. 
• 
Perform pre-vaccination reactogenicity assessment 
• 
Administer vaccine dose 2, per the randomization code and observing the required pre-
vaccination fasting for oral and rinsing for SL dosing 
• 
Provide memory aid and other 7-day solicited AE and reactogenicity instructions 
• 
Ask participant if they still have the digital thermometer for recording daily oral 
temperature for seven days following administration of the study product and offer re-
instruction on the use of the thermometer as needed. 
• 
Perform post-vaccination reactogenicity assessment after 90 minutes for oral cohorts and 
after 30 minutes for SL and ID cohorts. This assessment is to include unsolicited AEs and 
SAEs. 
• 
Provide stool sample collection container and instructions on how to collect stool sample 
• 
Confirm date and time for Visit 4 
7.3.3 
VISIT 4 (~1 week post-dose 2±1day) 
• 
Confirm ongoing consent prior to performing any study procedures. Conduct eligibility 
review. Review any AEs or SAEs that have occurred since the last visit as well as update 
the medical history and concomitant medications as necessary 
o Assess for injection site necrosis within 7 days of vaccination.    

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o Assess for urticaria, laryngospasm, bronchospasm, or anaphylaxis determined to be 
related to vaccine and within 7 days of vaccination. 
o Assess for participants experience facial nerve neuropathy, confirmed by an 
investigator and determined to be related to vaccine. 
• 
Record oral temperature, pulse, and blood pressure 
• 
Targeted physical examination, if indicated based on review of medical history 
• 
Collect approximately 21mL of venous blood: 
‒ 2 mL for serum ELISA and neutralization assays 
‒ 2 mL for ASC assay 
‒ 2 mL for ALS assay 
‒ 2 mL for ASC with homing 
‒ 13mL for cryopreservation of PBMC, for memory B cell (5 mL) and effector T cell 
assays (8 mL) 
‒ Collect plasma from PBMC tubes 
• 
Collect stool (approximately 4 gm) and saliva (approximately 2 mL) for IgA assay. 
• 
1-2 gm of stool frozen at -80°C±15°C for future use microbiome studies 
• 
Collect memory aid/reactogenicity information  
• 
Provide stool sample collection container and instructions on how to collect stool sample 
• 
Provide instructions to women participants to avoid pregnancy through the 28 days from 
receipt of the last dose of vaccine. 
• 
Confirm date and time for Visit 5 
7.3.4 
VISIT 5 - Third Vaccination (~14 days post dose-2 ±2 days) 
• 
This visit is ~14 days after the second dose for oral and SL cohorts and ~21 days after the 
second dose for the ID cohorts (window ±2 days) 
• 
Confirm ongoing consent and review eligibility criteria with the participant prior to 
performing any study procedures, to include updating the medical history and 
concomitant medications 
• 
Record oral temperature, pulse, and blood pressure 
• 
Targeted physical examination, if indicated based on review of medical history 
• 
Collect approximately 6 mL of venous blood: 
‒ 2 mL for serum ELISA and neutralization assays 
‒ 2 mL for ASC assay 
‒ 2 mL for ALS assay 
• 
Collect stool (approximately 4 gm) and saliva (approximately 2 mL) for IgA assay. 

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• 
1-2 gm of stool frozen at -80°C±15°C for future use microbiome studies 
• 
Urine pregnancy test for women will be performed and must be negative prior to dosing. 
Provide instructions to women participants to avoid pregnancy through the 28 days from 
receipt of the last dose of vaccine. 
• 
Perform Pre-vaccination reactogenicity assessment 
• 
Administer vaccine dose 3, per the randomization code and observing the required pre-
vaccination fasting for oral and rinsing for SL dosing 
• 
Provide memory aid and other 7-day solicited AE and reactogenicity instructions 
• 
Ask participant if they still have the digital thermometer for recording daily oral 
temperature for seven days following administration of the study product and offer re-
instruction on the use of the thermometer as needed. 
• 
Perform post-vaccination reactogenicity assessment after 90 minutes for oral cohorts and 
after 30 minutes for SL and ID cohorts. This assessment is to include unsolicited AEs and 
SAEs. 
• 
Provide stool sample collection container and instructions on how to collect stool sample 
• 
Confirm date and time for Visit 6 
7.3.5 
VISIT 6 (~1 week post-dose 3±1 day) 
• 
Confirm ongoing consent prior to performing any study procedures. Conduct eligibility 
review. Review any AEs or SAEs that have occurred since the last visit as well as update 
the medical history and concomitant medications as necessary 
o Assess for injection site necrosis within 7 days of vaccination.    
o Assess for urticaria, laryngospasm, bronchospasm, or anaphylaxis determined to be 
related to vaccine and within 7 days of vaccination. 
o Assess for participants experience facial nerve neuropathy, confirmed by an 
investigator and determined to be related to vaccine. 
• 
Record oral temperature, pulse, and blood pressure 
• 
Targeted physical examination, if indicated based on review of medical history 
• 
Collect approximately 22 mL of venous blood: 
‒ 2 mL for clinical safety labs: ALT (also known as SGPT), total bilirubin, and 
creatinine 
‒ 2 mL for serum ELISA and neutralization assays 
‒ 2 mL for ASC assay 
‒ 2 mL for ALS assay 
‒ 2 mL for ASC with homing 

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‒ 12 mL for cryopreservation of PBMC, for memory B cell (5 mL) and effector T cell 
assays (7 mL) 
‒ Collect plasma from PBMC tubes 
• 
Collect stool (approximately 4 gm) and saliva (approximately 2 mL) for IgA assay. 
• 
1-2 gm of stool frozen at -80°C±15°C for future use microbiome studies 
• 
Collect memory aid/reactogenicity information  
• 
Provide stool sample collection container and instructions on how to collect stool sample 
• 
Provide instructions to women participants to avoid pregnancy through the 28 days from 
receipt of the last dose of vaccine. 
• 
Confirm date and time for Visit 7 
7.3.6 
VISIT 7 (~4 weeks post-dose 3 (window ±3 days) 
• 
Confirm ongoing consent prior to performing any study procedures. Conduct eligibility 
review. Review any AEs or SAEs that have occurred since the last visit as well as update 
the medical history and concomitant medications as necessary 
o Assess for injection site necrosis within 7 days of vaccination.    
o Assess for urticaria, laryngospasm, bronchospasm, or anaphylaxis determined to be 
related to vaccine and within 7 days of vaccination. 
o Assess for participants experience facial nerve neuropathy, confirmed by an 
investigator and determined to be related to vaccine. 
• 
Record oral temperature, pulse, and blood pressure 
• 
Targeted physical examination, if indicated based on review of medical history 
• 
Collect approximately 17mL of venous blood: 
‒ 2 mL for clinical safety labs: CBC with differential for WBC, Hg, ANC, platelets; 
creatinine, albumin, and ALT 
‒ 3 mL for serum ELISA and neutralization assays 
‒ 12 mL for cryopreservation of PBMC, for memory B cell (5 mL) and effector T cell 
assays (7 mL) 
‒ Collect plasma from PBMC tubes 
• 
Collect stool (approximately 4 gm) and saliva (approximately 2 mL) for IgA assay. 
• 
1-2 gm of stool frozen at -80°C±15°C for future use microbiome studies 
• 
Provide stool sample collection container and instructions on how to collect stool sample 
• 
Provide instructions to women participants to avoid pregnancy through the 28 days from 
receipt of the last dose of vaccine. 
• 
Confirm date and time for Visit 8 

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7.3.7 
VISIT 8 (~12weeks post-dose 3 (window ±1week) 
• 
Confirm ongoing consent prior to performing any study procedures. Conduct eligibility 
review. Review any SAEs that have occurred since the last visit as well as update the 
medical history and concomitant medications as necessary, to support an SAE. 
• 
Record oral temperature, pulse, and blood pressure 
• 
Targeted physical examination, if indicated based on review of medical history 
• 
Collect approximately 15 mL of venous blood: 
‒ 2 mL for serum ELISA and neutralization assays 
‒ 13 mL for cryopreservation of PBMC, for memory B cell (5 mL) and effector T cell 
assays (8 mL) 
‒ Collect plasma from PBMC tubes 
• 
Confirm date and time for Visit 9 
7.4 
Final Study Visit 
7.4.1 
VISIT 9 (~6 months post-dose 3, window ±2 weeks) 
• 
Confirm ongoing consent prior to performing any study procedures. Review any 
SAEs that have occurred since the last visit as well as update the medical history 
and concomitant medications as necessary, to support an SAE. 
• 
Record oral temperature, pulse, and blood pressure 
• 
Collect approximately 15 mL of venous blood: 
‒ 15 mL for cryopreservation of PBMC, for memory B cell (5 mL) and 
effector T cell assays (10mL) 
‒ Collect plasma from PBMC tubes 
7.5 
Early Termination Visit 
Upon voluntary withdrawal of participation or any other reason for early termination from the 
study attempt to complete a clinic visit to include: 
• 
Obtain an interim medical history and concomitant medication information and 
record on the appropriate case report form. 
• 
Assess reactogenicity and review memory aid if early termination visit occurs 
within 7 days of any vaccination.  
• 
Assess for unsolicited AEs if visit occurs within 28 days of third vaccination 

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• 
Assess for SAE during all early termination visits.  Participants will be asked to 
be followed until resolution of any AEs. 
• 
Record oral temperature, pulse, and blood pressure 
• 
Perform a targeted physical examination, if indicated based on review of medical 
history 
• 
Obtain serum samples for laboratory safety assays if visit occurs ≤28 days after 
third vaccination  
7.6 
Unscheduled Visit 
If an unscheduled visit occurs, a member of the clinical study team (PI, Co-Investigator, Study 
Coordinator, or Clinical Nurse) will interview and evaluate the participant to determine the cause 
of the visit and provide care as needed, and information will be documented on a supplemental 
visit eCRF. 
Any of the following activities may be performed during this visit: 
• 
Review of reactogenicity (if within 7 days of last vaccination), unsolicited non-serious 
AE information (if within 28 days of last vaccination), or any SAE information (anytime 
within study) 
• 
Review of concomitant medications (if within 28 days of last vaccination) 
• 
Obtain interim medical history 
• 
Targeted physical examination, if indicated 
• 
If the unscheduled visit is for eligible diarrhea (section 8.2.2), then a stool specimen will 
be collected for culture. 
• 
If the unscheduled visit is grade 3 local injection site reactogenicity event in a participant 
in the ID cohort and the event is within 7 days of a dose administration (as described at 
the end of section 8.1), then measurements of the reaction and a digital photo will be 
performed. Unscheduled daily visits by the study team will continue until the injection 
site reaction is deemed stable or returns to a grade 2 or lower severity.  
• 
Upon the judgement of the clinical investigator, clinical laboratory assessments may be 
performed to evaluate an AE and medications (e.g., analgesics) may be recommended to 
treat an AE 

