Phase 1 Study of the Safety, Tolerability, and Immunogenicity of Oral Doses of CVD 1208S-122, a Prototype Attenuated Shigella Flexneri 2a Live Vector Expressing Enterotoxigenic Escherichia Coli Antigens
Phase: PHASE1
Conditions: Shigella Infection, Enterotoxigenic Escherichia Coli Infection
Sponsor: University of Maryland, Baltimore

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Shigella CVD 31000 
 
 
Phase 1 Study of the Safety, Tolerability, and Immunogenicity of Oral Doses 
of CVD 1208S-122, a Prototype Attenuated Shigella flexneri 2a Live Vector 
Expressing Enterotoxigenic Escherichia coli antigens 
Protocol Shigella CVD 31000 
Sponsored by: 
University of Maryland, Baltimore (UMB) 
 
 
Funded by