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8 
STUDY PROCEDURES/EVALUATIONS 
8.1 
Clinical Evaluations 
Medical History: Will be obtained by interview of the participants. Participants will be queried 
regarding a history of significant medical disorders of the head, eyes, ears, nose, throat 
(HEENT), mouth, cardiovascular system, lungs, gastrointestinal tract, liver, pancreas, kidney, 
urologic, nervous system, blood, lymph glands, endocrine system, musculoskeletal system, skin, 
and genital/reproductive tract. A history of any allergies, cancer, immunodeficiency, psychiatric 
illness, substance abuse, and autoimmune disease will be solicited. 
Medication History: All concomitant medications (e.g., vitamins, antacids, analgesics, 
prescription medications) taken 28 days prior to vaccination will be recorded in the concomitant 
medication data collection form.  Any medications taken during the 7 days following a dose of 
vaccine are to be recorded on the participant’s memory aid and/or reported to a research team 
member upon follow up clinic visit.  All analgesics and prescription medications from 8 to 28 
days post-vaccination will be recorded in the concomitant medication data collection form.   
Assessment of eligibility also will include a review of permitted and prohibited medications (per 
the exclusion criteria).  
Physical Examination: At screening (Days -7 through -4) appropriate study personnel will record 
oral temperature, pulse and blood pressure and perform an abbreviated physical examination to 
assess general wellness which will include the following areas/systems:  HEENT; lymph nodes; 
skin; pulmonary; cardiovascular; abdominal; neurological; and musculoskeletal systems. The 
physical examination should specifically address issues identified by the medical history of the 
participant. At following visits including enrollment, oral temperature, pulse, and blood pressure 
will be recorded and a targeted physical examination may be conducted if indicated based on 
review of medical history.  
Reactogenicity Assessments: Will include brief history for assessment of AE/SAEs just prior to 
and following vaccination (Days 1, 15, and 29 for oral/SL; Days 1, 22, and 43 for ID), which 
includes an assessment of systemic and local reactions according to section 9.1.  Participants will 
be examined at the end of a 30 minute observation period following each vaccination for the SL 
and ID cohorts, and at the end of a 90 minute observation period for oral cohorts. 
Memory Aids: All participants will complete a subject memory aid distributed after each 
vaccination. Subject memory aids will be reviewed with the participant for solicited AEs. 
Participants will be instructed to record daily oral temperature on the memory aid. Participants 
will be asked about the occurrence of unsolicited AE/SAEs during visits 2 (Day 8, oral/SL/ID), 4 

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(Day 22, oral/SL; Day 29, ID), and 6 (Day 36, oral/SL; Day 50, ID). Following review, the 
appropriate source document and electronic case report form (eCRF) will be completed by the 
study staff. For illiterate participants, either a literate member of the household will be trained to 
help to fill out the memory aid or a field staff member will be identified to visit the household to 
help with the completion of the memory aid. Memory aids will not be retained as a source 
document.  
Adverse Event Review: It is not anticipated that AEs of diarrhea and/or vomiting (or other signs 
of enterotoxicity) will be common or severe in this study, based on the previous experience with 
oral and SL dmLT or ID dmLT clinical trials. However, an evaluation of any participant who 
experiences diarrhea will be performed by the study staff, if it occurs within 7 days of dosing.  
The determination of severity diarrhea is based as follows: 
Clinical Sign 
Normal 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Diarrhea 
Normal or 1-2 
loose stool per 
24 hours 
3 loose stools 
per 24 hours 
4-7 loose stools 
per 24 hours 
8 or more 
loose/watery 
stools per 24 hours 
or require 
outpatient IV 
hydration, ED visit 
or hospitalization 
for diarrhea 
The assessment of dehydration will be performed in clinic by a study investigator, according to 
Table 2 below. The management of dehydration will be guided according to Table 2 below, but 
is ultimately to be managed at the discretion of the investigator. The assessment of severity of 
diarrhea and level of dehydration and treatment will be documented in the appropriate source 
document. A description for the conditions leading to the performance of stool cultures is 
described in section 8.2. 
 
 

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Table 2: Assessment of Dehydration and Treatment Guidance 
 
 
 
 
 
Clinical 
Assessment 
General 
Appearance 
Well, Alert 
Restless, Irritable 
Lethargic or Unconscious 
Eyes 
Normal 
Sunken 
Sunken 
Thirst 
Normal 
Thirsty, drinks eagerly 
Unable to Drink 
Skin Turgor 
Normal 
Slow Return 
Very Slow Return 
Degree of Dehydration 
No signs of 
dehydration 
Some Dehydration 
Severe Dehydration 
Treatment Guidance 
Reassure and 
educate on 
signs of 
dehydration 
Provide ORS and 
reassess after ORS 
completed, until no 
dehydration 
Provide ORS and consider 
IVF, reassess and repeat 
until no dehydration  
ORS = oral rehydration solution 
IVF = intravenous fluid 
For participants in the ID cohorts, should there be any grade 3 local injection site reactogenicity 
(i.e., within 7 days of dose administration); the research staff will request the participant be 
evaluated by the study team to mark the widest edges of the respective reaction (e.g., redness, 
induration, or bruising), measure, and record on the CRF.  Furthermore, a digital photo of the site 
will be taken by the study team.  Additional measurements and a photo should be taken on a 
daily basis by the study team until the injection site reaction is deemed stable or returns to a 
grade 2 or lower severity. 
8.2 
Laboratory Evaluations 
8.2.1 
Clinical Laboratory Evaluations 
A total of 2 mL blood will be collected for screening clinical labs for the evaluation of eligibility.  
If screening laboratory results are out of range value, they may be repeated once provided there 
is an alternative explanation for the out of range value. Participants must have acceptable 
screening laboratory findings within 7 days prior to enrollment.  The acceptable ranges for study 
eligibility are listed in Appendix B. 
For the assessment of safety, a total of 2 mL of blood will be collected for clinical safety labs on 
visit 7, about 4 weeks after the third dose of vaccine. These include a CBC with differential for 
WBC, Hg, ANC, Platelet count, creatinine, albumin, and ALT.  In addition, 2 mL of blood will 
be collected at visit 6 (7-days post-third dose of vaccine) for ALT, serum bilirubin, and 
creatinine. The grading of toxicity for clinical safety lab values that are not in the normal range is 

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provided in Appendix C. 
All women will have a urine pregnancy test performed during screening and a urine pregnancy 
test on the vaccination days. The results of the urine pregnancy test must be negative prior to 
enrollment in the study, and prior to each vaccination. 
For those laboratory tests run as part of a panel of tests, but not required by the protocol, the site 
is required to do the following:  
• Enter all non-required lab results into the database in the comments section of the eCRF.  
• When a result is abnormal per the site normal laboratory reference range, the result must be:  
 Entered in the comments section of the database including assessment for clinical 
significance by site PI,  
 An AE generated and followed until resolution if clinically significant based on 
assessment by the site PI,  
 If clinically significant, graded according to either the toxicity tables in Appendix C 
or if a grading scale is not available, the PI should use the guidance for AE grading.   
8.2.2 
Clinical Microbiology Evaluation 
For any participant who experiences any diarrhea within 3 days (72 hours) of receipt of any dose 
of study product will be requested to provide a stool specimen for stool culture. 
For any participant (irrespective of the route of administration) that experiences moderate or 
severe diarrhea within 28 days of receipt of any dose of study product will be requested to 
provide a stool specimen for stool culture.  If the participant experiences further episodes of 
moderate or severe diarrhea, there will be a maximum of one stool culture to be performed per 7 
day period.   
Upon notification of eligible diarrhea, study staff will instruct participants to come to the 
research clinic to collect a stool specimen or a study staff member will go to the household of the 
participant for the collection of a stool specimen. The stool will be cultured, using standard 
bacteriological methods, for the presence of: ETEC, Vibrio cholerae, Shigella sp., Salmonella 
sp., and Campylobacter sp. 
8.2.3 
Special Assays or Procedures 
Detailed procedures for the conduct of each of the immunology assays are described in the 
Manual of Procedures. 
The following immunology assays will be performed at icddr,b (Dhaka, Bangladesh): 

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Table 3: Assays to be performed at icddr,b 
Immunology Assay: 
Visit(s) collected at: 
Serum anti-dmLT IgG and IgA by ELISA   
Visits 1, 2, 3, 4, 5, 6, 7, and 8 
dmLT-specific IgG and IgA ASC assays    
Visits 1, 2, 3, 4, 5, and 6 
dmLT-specific IgG and IgA ALS assays    
Visits 1, 2, 3, 4, 5, and 6 
ASC homing studies 
Visits 2, 4, and 6 
Fecal total IgA and anti-dmLT IgA by ELISA Visits 1, 2, 3, 4, 5, 6 and 7 
Saliva total IgA and anti-dmLT IgA by 
ELISA 
Visits 1, 2, 3, 4, 5, 6 and 7 
dmLT-specific memory B cell responses    
Visits 1, 4, 6, 7, 8, and 9 
The following immunology assay will be performed at CVD (Baltimore, MD, U.S.): 
• 
Serum toxin (LT) neutralization assay 
• 
dmLT-specific effector and memory T cell responses, by CyTOF--including the 
characterization of: homing potential, cytokine/chemokine production profile, 
degranulation capacity, and activation potential 
o Collected at visits screening, 4, 6, 7, 8, and 9 
8.2.4 
Specimen Preparation, Handling, and Shipping 
 
All sample collection time are described in the Table 3 and Appendix A.  
8.2.4.1 
Instructions for Specimen Preparation, Handling, and Storage 
Instructions for specimen preparation, handling, and storage are described in the Manual of 
Procedures.  
8.2.4.2 
Specimen Shipment 
Instructions for specimen shipping are described in the Manual of Procedures. 
 

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9 
ASSESSMENT OF SAFETY 
9.1 
Specification of Safety Parameters 
The Investigator is responsible for reporting all AEs that are observed or reported during the 
study, regardless of their relationship to investigational product or their clinical significance. 
All events will be recorded on appropriate clinical report forms with notation of duration, 
severity, and outcome. 
Safety and tolerability will be assessed by the frequency and severity of: 
1. Study vaccine-related SAEs occurring from first vaccination through 6 months after the 
last study vaccination. 
2. Solicited AEs – reactogenicity events occurring within 7 days of each dose of vaccine: 
a. Systemic Reactions to include: feverishness (chills/shivering/sweating), fatigue 
(tiredness), malaise (general unwell feeling), myalgia (body aches, muscular 
pain), or headache, diarrhea, nausea, vomiting, or abdominal discomfort. Note: for 
the oral and SL routes of administration, diarrhea, nausea, vomiting, and 
abdominal discomfort will be evaluated as local reactions as described below. 
b. Systemic Reactions to also include the measurement of daily oral temperature 
c. Local Reactions 
i. For oral route of administration: irritation of the oral cavity or tongue, plus 
diarrhea, nausea, vomiting, and abdominal discomfort 
ii. For SL route of administration: irritation of the oral cavity or tongue, or 
facial nerve disturbance, plus diarrhea, nausea, vomiting, and abdominal 
discomfort 
iii. For ID route of administration: injection site pain, redness, swelling, 
bruising, itching, hypo/hyper pigmentation and induration, vesicles or 
hardened mass. 
3. Unsolicited AEs – study vaccine-related non-serious AEs from first vaccination through 
28 days after the last (third) dose of vaccine 

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4. Withdrawals and discontinuation of study vaccinations will be assessed as a part of 
determining tolerability  
9.2 
Methods and Timing for Assessing, Recording, and Analyzing Safety 
Parameters 
9.2.1 
Adverse Events 
Adverse Event (AE):  International Conference on Harmonization (ICH) E6 defines an AE as 
any untoward medical occurrence in a patient or clinical investigation participants administered a 
pharmaceutical product regardless of its causal relationship to the study treatment.  An AE can 
therefore be any unfavorable and unintended sign (including an abnormal laboratory finding), 
symptom, or disease temporally associated with the use of a medicinal (investigational) product.  
The occurrence of an AE may come to the attention of study personnel during study visits and 
interviews of a study recipient presenting for medical care, or upon review by a study monitor. 
AEs, including local and systemic (subjective and quantitative) reactions, not meeting the 
protocol-defined criteria for SAEs will be captured on the appropriate data collection form and 
eCRF.  Information to be collected for unsolicited non-serious AEs includes event description, 
date of onset, licensed study physician’s assessment of severity and relationship to study product 
and alternate etiology (if not related to study product) (assessed only by those with the training 
and authority to make a diagnosis and listed on the Form FDA 1572 as the site principal 
investigator or sub-investigator), date of resolution of the event, seriousness and outcome.  AEs 
occurring during the collection and reporting period will be documented appropriately regardless 
of relationship.  AEs will be followed through resolution. 
Any medical condition that is present at the time that the participant is screened will be 
considered as baseline and not reported as an AE.  However, if the severity of any pre-existing 
medical condition increases, it will be recorded as an AE. 
AEs must be graded for severity and assessed for relationship to study product (see definitions 
below).  Adverse events characterized as intermittent require documentation of onset and 
duration of each episode.  The start and stop date of each reported AE will be recorded on the 
appropriate data collection form and eCRF. 
FDA defines an AE as any untoward medical occurrence associated with the use of a drug in 
humans, whether or not considered drug related. 
Severity of Event:  AEs will be assessed by a licensed study physician listed on the Form FDA 
1572 as the site principal investigator or appropriate sub-investigator the “Laboratory and 

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Clinical Toxicity Grading Scales” in Appendix C.  For events not included in the protocol-
defined grading system, the following guidelines will be used to quantify severity: 
• 
Mild (Grade 1):  Events require minimal or no treatment and do not interfere with the 
participant’s daily activities. 
• 
Moderate (Grade 2):  Events result in a low level of inconvenience or concern with 
therapeutic measures.  Moderate events may cause some interference with functioning 
and daily activities. 
• 
Severe (Grade 3):  Events interrupt the participant’s usual daily activities and may require 
systemic drug therapy or other treatment.  Severe events are usually incapacitating. 
Relationship to Study Product:  The study physician’s assessment of an AE's relationship to 
study product is part of the documentation process, but it is not a factor in determining what is or 
is not reported in this study.  If there is any doubt as to whether a clinical observation is an AE, 
the event should be reported.  The relationship to study product must be assessed for AEs using 
the terms: related or not related.  In a clinical trial, the study product must always be suspect.  To 
help assess, the following guidelines are used: 
• Related – There is a reasonable possibility that the study product caused the adverse 
event.  Reasonable possibility means that there is evidence to suggest a causal 
relationship between the study product and the adverse event. 
• Not Related – There is not a reasonable possibility that the administration of the study 
product caused the event. 
9.2.2 
Solicited Events 
The Toxicity Grading Scales to be used to guide the grading of solicited AEs is in Appendix C. 
9.2.3 
Serious Adverse Events 
Serious Adverse Event (SAE):  An adverse event or suspected adverse reaction is considered 
“serious” if, in the view of either the site principal investigator or sponsor, it results in any of the 
following outcomes: 
• Death, 
• a life-threatening adverse event*, 
• inpatient hospitalization (defined by local practice as the study participant being present in 
the hospital greater than 24 hours) or prolongation of existing hospitalization, 

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• a persistent or significant incapacity or substantial disruption of the ability to conduct normal 
life functions, or 
• a congenital anomaly/birth defect. 
• Important medical events that may not result in death, be life-threatening, or require 
hospitalizations may be considered serious when, based upon appropriate medical judgment 
they may jeopardize the patient or participant 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. 
* Life-threatening adverse event.  An adverse event is considered “life-threatening” if, in the 
view of either the site principal investigator or sponsor, its occurrence places the patient or 
participant at immediate risk of death.  It does not include an adverse event that, had it occurred 
in a more severe form, might have caused death. 
SAEs will be: 
• 
Assessed for severity and relationship to study product and alternate etiology (if not 
related to study product) by a licensed study physician listed on the Form FDA 1572 as 
the site principal investigator or sub-investigator. 
• 
Recorded on the appropriate SAE forms and the eCRF. 
• 
Followed through resolution by a licensed study physician listed on the Form FDA 1572 
as the site principal investigator or sub-investigator. 
• 
Reviewed and evaluated by an Independent Safety Monitor (ISM), Independent Protocol 
Safety Team (IPST), both the DMID DSMB and local DSMB (periodic review unless 
related), DMID, and the IRB. 
9.2.4 
Procedures to be followed in the Event of Abnormal Laboratory Test Values or 
Abnormal Clinical Findings 
The site principal investigator or appropriate sub-investigator is responsible for recording all 
AE/SAEs that are observed or reported during this study, regardless of the relationship to study 
product.  AE/SAEs or abnormal clinical findings will be collected, assessed, documented, 
reported, and followed until resolved or considered stable. 

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9.3 
Reporting Procedures 
9.3.1 
Serious Adverse Events 
If in the opinion of a study physician Investigator the event meets the criteria of a SAE, the 
following procedures will occur: 
All SAEs will be: 
• 
recorded on the appropriate AE eCRF, DMID SAE form, and icddr,b SAE form 
• 
followed by a study physician until satisfactory resolution or until the PI or  
co-Investigator deems the event to be chronic or the participant to be stable as reviewed 
by a study physician 
Any AE that meets a protocol-defined serious criterion must be submitted immediately (within 
24 hours of site awareness) on the DMID SAE form to the DMID Pharmacovigilance Group at 
the following address: 
DMID Pharmacovigilance Group 
Clinical Research Operations and Management Support (CROMS) 
6500 Rock Spring Dr. Suite 650 
Bethesda, MD 20817, USA 
SAE Hot Line: 1-800-537-9979 (US) or 1-301-897-1709 (outside US) 
SAE FAX: 1-800-275-7619 (US) or 1-301-897-1710 (outside US) 
SAE Email Address: PVG@dmidcroms.com 
In addition to the SAE form, selected SAE data fields must also be entered into Advantage 
eClinicalSM.  Please see the protocol-specific MOP for details regarding this procedure. 
Other supporting documentation of the event may be requested by the DMID Pharmacovigilance 
Group and should be provided as soon as possible. 
The site will send a copy of the SAE report(s) to the ISM when they are provided to the DMID 
Pharmacovigilance Group.  The DMID Medical Monitor and DMID Clinical Project Manager 
will be notified of the SAE by the DMID Pharmacovigilance Group.  The DMID Medical 
Monitor will review and assess the SAE for regulatory reporting and potential impact on study 
participant safety and protocol conduct. 
At any time after completion of this study, if the site principal investigator or appropriate sub-
investigator becomes aware of an SAE that is suspected to be related to study product, the site 
principal investigator or appropriate sub-investigator will report the event to the DMID 
Pharmacovigilance Group. 

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In addition, such events will be reported to the local IRB/ERC in accordance with IRB/ERC 
policy. 
All SAEs will be followed until satisfactory resolution or until the PI or Co-Investigator deems 
the event to be chronic or the participant to be stable. 
9.3.2 
Regulatory Reporting for Studies Conducted Under DMID-Sponsored IND 
Following notification from the investigator, DMID, the IND sponsor, will report any suspected 
adverse reaction that is both serious and unexpected.  DMID will report an AE as a suspected 
adverse reaction only if there is evidence to suggest a causal relationship between the study 
product and the adverse event.  DMID will notify FDA and all participating investigators (i.e., all 
investigators to whom the sponsor is providing drug under its INDs or under any investigator’s 
IND) in an IND safety report of potential serious risks from clinical trials or any other source, as 
soon as possible, but in no case later than 15 calendar days after the sponsor determines that the 
information qualifies for reporting as specified in 21 CFR Part 312.32. DMID will also notify 
FDA of any unexpected fatal or life-threatening suspected adverse reaction as soon as possible 
but in no case later than 7 calendar days after the sponsor’s initial receipt of the information.  
Relevant follow-up information to an IND safety report will be submitted as soon as the 
information is available. Upon request from FDA, DMID will submit to FDA any additional data 
or information that the agency deems necessary, as soon as possible, but in no case later than 15 
calendar days after receiving the request. All serious events designated as “not related” to study 
product(s), will be reported to the FDA at least annually in a summary format. 
9.3.3 
Reporting of Pregnancy 
Reproductive animal toxicity studies have not been conducted with dmLT vaccine. Therefore, 
the risk to the unborn fetus or pregnant mother is unknown. Consequently, all women will be 
instructed to avoid pregnancy through the 28 days from receipt of the last dose of vaccine.  
Female participants must be non-pregnant at enrollment and prior to the receipt of each dose of 
vaccine.  All women presenting for screening will have urine pregnancy testing.  “Females of 
childbearing potential must agree to use an efficacious hormonal or barrier method of birth 
control from screening and through 28 days post last dose of vaccine. Abstinence is also 
acceptable.”  
If an eligible female participant has a positive urine pregnancy test at any time, she will not 
receive any further study treatment. Should a female participant become pregnant subsequent to 
Dose 1, the Investigator will obtain permission from the participant to follow the participant’s 
pregnancy. If permission is obtained, the Investigator should report to DMID the course of 
pregnancy including perinatal and neonatal outcome on the appropriate eCRF. 

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Although pregnancy is not a SAE, all pregnancies occurring during this study will be reported in 
the same time frame as SAEs. The Investigator will immediately notify the DMID MM and CPM 
about the pregnancy and complete a Pregnancy Report eCRF. If an SAE occurs during the 
pregnancy, the SAE will be reported on the appropriate SAE form and faxed to DMID-CROMS 
if appropriate. 
9.4 
Type and Duration of Follow-up of Subjects after Adverse Events 
All AEs and SAEs will be followed until resolved or considered stable. 
9.5 
Halting Rules  
Further dosing of any participant only for the route (oral, SL, ID) will be halted if any of the 
following criteria are met. The study will be halted, and all further vaccine administration will be 
halted for DSMB review/recommendation if any of the following criteria are met for each route 
of administration: 
• 
Any participant experiences vaccine-related SAE (e.g., excluding trauma or accident) 
from the time of the first study vaccination through the participant’s last study visit.  
• 
Any participant experiences an injection site necrosis within 7 days of vaccination. 
• 
Any participant experiences an anaphylactic reaction related to vaccine occurring within 
24 hours after vaccination.  
• 
Any participant experiences urticaria, laryngospasm, bronchospasm, or anaphylaxis 
determined to be related to vaccine and within 7 days of vaccination. 
• 
Any participant experiences facial nerve neuropathy, within 28 days of vaccination, 
confirmed by an investigator and determined to be related to vaccine. 
• 
Any participant experiences a death for any reason excluding trauma or accident. 
In addition, the study will be halted if two or more participants in the same cohort or three 
participants across all cohorts within a route of administration (who received at least one dose of 
study product) experience: 
• 
The same severe (Grade 3) study vaccine-related local solicited event, 
• 
The same severe (Grade 3) study vaccine-related systemic solicited event, 
• 
The same severe (Grade 3) study vaccine-related clinical laboratory test, 
• 
The same severe (Grade 3) study vaccine-related vital sign, or 
• 
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Exceptions to this would be: 
• 
If there are obvious and acceptable physiological explanation for a Grade 3 abnormality 
(example, grade 3 hematuria in menstruating females) 
Once a halting rule is met, further dosing of the respective route will cease, but follow-up visits 
may continue.  An ad hoc DMID DSMB meeting and local DSMB meeting will be convened to 
discuss the halting event.  If, following review by both the DMID DSMB and local DSMB, this 
is deemed acceptable to restart the study, the study will resume, upon DMID’s approval and 
authorization. 
9.6 
Safety Oversight 
There will be two independently functioning Data and Safety Monitoring Boards (DSMB) which 
will provide safety oversight for this study. 
9.6.1 
DMID DSMB 
The DMID DSMB is convened by authority of DMID and is advisory to DMID and the study 
team. The DMID DSMB must consist of at least three voting members including a biostatistician 
experienced in statistical methods for clinical trials and a clinician with relevant expertise. 
Selection of DMID DSMB members should include consideration of clinical trials experience, 
relevant expertise, prior DSMB service and absence of significant conflict of interest. DMID is 
responsible for deciding whether consultancies or the financial interests of the members 
materially affect their objectivity. Members will notify DMID promptly if a change occurs that 
may create a potential conflict of interest. 
The DMID DSMB will operate under the rules of a DMID-approved charter that will be 
reviewed and finalized following the organizational meeting of the DSMB.  The DMID DSMB 
Charter serves as the standard operating procedure and defines the primary responsibilities of the 
DSMB, its membership, the purpose and timing of its meetings, data to be reviewed, and 
procedures for ensuring confidentiality and proper communication.  The following times are the 
proposed scheduled meetings of the DMID DSMB: 
• 
First/Organizational DMID DSMB meeting –The first meeting of the DMID DSMB will 
be primarily organizational and to review the study protocol, safety data shells, and 
frequency of scheduled meetings. This meeting will occur prior to the initiation of study 
enrollment.  
Second Scheduled DMID DSMB meeting – to review the available safety data through 7 
days post-third dose of vaccine for cohorts A-F prior to progressing to cohorts G-I, 
and make a recommendation whether to proceed.   

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• 
Third Scheduled DMID DSMB meeting – to review to aggregate safety data through 
28 days post-third dose of vaccine for all nine cohorts (can be before database lock). 
• 
Final DMID DSMB meeting - 6 to 8 months after clinical database lock to review the 
cumulative unblinded safety and efficacy data for the study.  The data will be 
provided in a standard summary format.  The DSMB may be asked to provide 
recommendations in response to questions posed by DMID. 
The DMID DSMB may also be convened for an ad hoc meeting, an unplanned meeting that is 
called for a specific purpose such as when a study halting rule is met.  The meeting can be 
requested by any party with the responsibility of overseeing the trial (such as the PI, ISM, 
DSMB, DMID, industry collaborator).  In the case of an ad hoc meeting, the DMID DSMB may 
request special reports on an as-needed basis.  
If the study is discontinued, additional participants will not receive study vaccine. If the study is 
discontinued, follow-up visits for safety would continue. The DMID DSMB will be notified by 
DMID when the decision is made to progress to the next cohort. 
9.6.2 
DMID Independent Safety Monitor (ISM) 
The DMID ISM is a local physician with relevant expertise whose primary responsibility is to 
provide independent safety monitoring in a timely fashion.  The ISM will review SAEs in real 
time and other AEs as needed and provide an independent assessment to the DMID DSMB, 
when requested.  The local study team will identify a DMID ISM and back-up ISM with 
experience in infectious diseases or gastrointestinal diseases, in close proximity to the 
participating site, and have the authority to readily access study participant records. 
9.6.3 
Local DSMB 
The icddr,b has its own regulations and standards for safety oversight, which are independent 
from the DMID DSMB.  The Ethical Review Committee (ERC) of icddr,b will arrange for the 
formation of a local DSMB for this study. The local DSMB is an independent safety oversight 
body from the DMID DSMB. The membership of the local DSMB consists of at least 2 members 
from the ERC, as well as 1-2 members from icddr,b  who are not the study investigators (~4-5 
members); there may also be 1-2 external members (experts) invited to participate in the local 
DSMB review—thus there are typically 5-6 individuals participating in the local DSMB for a 
particular study.  The study site PI, together with the key investigators, is expected to formally 
present the study safety data directly to the local DSMB at specified time points during the 
conduct of the study.  The safety data will be generated from web-reports that are updated daily 
in the electronic data system. 

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The first anticipated time for a local DSMB review will be prior to initiation of the study and will 
be followed by meetings for presentation of safety results as soon as it becomes available after 
the completion of the 7 days follow up visits after the third dose of vaccine for each dosage and 
route of administration cohort (cohort A-I). Following each local DSMB meeting, a 
recommendation will be provided to proceed with the next cohort. Ad hoc local DSMB meetings 
will be convened whenever there are any unexpected safety events. Following completion of the 
study, a final DSMB meeting is held to update the committee on the results. 
9.6.4 
Independent Protocol Safety Team (IPST) 
The IPST will consist of two icddr,b physicians with relevant expertise in infectious and/or 
gastrointestinal diseases and whose primary responsibility is to provide independent safety 
monitoring in a timely fashion. The IPST members are located in close proximity to the 
participating clinical site and have the authority to readily access study participant records. The 
IPST will review AE and SAEs in real time during the study period and provide an independent 
assessment of AEs and unexpected events, as needed. The IPST are required to be present when 
the local icddr,b DSMB meets; they will also be available for the DMID DSMB, when 
requested. The IPST members may be the same individuals that serve the capacity of DMID ISM 
and back-up ISM. 
 

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10 
CLINICAL MONITORING 
10.1 
Site Monitoring Plan 
Site monitoring is conducted to ensure that human subject protection, study procedures, clinical 
laboratory, study intervention administration, and data collection processes are of high quality 
and meet sponsor, ICH E6, and other appropriate, regulatory guidelines and that the study is 
conducted in accordance with the protocol and sponsor standard operating procedures (SOPs). 
Site visits may be conducted by an authorized representative of DMID or other regulatory 
agencies to inspect study data, participants’ medical records, and CRFs in accordance with ICH 
guidelines, GCP, and the respective local and national government regulations and guidelines. 
The investigator will permit authorized representatives of DMID and the respective local and 
national health or regulatory authorities to inspect facilities and records relevant to this study if 
needed. The site PI will provide direct access to all trial-related sites, source data/documents, and 
reports for the purpose of monitoring and auditing by the sponsor, and inspection by local and 
regulatory authorities.  
DMID-designated clinical monitors will verify that the clinical trial is conducted and data are 
generated, documented (recorded), and reported in compliance with the protocol, GCP, and the 
applicable regulatory requirements. Clinical monitoring reports will be submitted to DMID. 
A Clinical Monitoring Plan (CMP) will be written and implemented by DMID and its monitoring 
contractor, ICON. 
ICON Clinical Research 
212 Church Road 
North Wales PA 19454, USA 
 

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11 
STATISTICAL CONSIDERATIONS 
11.1 
Study Hypotheses 
The study is not designed to test a specific hypothesis for the primary safety objectives, so no 
formal hypothesis testing is planned. Rather the study is designed to assess the safety and 
immune responses of a range of doses of dmLT when administered by the oral, SL, or ID route; 
in volunteers residing in an endemic country for ETEC disease.   
11.2 
Sample Size Considerations 
The sample size for each cohort was chosen based on the number of participants deemed 
appropriate for a Phase 1 study in which the vaccine has had limited experience by the different 
routes of administration in humans. We have limited the sample size to at least 11evaluable 
participants and up to 15 participants for each cohort (each of the nine cohorts A through I).  
The DMID DSMB is scheduled to review cumulative data after Cohorts A through F have 
completed safety data through 7 days post-third dose of vaccine. At which time each cohort is 
expected to have at least 10 evaluable vaccinees (10 per cohort). Among the 10 vaccinees per 
dose, the absence of a dose-limiting AE provides for an upper 95% confidence bound of 30.8%. 
The absence of a dose-limiting AE among 20 vaccinees per route (e.g., combining safety data 
from Cohort A and B) provides for an upper 95% confidence bound of 16.8%. If at the time of 
data review, there are no dose-limiting AEs among 60 vaccinees (e.g., combining safety data 
from Cohorts A through F), this would provide an upper 95% confidence bound of 6.0%. 
11.3 
Planned Interim Analyses 
11.3.1 
Safety Review 
There is no formal statistical interim analysis based on the safety data. The DMID DSMB will 
review all safety data through 7 days after the third dose of vaccine from Cohorts A through F, 
prior to making a recommendation to DMID on proceeding to Cohorts G, H, and I.  Data for 
review will have any outstanding queries resolved but the database will not be locked for this 
review. The DMID DSMB will advise DMID of its findings. If a halting rule is reached, more 
frequent meetings may be held. The DMID DSMB will be notified when the decision is made to 
progress to the next cohort. 

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A local DSMB will also review safety data and will provide a recommendation with enrollment 
and vaccination of the next successive dosage and route of administration cohort.   
11.3.2 
Immunogenicity Review 
The immunology data will not be a required element of the data for the DMID DSMB or local 
DSMB to review. If available, it may be incorporated into the interim safety review.  There is no 
formal statistical interim analysis based on the immunology data. 
11.4 
Final Analysis Plan 
The final analysis will be performed and clinical study report completed when all primary safety 
endpoint data and all secondary immunogenicity data are available.  
11.4.1 
Analysis Populations 
The Safety Analysis population will include all eligible participants who received at least one 
dose of study product. 
The modified-intention-to-treat (mITT) immunogenicity population includes all eligible 
participants who received at least one dose of study product and contributed both pre- and at 
least one post-vaccination samples for immunogenicity testing for which valid results were 
reported. 
The per protocol (PP) immunogenicity population includes all participants in the mITT subset 
with the following exclusions: 
• 
Data from all available visits for participants found to be ineligible at baseline 
• 
Data from all visits subsequent to major protocol deviations, such as: 
o Second or third vaccination not received 
o Second or third vaccination received out of window 
o Receipt of non-study vaccines during the timeframe prohibited by the protocol 
• 
Data from any visit that occurs substantially out of window   
In the case of miss-randomization, participants will be analyzed according to the study product 
actually received for all analysis populations. 

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11.4.2 
Safety Data 
Summaries and analysis of safety data will be presented for the Safety Analysis Population. 
Solicited AEs will be summarized by severity for each day after each study vaccination (Days 1-
7 post each study vaccination) and as the maximum severity over all 8 days. Additionally, 
solicited AEs will be analyzed by taking the most severe response over the follow-up period, 
dichotomizing into a binary variable (none versus mild, moderate, or severe) and using standard 
techniques, such as exact confidence intervals, to summarize the percentage of participants 
reporting each symptom, any local symptom, and any systemic symptom. Summaries of solicited 
AEs will be presented separately for each study vaccination as well as overall study vaccinations 
by group.  
Unsolicited AEs will be coded by Medical Dictionary for Regulatory Activities (MedDRA) for 
preferred term and system organ class. The number of SAEs is expected to be small in this study 
and will be reported by a detailed listing showing the event description, MedDRA coding, 
relevant dates (vaccinations and AEs), severity, relatedness, and outcome for each event. Non-
serious unsolicited AEs will be summarized as number and percentage of participants reporting 
at least one event in each MedDRA preferred term and SOC, cross tabulated by severity and 
relationship to study product. Additionally, the percentage of participants and exact 95% 
confidence intervals of AEs in aggregate and by MedDRA categories will be computed. 
Descriptive statistics for the actual values of clinical laboratory parameters and changes from 
baseline in clinical laboratory parameters will be presented for all measurements over time. If a 
participant has repeated laboratory values for a given time point, the value from the last 
evaluation will be used for analysis.   
The number and percentage of participants who terminate the study early (withdraw) or 
discontinued treatment will be included in the subject disposition summary table. The reason for 
early termination and discontinuation of treatment will be tabulated if the number of occurrences 
permits. All early terminations and discontinuations will be listed showing the category (“Early 
Termination” or “Treatment Discontinuation”), reason, and study day. 
11.4.3 
Immunogenicity Data  
Summaries and analysis of immunogenicity data will be presented for the mITT and PP 
populations. 
Immune responses will be summarized at each time point within each route by treatment, 
pooling participants who received placebo. Analyses will include number and percentage of 
participants with responses, as defined by each outcome measure. Descriptive summary statistics 

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will be provided for all assays and time points. Measurements that are titers or concentrations 
will be summarized by geometric means and 95% confidence intervals.  
 

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12 
SOURCE DOCUMENTS AND ACCESS TO SOURCE 
DATA/DOCUMENTS 
The site will maintain appropriate medical and research records for this trial, in compliance with 
ICH E6, Section 4.9 and regulatory and institutional requirements for the protection of 
confidentiality of participants.  The site will permit authorized representatives of the DMID, its 
designees, and appropriate regulatory agencies to examine (and when required by applicable law, 
to copy) clinical study records for the purposes of quality assurance reviews, audits, monitoring 
and evaluation of the study safety and progress.  These representatives will be permitted access 
to all source data, which include, but are not limited to, hospital records, clinical and office 
charts, laboratory notes, memoranda, evaluation checklists, pharmacy dispensing records, 
recorded data from automated instruments, copies or transcriptions certified after verification as 
being accurate and complete, microfiches, photographic negatives, digital photos, microfilm or 
magnetic media, x-rays, and participant files and records kept at the pharmacy, at the 
laboratories, and medico-technical departments involved in the clinical trial.  Source data are all 
information, original records of clinical findings, observations, or other activities in a clinical 
trial necessary for the reconstruction and evaluation of the trial. Data collection forms will be 
derived from the eCRFs and be provided by the Statistical and Data Coordinating Center 
(SDCC). 
 
 

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13 
QUALITY CONTROL AND QUALITY ASSURANCE 
Following a written DMID-accepted protocol-specific quality management plan, the 
investigational site is responsible for conducting routine quality assurance (QA) and quality 
control (QC) activities to internally monitor study progress and protocol compliance.  The PI will 
provide direct access to all source data/documents, and reports for the purpose of monitoring and 
auditing by the sponsor, and inspection by local and regulatory authorities. The PI will ensure all 
study personnel are appropriately trained, and applicable documentations are maintained on site.  
DMID-designated clinical monitors will verify the clinical trial is conducted and data are 
generated, documented (recorded), and reported in compliance with the protocol, GCP, and the 
applicable regulatory requirements.  Clinical monitoring reports will be submitted to DMID.  
The SDCC will implement quality control procedures beginning with the data entry system and 
generate data quality control checks that will be run on the database. Any missing data or data 
anomalies will be communicated to the site for clarification and resolution. 
 
 

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14 
ETHICS/PROTECTION OF HUMAN SUBJECTS 
Participants in this study will receive no assurance of benefit from their participation. However, 
participants will receive medical care or referral according to the local standard of care anytime 
they are sick while enrolled in the study. Those who receive the vaccine, but not those receiving 
the placebo, might obtain some protection from ETEC diarrhea. If the results of this research 
study show that the vaccine is safe and likely to be effective in preventing ETEC diarrhea, the 
vaccine will be further developed and thus there is a societal benefit of this study which may 
result in preventing ETEC infections.  
Nonetheless, the education and training of participants about the rationale, procedures, and risks 
is critical for the protection of human participants. Prospective participants must demonstrate 
their persistent interest verbally in the study and compliance with study requirements. There will 
be multiple opportunities for discussion and questions and answers during the recruiting and 
screening period and throughout the study period, including time with the investigators. 
14.1 
Ethical Standard 
The investigator will ensure that this study is conducted in full conformity with the principles set 
forth in The Belmont Report: Ethical Principles and Guidelines for the Protection of Human 
Subjects of Research of the US National Commission for the Protection of Human Subjects of 
Biomedical and Behavioral Research (April 18, 1979) and codified in 45 CFR Part 46, 21 CFR 
50 and 56 and/or the ICH E6; 62 Federal Regulations 25691 (1997). The study will also be 
conducted to conform with the ethical standards of Bangladesh, which is to include the Research 
Review Committee (RRC), Ethical Review Committee (ERC), and the icddr,b DSMB convened 
at the behest of icddr,b.   
14.2 
Institutional Review Board 
Prior to enrollment of participants into this trial, the approved protocol and informed consent 
forms, as well as any other participant recruitment materials, or any other materials provided to 
participants, will be reviewed and approved by the appropriate IRB listed on the institutions 
FWA. Should amendments to the protocol be required, the amendments will be written by the 
Sponsor and provided to the site principal investigator for submission to the IRB. Any 
amendments to the protocol and any materials provided to study participants will be approved by 
the following IRBs prior to being placed into use:  
 
 

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University of Maryland School of Medicine 
Human Research Protections Office 
Lexington Building 
620 W. Lexington Street, Second Floor 
Baltimore, MD 21201 
410-706-5037 
 
Icddr,b Ethical Review Committee 
 Contact-Mr. M.A. Salam Khan 
icddr,b, Coordination Committee Secretariat 
68, Shaheed Tajuddin Ahmed Sarani, Mohakhali, Dhaka-1212, Bangladesh 
Phone +88029886098 
 
14.3 
Informed Consent Process 
Before any study procedures are performed, participants must sign an informed consent form that 
complies with the requirements of 21 CFR Part 50 and 45 CFR 46 and the local IRB.  The site 
principal investigator will choose participants in accordance with the eligibility criteria detailed 
in Section 5.   
Participants for this study will be recruited from the Mirpur field site. Study staff will visit 
nearby households of Mirpur field clinic and have preliminary discussions with adults regarding 
the study and study procedures. During this process of recruitment, study staff will identify 
potential participants who are willing to hear more about the study and further invite them to 
come to our field clinic for a scheduled time of screening. Interested individuals will sign the 
informed consent forms (ICFs) before any protocol-specific screening procedures are conducted.  
The consent forms will be utilized by the field staff throughout the recruitment process.   
Study personnel may employ this type of recruitment effort prior to obtaining study consent if a 
patient-specific screening consent is on record or if the IRB has agreed that chart review is 
allowed without a fully executed screening consent.  In cases where there is not a patient-specific 
screening consent on record, site clinical staff may pre-screen via chart review and refer potential 
participants to the research staff. Research staff would obtain written consent per the standard 
informed consent process before conducting protocol-specific screening activities. 
Informed consent is an ongoing process that is initiated before the individual’s agreeing to 
participate in the study and continuing throughout the individual’s study participation.  Extensive 
discussion of risks and possible benefits of this therapy will be provided to the participants.  
Consent forms describing in detail the study interventions/products, study procedures, and risks 
are given to the participant and written documentation of informed consent is required before 
performing screening activities and enrolling participants on the study. 

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A delegated consent taker will explain every detail to the potential participant. Consent forms 
will be translated into Bengali and approved by the local Ethics Board and UMB IRB and the 
study participant will be asked to read and review the document.  Upon reviewing the document, 
the delegated consent taker or investigator will explain the research study to the participant and 
answer any questions that may arise.  The participants should have the opportunity to discuss the 
study with their surrogates or consider participation before agreeing to enroll in the study.  When 
the participant has voluntarily accepted all aspects of the study, the consent document will be 
signed by the participant. The participants will sign the informed consent document before any 
procedures are done specifically for the study.   
A literacy rate of approximately 40% is expected in the Mirpur study area. Bengali is the most 
commonly spoken language. So there is a chance that a number of illiterate participants will be 
enrolled.  For illiterate participants, an impartial witness will be present during all informed 
consent discussions between the participant and the delegated consent taker. A person who can 
only write his/her name but cannot read or write in Bangla will also be considered as illiterate. 
The Witness must be literate, adult and impartial (i.e., have no relation with study by any 
means).  The Witness will be present during the consent process with the potential participant 
and will help him/her to understand the study process, study procedures, risk-benefit, etc. The 
Witness will write the name of study participant on the ICF in the case of an illiterate participant. 
The Witness will also write his/her name and provide a signature on the appropriate section of 
ICF. Illiterate participants will need to give a thumbprint for their signature and the Witness will 
date the form next to the thumbprint. An impartial Witness will be made available to also 
confirm ongoing consent. 
The impartial witness can read, write and understand the ICF, whereas the delegated consent 
taker (a member of the study team) will explain the ICF and answer any questions to the study 
participant in the presence of the witness. 
DMID will provide the site principal investigator, in writing, any new information that 
significantly impacts the participants' risk of receiving the investigational product.  This new 
information will be communicated by the site principal investigator to participants who consent 
to participate in this trial in accordance with IRB requirements.  The informed consent document 
will be updated and participants will be re-consented per IRB requirements, if necessary. 
The participants may withdraw consent at any time throughout the course of the trial.  The verbal 
withdrawal of consent will be confirmed by the investigator and reasons for withdrawal if 
known, and whether the withdrawal is from the entire research study or just the primary 
interventional component will be documented. A copy of the informed consent document will be 
given to the participants for their records. The rights and welfare of the participants will be 
protected by emphasizing that the quality of their medical care will not be adversely affected if 
they decline to participate in this study. No individual group will be targeted for enrollment. 

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Every effort will be made to ensure that volunteers do not feel there is any secondary gain for 
themselves from participation.   
14.4 
Subject Confidentiality 
All information collected about participants will be kept confidential to the extent required by 
federal, state, and local law.   
Participant confidentiality is strictly held in trust by the participating investigators, their staff, the 
sponsor(s), and their agents. This confidentiality is extended to cover testing of biological 
samples in addition to the clinical information relating to participating participants.  The study 
protocol, documentation, data, and all other information generated will be held in strict 
confidence. No information concerning the study or the data will be released to any unauthorized 
third party without prior written approval of the sponsor.  All information provided by the 
Sponsor and all data and information generated by the site as part of the study (other than a 
participant’s medical records) will be kept confidential by the Investigator and other site staff. 
This information and data will not be used by the Investigator or other site personnel for any 
purpose other than conducting the study. These restrictions do not apply to: (1) information 
which becomes publicly available through no fault of the Investigator or site staff; (2) 
information which it is necessary to disclose in confidence to an IRB solely for the evaluation of 
the study; (3) information which it is necessary to disclose in order to provide appropriate 
medical care to a study participant; or (4) study results which may be published as described in 
Section 16.  
The study monitor or other authorized representatives of the sponsor and members of the ethics 
committee and data and safety monitoring committee may inspect all documents and records 
required to be maintained by the Investigator, including but not limited to, medical records 
(office, clinic, or hospital) and pharmacy records for the participants in this study. The FDA or 
local National Regulatory Authority (Directorate General of Drug Administration, DGDA) may 
also access participant records. The clinical study site will permit access to such records. 
14.5 
Future Use of Stored Specimens 
Aliquots of unused sera from immunogenicity samples of participants who consented to 
specimen future use will be stored at DMID CMS for an indefinite period of time, according to 
the approval policy of the local site’s IRB equivalent. Note: the local institution’s (icddr,b) 
Ethical Review Committee (ERC) requires for specimens which are stored outside of Bangladesh 
to have a limit of 5 years of storage, nonetheless requests to extend the storage for additional 5 
year increments is planned indefinitely. Unused Stool collected for future use microbiome 

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studies and unused residual stool and saliva specimens will be stored at DMID CMS for 
participants who consented to stool and saliva specimen future use, for at least 2 years. However, 
if the participant originally consents to future use and subsequently changes his/her decision, any 
data obtained prior to the withdrawal of consent may still be used for research. The withdrawal 
of future use consent must be documented; for illiterate participants, this can be completed by an 
investigator upon confirmation with the study participant. UMB, icddr,b, and investigators in 
other NIAID approved studies may use the archived specimens for future studies if approved by 
the clinical site IRB and DMID. Specimens will have a barcode label attached so that any 
information derived from these research studies will remain confidential. The PBMC will be 
isolated from the whole blood collected and cryopreserved for memory B cell and T cell assays. 
No human genetic testing will be done on these specimens.  Specimens will not be sold or used 
for production of any commercial product. 
An aliquot of 1-2 grams of stool from each participant as described in the Section 7 and appendix 
A will be collected and kept frozen at -80°C±15°C until used in the future for microbiome 
studies. 
 

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15 
DATA HANDLING AND RECORD KEEPING 
The Statistical and Data Coordinating Center at The Emmes Corporation will prepare source 
documents to match the Advantage eClinicalSM eCRF screens. These forms are posted to the 
DMID study web site. The Investigator is responsible for ensuring the accuracy, completeness, 
legibility, and timeliness of the data reported. All source documents will be completed in a neat, 
legible manner to ensure accurate interpretation of data. Black or blue ink will be used to ensure 
clarity of reproduced copies. When making changes or corrections, the original entry will be 
crossed out with a single line, and the change initialed and dated. No entry will be erased, 
overwritten, or covered with correction fluid or tape.  
Copies of the eCRF will be provided for use as source documents and maintained for recording 
data for each participant enrolled in the study. These will be maintained in binders for each 
participant, separated by visit for easy use. The study records will be stored in a locked office or 
in a password-protected secure database. The study records will be stored indefinitely. 
Data reported in the eCRF derived from source documents must be consistent with the source 
documents or the discrepancies must be explained 
15.1 
Data Management Responsibilities 
Data management will be the responsibility of The Emmes Corporation.  All source documents 
and laboratory reports will be reviewed for accuracy and completeness by the clinical team. AEs 
will be graded, assessed for severity and causality, and reviewed by the site PI. 
Data collection is the responsibility of the clinical trial staff at icddr,b under the supervision of 
the PI and with oversight from CVD. During the study, the investigators will maintain complete 
and accurate documentation for the study. 
15.2 
Data Capture Methods 
Clinical data (including AEs, concomitant medications, and solicited events data) will be entered 
into a 21 CFR Part 11-compliant internet data entry system provided by The Emmes 
Corporation. The data system includes password protection and internal quality checks, such as 
automatic range checks, to identify data that appear inconsistent, incomplete, or inaccurate. 
Clinical data will be entered directly from the data collection forms/source documents.  
15.3 
Types of Data 
Data for this study will include safety, clinical laboratory, and outcome measures (e.g., solicited 
events, immunogenicity). 

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15.4 
Timing/Reports  
Safety review reports will be prepared by Emmes before each DSMB review. After all 
participants have completed the study, the final report including safety and immunologic data 
will be prepared. 
15.5 
Study Records Retention 
Study documents will be retained for a minimum of 2 years after the last approval of a marketing 
application in an ICH region and until there are no pending or contemplated marketing 
applications in an ICH region or at least 2 years have elapsed since the formal discontinuation of 
clinical development of the investigational product and the FDA has been notified. These 
documents will be retained for a longer period, however, if required by local regulations. No 
records will be destroyed without the written consent of the sponsor. It is the responsibility of the 
sponsor to inform the investigator when these documents no longer need to be retained. Informed 
consent forms for future use will be maintained as long as the samples exist. 
15.6 
Protocol Deviations 
A protocol deviation is any noncompliance with the clinical trial protocol, GCP, or protocol-
specific MOP requirements.  The noncompliance may be either on the part of the participant, the 
site principal investigator, or the VTEU site personnel.  As a result of deviations, corrective 
actions are to be developed by the site and implemented promptly. 
These practices are consistent with ICH E6: 
4.5 
Compliance with Protocol, Sections 4.5.1, 4.5.2, and 4.5.3 
5.1 
Quality Assurance and Quality Control, Section 5.1.1 
5.20 
Noncompliance, Sections 5.20.1, and 5.20.2. 
It is the responsibility of the site principal investigator and personnel to use continuous vigilance 
to identify and report deviations within five working days of identification of the protocol 
deviation, or within five working days of the scheduled protocol-required activity.  All 
deviations must be promptly reported to DMID, via the DCC’s IDES. 
All protocol deviations, as defined above, must be addressed in study participant data collection 
forms.  A completed copy of the DMID Protocol Deviation Form must be maintained in the 
Regulatory File, as well as in the participant's chart.  Protocol deviations must be sent to the local 
IRB/IEC per their guidelines.  The site principal investigator and personnel are responsible for 
knowing and adhering to their IRB requirements. 
 

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16 
PUBLICATION POLICY 
All investigators funded by the National Institutes of Health (NIH) must submit, or have 
submitted for them, to the National Library of Medicine’s (NLM) PubMed Central an electronic 
version of their final, peer-reviewed manuscripts upon acceptance for publication, to be made 
publicly available no later than 12 months after the official date of publication. The NIH Public 
Access Policy ensures the public has access to the published results of NIH funded research. It 
requires investigators to submit final peer-reviewed journal manuscripts that arise from NIH 
funds to the digital archive PubMed Central upon acceptance for publication. Further, the policy 
stipulates that these papers must be accessible to the public on PubMed Central no later than 12 
months after publication. All publications resulting from this study shall be reviewed by DMID 
prior to submittal for publication. NIH contract support shall be acknowledged in all 
publications. (refer to NOT-OD-08-033; http://grants.nih.gov/grants/guide/notice-files/NOT-OD-
08-033.html) 
The International Committee of Medical Journal Editors (ICMJE) member journals have adopted 
a trials-registration policy as a condition for publication.  This policy requires that all clinical 
trials be registered in a public trials registry such as ClinicalTrials.gov, which is sponsored by the 
National Library of Medicine (NLM).  Other biomedical journals are considering adopting 
similar policies.  This trial will be registered in NLM in accordance with the new NLM 
requirements under the FDAAA. (refer to FDAA 801 requirements; 
https://www.clinicaltrials.gov/ct2/manage-recs/fdaaa)  
 
 

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17 
LITERATURE REFERENCES 
1. 
Kotloff KL, Nataro JP, Blackwelder WC, et al. Burden and aetiology of diarrhoeal 
disease in infants and young children in developing countries (the Global Enteric 
Multicenter Study, GEMS): a prospective, case-control study. Lancet 2013;382:209-22. 
2. 
Allen KP, Randolph MM, Fleckenstein JM. Importance of heat-labile enterotoxin in 
colonization of the adult mouse small intestine by human enterotoxigenic Escherichia 
coli strains. Infect Immun 2006;74:869-75. 
3. 
Berberov EM, Zhou Y, Francis DH, Scott MA, Kachman SD, Moxley RA. Relative 
importance of heat-labile enterotoxin in the causation of severe diarrheal disease in the 
gnotobiotic piglet model by a strain of enterotoxigenic Escherichia coli that produces 
multiple enterotoxins. Infect Immun 2004;72:3914-24. 
4. 
Glenn GM, Francis DH, Danielsen EM. Toxin-mediated effects on the innate mucosal 
defenses: implications for enteric vaccines. Infect Immun 2009;77:5206-15. 
5. 
Walker RI, Steele D, Aguado T. Analysis of strategies to successfully vaccinate infants in 
developing countries against enterotoxigenic E. coli (ETEC) disease. Vaccine 
2007;25:2545-66. 
6. 
Ahren CM, Svennerholm AM. Synergistic protective effect of antibodies against 
Escherichia coli enterotoxin and colonization factor antigens. Infect Immun 1982;38:74-
9. 
7. 
Zhang W, Sack DA. Progress and hurdles in the development of vaccines against 
enterotoxigenic Escherichia coli in humans. Expert Rev Vaccines 2012;11:677-94. 
8. 
Frech SA, Dupont HL, Bourgeois AL, et al. Use of a patch containing heat-labile toxin 
from Escherichia coli against travellers' diarrhoea: a phase II, randomised, double-blind, 
placebo-controlled field trial. Lancet 2008;371:2019-25. 
9. 
Ellingsworth LR. Transcutaneous immunization and the traveler's diarrhea vaccine 
system: A phase II pivotal efficacy study. In: Vaccines for Enteric Diseases. Cannes, 
France; 2011. 
10. 
Torrell JM, Aumatell CM, Ramos SM, Mestre LG, Salas CM. Reduction of travellers' 
diarrhoea by WC/rBS oral cholera vaccine in young, high-risk travellers. Vaccine 
2009;27:4074-7. 
11. 
Peltola H, Siitonen A, Kyronseppa H, et al. Prevention of travellers' diarrhoea by oral B-
subunit/whole-cell cholera vaccine. Lancet 1991;338:1285-9. 
12. 
Clemens JD, Sack DA, Harris JR, et al. Cross-protection by B subunit-whole cell cholera 
vaccine against diarrhea associated with heat-labile toxin-producing enterotoxigenic 
Escherichia coli: results of a large-scale field trial. J Infect Dis 1988;158:372-7. 
13. 
da Hora VP, Conceicao FR, Dellagostin OA, Doolan DL. Non-toxic derivatives of LT as 
potent adjuvants. Vaccine 2011;29:1538-44. 
14. 
Freytag LC, Clements JD. Mucosal adjuvants. Vaccine 2005;23:1804-13. 
15. 
Sack DA, Qadri F, Svennerholm AM. Determinants of Responses to Oral Vaccines in 
Developing Countries. Annales Nestle 2008;66:71-9. 

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16. 
Michetti P, Kreiss C, Kotloff KL, et al. Oral immunization with urease and Escherichia 
coli heat-labile enterotoxin is safe and immunogenic in Helicobacter pylori-infected 
adults. Gastroenterology 1999;116:804-12. 
17. 
Banerjee S, Medina-Fatimi A, Nichols R, et al. Safety and efficacy of low dose 
Escherichia coli enterotoxin adjuvant for urease based oral immunisation against 
Helicobacter pylori in healthy volunteers. Gut 2002;51:634-40. 
18. 
Dickinson BL, Clements JD. Dissociation of Escherichia coli heat-labile enterotoxin 
adjuvanticity from ADP-ribosyltransferase activity. Infect Immun 1995;63:1617-23. 
19. 
Oplinger M, Baqar S, Trofa A, et al. Safety and immunogenicity in volunteers of a new 
candidate oral mucosal adjuvant, LT(R192G). In: 37th Intersci Conf Antimicrob Agents 
Chemother Washington, MD: American Societ for Microbiology; 1997:193. 
20. 
Kotloff KL, Sztein MB, Wasserman SS, Losonsky GA, DiLorenzo SC, Walker RI. Safety 
and immunogenicity of oral inactivated whole-cell Helicobacter pylori vaccine with 
adjuvant among volunteers with or without subclinical infection. Infect Immun 
2001;69:3581-90. 
21. 
Tribble D, Baqar S, Faix D, et al. Modified Escherichia coli heat-labile toxin 
(LT[R192G]) adjuvant dose-range study of an oral, inactivated whole cell Campylobacter 
(CWC) vaccine in volunteers. In: Modern Vaccines, Adjuvants and Delivery System. 
Dublin, Ireland; 2004. 
22. 
Lapa JA, Sincock SA, Ananthakrishnan M, et al. Randomized clinical trial assessing the 
safety and immunogenicity of oral microencapsulated enterotoxigenic Escherichia coli 
surface antigen 6 with or without heat-labile enterotoxin with mutation R192G. Clin 
Vaccine Immunol 2008;15:1222-8. 
23. 
Norton EB, Lawson LB, Freytag LC, Clements JD. Characterization of a mutant 
Escherichia coli heat-labile toxin, LT(R192G/L211A), as a safe and effective oral 
adjuvant. Clin Vaccine Immunol 2011;18:546-51. 
24. 
Song JH, Nguyen HH, Cuburu N, et al. Sublingual vaccination with influenza virus 
protects mice against lethal viral infection. Proc Natl Acad Sci U S A 2008;105:1644-9. 
25. 
Cuburu N, Kweon MN, Song JH, et al. Sublingual immunization induces broad-based 
systemic and mucosal immune responses in mice. Vaccine 2007;25:8598-610. 
26. 
Amuguni H, Lee S, Kerstein K, et al. Sublingual immunization with an engineered 
Bacillus subtilis strain expressing tetanus toxin fragment C induces systemic and mucosal 
immune responses in piglets. Microbes Infect 2012;14:447-56. 
27. 
Amuguni JH, Lee S, Kerstein KO, et al. Sublingually administered Bacillus subtilis cells 
expressing tetanus toxin C fragment induce protective systemic and mucosal antibodies 
against tetanus toxin in mice. Vaccine 2011;29:4778-84. 
28. 
Kotloff KL, Wasserman SS, Losonsky GA, et al. Safety and immunogenicity of 
increasing doses of a Clostridium difficile toxoid vaccine administered to healthy adults. 
Infect Immun 2001;69:988-95. 
29. 
Lu YJ, Yadav P, Clements JD, et al. Options for inactivation, adjuvant, and route of 
topical administration of a killed, unencapsulated pneumococcal whole-cell vaccine. Clin 
Vaccine Immunol 2010;17:1005-12. 
30. 
Cuburu N, Kweon MN, Hervouet C, et al. Sublingual immunization with nonreplicating 
antigens induces antibody-forming cells and cytotoxic T cells in the female genital tract 

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mucosa and protects against genital papillomavirus infection. J Immunol 2009;183:7851-
9. 
31. 
Raghavan S, Ostberg AK, Flach CF, et al. Sublingual immunization protects against 
Helicobacter pylori infection and induces T and B cell responses in the stomach. Infect 
Immun 2010;78:4251-60. 
32. 
Holmgren J, Czerkinsky C. Mucosal immunity and vaccines. Nat Med 2005;11:S45-53. 
33. 
Mutsch M, Zhou W, Rhodes P, et al. Use of the inactivated intranasal influenza vaccine 
and the risk of Bell's palsy in Switzerland. N Engl J Med 2004;350:896-903. 
34. 
El-Kamary SS, Cohen MB, Bourgeois AL, et al. Safety and immunogenicity of a single 
oral dose of recombinant double mutant heat-labile toxin derived from enterotoxigenic 
Escherichia coli. Clin Vaccine Immunol 2013;20:1764-70. 
35. 
Lundgren A, Bourgeois L, Carlin N, et al. Safety and immunogenicity of an improved 
oral inactivated multivalent enterotoxigenic Escherichia coli (ETEC) vaccine 
administered alone and together with dmLT adjuvant in a double-blind, randomized, 
placebo-controlled Phase I study. Vaccine 2014;32:7077-84. 
36. 
Harro C, Sack DA, Chakraborty S, et al. Evaluation of a Lyophilized Formulation of 
Enterotoxigenic Escherichia coli (ETEC) ACE527 Vaccine Administered Alone or With 
a Double-Mutant E. coli Heat-Labile Toxin (dmLT) in Healthy Adult Subjects 
(NCT01739231). In: The Seventh International Conference on: Vaccines for Enteric 
Diseases. Bangkok, Thailand; 2013. 
37. 
Gutierrez R, Riddle M, Porter C, et al. Phase 1 Clinical Evaluation of Adhesin-based 
ETEC Vaccines. In: Vaccines for Enteric Diseases. Bangkok, Thailand; 2013. 
38. 
Harro C, Chakraborty S, Sack DA, et al. Live attenuated enterotoxigenic Escherichia coli 
(ETEC) vaccine candidate protects against virulent ETEC in a human ETEC challenge 
model. In: 8th Vaccine and ISV Congress. Philadelphia, PA; 2014. 
39. 
Lewis DJ, Huo Z, Barnett S, et al. Transient facial nerve paralysis (Bell's palsy) 
5following intranasal delivery of a genetically detoxified mutant of Escherichia coli heat 
labile toxin. PLoS One 2009;4:e6999. 
40. 
Couch RB. Nasal vaccination, Escherichia coli enterotoxin, and Bell's palsy. N Engl J 
Med 2004;350:860-1. 
41. 
van Ginkel FW, Jackson RJ, Yuki Y, McGhee JR. Cutting edge: the mucosal adjuvant 
cholera toxin redirects vaccine proteins into olfactory tissues. J Immunol 2000;165:4778-
82. 
 
 
 

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18 
SUPPLEMENTS/APPENDICES 
 
 

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APPENDIX A: SCHEDULE OF EVENTS 
Oral and Sublingual cohorts 
Study Visit (V) screen 
1 
2 
3 
4 
5 
6 
7 
8 
9 
Unscheduled 
visit, upon 
investigator 
judgement 
Early Termination10 
Study Day 
-7 to 
-4 
1 
8 
15 
22 
29 
36 
57 
114 
209 
Window 
 
 
±1d ±2d ±1d ±2d ±1d ±3d ±1w ±2w 
Study Week 
 
0 
1 
2 
3 
4 
5 
8 
16 
30 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
Confirm Ongoing Consent 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Eligibility Review 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Collection of demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
Medical History 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
[X] 
Concomitant Medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
[X] 
Physical Exam 
X 
X2 
X2 
X2 
X2 
X2 
X2 
X2 
X2 
 
X 
[X] 
Vitals Signs 
X3 
X3 
X3 
X3 
X3 
X3 
X3 
X3 
X3 
X3 
X 
[X] 
Urine pregnancy test4,9 
X 
X 
 
X 
 
X 
 
 
 
 
 
 
VACCINATION 
 
#1 
 
#2 
 
#3 
 
 
 
 
 
 
Pre-vaccination & Post-
vaccination assessment 
 
X5 
 
X5 
 
X5 
 
 
 
 
 
 
Memory Aid and thermometer 
(d)distribute, (c) collect Memory 
Aid 
 
d 
c 
d 
c 
D 
c 
 
 
 
 
[c]7 
Confirm date & time of next 
Visit 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Adverse Event (AE)/Serious AE 
review 
 
X 
X6 
X 
X6 
X 
X6 
X6 
X1 
X1 
X8 
X 
Screening stool 
X 
 
 
 
 
 
 
 
 
 
 
 
Saliva/Oral Fluid for Ab 
 
X 
X 
X 
X 
X 
X 
X 
 
 
Also may 
occur:  
 
Stool for ELISA Ab 
 
X 
X 
X 
X 
X 
X 
X 
 
 
 

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Stool for future use (Microbiome 
studies) 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Stool culture, 
as when 
indicated in 
section 8.2.2;  
Exam and 
Photo of local 
reactogenicity, 
as when 
indicated in 
section 8.1 
 
Clinical labs/chemistry 
2 mL 
 
 
 
 
 
2 mL 2 mL 
 
 
 
Blood-Serum for ELISA Ab & 
Neut. 
 
3 mL 2 mL 2 mL 2 mL 2 mL 2 mL 3 mL 2 mL 
 
 
Blood-PBMC for ASC 
 
2 mL 2 mL 2 mL 2 mL 2 mL 2 mL 
 
 
 
 
Blood-PBMC for ASC w/ 
homing 
 
 
2 mL 
 
2 mL 
 
2 mL 
 
 
 
 
Blood-PBMC for ALS 
 
2 mL 2 mL 2 mL 2 mL 2 mL 2 mL 
 
 
 
 
Blood-PBMC for Memory B  
assays 
 
8mL 
 
 
5 mL 
 
5 mL 5 mL 5 mL 5 mL 
 
Blood-PBMC for effector T cell 
assays 
13 mL 
 
 
 
8mL 
 
7 mL 7 mL 8 mL 
10 
mL 
 
Store plasma from PBMC 
 
X 
X 
 
X 
 
X 
X 
X 
X 
 
Total Blood Volume up to ~140 
mL/volunteer 
15 mL 
15 
mL 8 mL 6 mL 21 
mL 6 mL 22 
mL 
17 
mL 
15 
mL 
15 
mL 
 
10 mL 
[ ] Make every effort to collect at Early Termination visit.  If not collected, this will not result in a protocol deviation. 
1review for Serious Adverse Events only 
 
2Targeted physical examination, if indicated based on review of medical history 
 
3Vital signs: collect oral temperature, pulse, and blood pressure 
 
4Urine pregnancy test required for all women  
5Exam at end of 30 min observation period post-vaccination for SL and ID, at end of a 90 min observation period for Oral 
6Review to include assessments of:  1) injection site necrosis within 7 days of vaccination, 2) urticaria, laryngospasm, bronchospasm, or 
anaphylaxis determined to be related to vaccine and within 7 days of vaccination, 3) facial nerve neuropathy, confirmed by an investigator and 
determined to be related to vaccine. 
7Assess reactogenicity and review memory aid if early termination occurs within 7 days of any vaccination 
8 AE assessment to include (as applicable):  1) Assess reactogenicity (if within 7 days of last vaccination), unsolicited non-serious AE information 
(if within 28 days of last vaccination), or any SAE information (anytime within study) 2) Upon judgement of the clinical investigator, clinical 
laboratory assessments may be performed to evaluate an AE and medications (e.g analgesics) may be recommended to treat an AE. 
9Visits 1-7: Provide instructions to women participants to avoid pregnancy through the 28 days from receipt of the last dose of vaccine. 
10Early Termination Visit is to include: assess reactogenicity and review memory aid if early termination occurs within 7 days of any vaccination; 
assess for SAE during all early termination visits; obtain serum samples for laboratory safety assays if visit occurs <28 days after third vaccination. 
 
 

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Intradermal cohorts 
Study Visit (V) 
screen 
1 
2 
3 
4 
5 
6 
7 
8 
9 
Unschedule
d visit, 
upon 
investigator 
judgement 
Early 
Termination11 
Study Day 
-7 to -4 
1 
8 
22 
29 
43 
50 
71 
128 
223 
Window 
 
 
±1d 
±2d 
±1d 
±2d 
±1d 
±3d 
±1w 
±2w 
Study Week 
 
0 
1 
3 
4 
6 
7 
10 
18 
32 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
Confirm Ongoing Consent 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Eligibility Review 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Collection of demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
Medical History 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
[X] 
Concomitant Medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
[X] 
Physical Exam 
X 
X2 
X2 
X2 
X2 
X2 
X2 
X2 
X2 
 
X 
[X] 
Vitals Signs 
X3 
X3 
X3 
X3 
X3 
X3 
X3 
X3 
X3 
X3 
X 
[X] 
Urine pregnancy test4,10 
X 
X 
 
X 
 
X 
 
 
 
 
 
 
VACCINATION 
 
#1 
 
#2 
 
#3 
 
 
 
 
 
 
Pre-vaccination & Post-
vaccination assessment 
 
X6 
 
X6 
 
X6 
 
 
 
 
 
 
Memory Aid and thermometer 
(d)distribute, (c) collect Memory 
Aid 
 
d 
c 
d 
c 
d 
c 
 
 
 
 
[c]8 
Confirm date & time of next Visit 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Adverse Event (AE) / Serious AE 
review 
 
X 
X5,7 
X 
X5,7 
X 
X5,7 
X7 
X1 
X1 
X5,9 
X 
Screening stool 
X 
 
 
 
 
 
 
 
 
 
 
 
Saliva/Oral Fluid for Ab 
 
X 
X 
X 
X 
X 
X 
X 
 
 
Also may 
occur:  
Stool 
culture, 
as when 
indicated 
in section 
8.2.2;  
 
Stool for ELISA Ab 
 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Stool for future use (Microbiome 
studies) 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Clinical labs/chemistry 
2 mL 
 
 
 
 
 
2 mL 2 mL 
 
 
 
Blood-Serum for ELISA Ab & 
Neut. 
 
3 mL 2 mL 2 mL 2 mL 2 mL 2 mL 3 mL 2 mL 
 
 
Blood-PBMC for ASC 
 
2 mL 2 mL 2 mL 2 mL 2 mL 2 mL 
 
 
 
 

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106 
Blood-PBMC for ASC w/ homing 
 
 
2 mL 
 
2 mL 
 
2 mL 
 
 
 
Exam and 
Photo of 
local 
reactogen
icity, as 
when 
indicated 
in section 
8.1 
 
Blood-PBMC for ALS 
 
2 mL 2 mL 2 mL 2 mL 2 mL 2 mL 
 
 
 
 
Blood-PBMC for Memory B  
assays 
 
8mL 
 
 
5 mL 
 
5 mL 5 mL 5 mL 5 mL 
 
Blood-PBMC for effector T cell 
assays 
13 mL 
 
 
 
8 mL 
 
7 mL 7 mL 8 mL 10 mL 
 
Store plasma from PBMC 
 
X 
X 
 
X 
 
X 
X 
X 
X 
 
Total Blood Volume up to ~140 
mL/volunteer 
15 mL 
15 mL 8 mL 6 mL 21 mL 6 mL 22 mL 17 mL 15 mL 15 mL 
 
10 mL 
[ ] Make every effort to collect at Early Termination visit.  If not collected, this will not result in a protocol deviation. 
1review for Serious Adverse Events only 
 
2Targeted physical examination, if indicated based on review of medical history 
3Vital signs: collect oral temperature, pulse, and blood pressure 
 
4Urine pregnancy test required for all women  
5For grade 3 local site reactions, a photo should be taken on a daily basis until the injection site reaction is deemed stable or returns back to a grade 
2 or lower severity, if the event occurs within 7 days of an intradermal dose administration 
6Exam at end of 30 min observation period post-vaccination 
7Review to include assessments of:  1) injection site necrosis within 7 days of vaccination, 2) urticaria, laryngospasm, bronchospasm, or 
anaphylaxis determined to be related to vaccine and within 7 days of vaccination, 3) facial nerve neuropathy, confirmed by an investigator and 
determined to be related to vaccine. 
8 Assess reactogenicity and review memory aid if early termination occurs within 7 days of any vaccination 
9AE assessment to include (as applicable):  1) Assess reactogenicity (if within 7 days of last vaccination), unsolicited non-serious AE information 
(if within 28 days of last vaccination), or any SAE information (anytime within study) 2) Upon judgement of the clinical investigator, clinical 
laboratory assessments may be performed to evaluate an AE and medications (e.g analgesics) may be recommended to treat an AE. 
10Visits 1-7: Provide instructions to women participants to avoid pregnancy through the 28 days from receipt of the last dose of vaccine. 
11Early Termination Visit is to include: assess reactogenicity and review memory aid if early termination occurs within 7 days of any vaccination; 
assess for SAE during all early termination visits; obtain serum samples for laboratory safety assays if visit occurs <28 days after third vaccination. 

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APPENDIX B: ACCEPTABLE SCREENING LAB VALUES 
Test 
Reference Range 
Acceptable Range 
Units 
White Blood Cell count 
(WBC) 
4.0-11.0 
4-11 
cells x109 /Liter 
Absolute Neutrophil Count 
(ANC) 
2.0-7.5 
2-7.5 
cells x109 /Liter 
Hemoglobin (Hg) – Female 
11.5-16.5 
9.5-16.5 
gm/dL 
Hemoglobin (Hg) – Male 
12.5-17.5 
11.0-17.5 
gm/dL 
Platelets 
150-450 
150-450 
cells x 109 
/Liter 
Creatinine, Female 
44-97 
≤97 
µmol/Liter 
Creatinine, Male 
53-106 
≤106 
µmol/Liter 
Albumin 
34.0-50.0 
≥34 
g/L 
Alanine Aminotransferase 
(ALT), Female 
0.01 - 31.0 
≤31 
U/L 
Alanine Aminotransferase 
(ALT), Male 
0.01 - 41.0 
≤41 
U/L 
Hepatitis B surface antigen 
(HBsAg) 
Non Reactive 
Non Reactive 
n/a 
HCV antibody 
Non Reactive 
Non Reactive 
n/a 
Total Bilirubin 
5.0 - 21.0 
5.0 - 21.0 
µmol/Liter 
 
 
 

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APPENDIX C: LABORATORY AND CLINICAL TOXICITY 
GRADING SCALES 
 
Solicited Lab AEs 
 
Grade 1 – Mild 
 
Grade 2 – 
Moderate 
 
Grade 3 - Severe 
WBC, Decreased  
(cells x109 /Liter) 
2.0 – 3.9 
1.5 – 1.9 
≤1.4 
WBC, Increased  
(cells x109 /Liter) 
11.1 – 15.0 
15.1 – 20.0 
≥20.1 
ANC, Decreased 
 (cells x109 /Liter) 
1.0 – 1.9 
0.75 – 0.99 
≤0.74 
Hemoglobin female 
(gm/dL) 
9.0- 9.4 
8.0 - 8.9 
6.5 – 7.9 
Hemoglobin, male  
(gm/dL) 
10.0 - 10.9 
9.0 – 9.9 
7.0 – 8.9 
Platelets   
(cells x 109 /Liter) 
100 – 149 
50 – 99 
≤49 
Creatinine  
(µmol/Liter)  
1.1 – 1.3 x ULN 
>1.3 – 1.8 x ULN 
>1.8 – 3.4 x ULN 
ALT (SGPT) 
(U/L) 
1.25 – 2.5 x ULN 
>2.5 – 5.0 x ULN 
>5.0 – 10.0 x ULN 
Albumin  
(g/dL)  
2.8 - 3.1  
2.5 - 2.7  
< 2.5  
Total Bilirubin 
1.1 to <1.6 x ULN 
1.6 to <2.6 x ULN 
≥2.6 x ULN 
ULN = upper limit of normal, from the laboratory reference range 
Clinical Sign 
Normal 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Temperature, °C 
35-37.9 
38.0 to 38.4 
38.5 to 38.9 
≥39.0 
Tachycardia, bpm 
≤100 
101 to 115 
116 to 130 
>130 
Bradycardia, bpm 
≥50 
45 to 49 
40 to 44 
<40 
Hypertension, systolic, 
mmHg 
≤140 
141 to 150 
151 to 155 
>155 

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Clinical Sign 
Normal 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Hypotension, systolic, 
mmHg 
≥90 
85-89 
80-84 
<80 
Hypertension, diastolic, 
mmHg 
≤90 
91-95 
96-100 
>100 
Hypotension, diastolic, 
mmHg 
≥60 
45-59 
35-44 
<35 
Feverishness 
(Chills/Shivering/Sweating) 
none 
Mild, but no 
interference 
with function 
Some 
interference 
with daily 
activity 
Significant 
interference, 
prevents daily 
activity 
Fatigue 
(Tiredness) 
Normal 
activity 
reduced 
slightly 
Normal 
activity 
decreased 25-
50% 
Unable to 
perform daily 
activity 
Urgent care 
visit or 
hospitalization 
Malaise 
(General Unwell Feeling) 
No 
interference 
with daily 
activity 
Some 
interference 
with daily 
activity 
Significant 
interference 
with daily 
activity 
Urgent care 
visit or 
hospitalization 
Myalgia 
(Body/Muscle Aches) 
Mild, but no 
interference 
with function 
Moderate, 
interferes 
with 
function, but 
not with daily 
activity 
Severe, 
interferes 
with daily 
activity 
Severe and 
disabling, 
unable to 
perform daily 
activity 
Headache 
Mild and 
brief or easily 
tolerated 
Moderate 
with limited 
effect to daily 
activity 
Severe and 
prevents 
daily activity 
Urgent care 
visit or 
hospitalization 
Allergic reaction 
Pruritus, no 
rash 
Localized 
urticaria 
Generalized 
urticarial, 
angioedema 
Anaphylaxis 
Diarrhea 
Normal or 1-
2 loose stool 
per 24 hr 
3 loose stools 
per 24 hr 
4-7 loose 
stools per 24 
hr 
8 or more 
loose/watery 
stools per 24 hr 
or requires 
outpatient IV 
hydration, ED 
visit or 
hospitalization 
for diarrhea 

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Clinical Sign 
Normal 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Nausea 
none 
Mild, but no 
interference 
with activity 
or intake 
Moderate, 
interferes 
with activity 
or intake 
Severe, unable 
to perform 
activity or no 
intake 
Vomiting 
none 
1-2 episodes  
per 24 hr. 
3-4 episodes  
per 24 hr. 
≥5 episodes  
per 24 hr. 
Abdominal Discomfort 
No 
interference 
with daily 
activity 
Some 
interference 
with daily 
activity 
Significant 
interference 
with daily 
activity 
Urgent care 
visit or 
hospitalization 
Irritation of Oral Cavity 
(Mouth Pain/Sores) 
none 
Mild or 
transient, no 
limitation of 
oral intake 
Moderate, 
slight 
interference 
with oral 
intake  
Severe, unable 
to tolerate oral 
intake 
Facial Nerve Disturbance 
none 
Mild or 
transient, no 
limitation 
Moderate 
disturbance 
but no overt 
facial nerve 
palsy 
Severe, obvious 
facial nerve 
palsy 
Injection Site Pain 
none 
Aware of 
pain but no 
interference 
with activity 
Slight 
interference 
with activity, 
may require 
analgesic 
Pain that 
prevents daily 
activity 
Injection Site Redness 
none 
Less than 5x5 
cm 
Greater than 
5x5 cm but 
less than 9x9 
cm 
Greater than 
9x9 cm 
Injection Site Swelling 
none 
Less than 5x5 
cm 
Greater than 
5x5 cm but 
less than 9x9 
cm 
Greater than 
9x9 cm or 
ulceration or 
necrosis 
Injection Site Bruising 
(Ecchymosis) 
none 
Less than 5x5 
cm 
Greater than 
5x5 cm but 
less than 9x9 
cm 
Greater than 
9x9 cm 
Injection Site Itching 
none 
Aware of 
sensation but 
no 
Slight 
interference 
with activity, 
Itching that 
prevents daily 
activity 

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Clinical Sign 
Normal 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
interference 
with activity 
may require 
medication 
Injection Site Induration 
(limited to the dermal 
layer) 
none 
Less than 5x5 
cm 
Greater than 
5x5 cm but 
less than 9x9 
cm 
Greater than 
9x9 cm 
Injection Site 
Hypo/Hyperpigmentation 
none 
Less than 1x1 
cm 
Greater than 
1x1 cm but 
less than 5x5 
cm 
Greater than 
5x5 cm 
Injection Site Vesicles 
none 
≤5 vesicles 
and localized 
to injection 
site 
>5 vesicles 
and localized 
to injection 
site 
Any number of 
vesicles which 
are generalized 
in distribution 
Injection Site Hardened 
Mass (deeper than dermal 
layer) 
none 
Less than 1x1 
cm 
Greater than 
1x1 cm but 
less than 5x5 
cm 
Greater than 
5x5 cm 
Other illness 
No 
interference 
with daily 
activity 
Some 
interference 
with daily 
activity 
Significant 
interference 
with daily 
activity 
Urgent care 
visit or 
hospitalization 
bpm = beats per minute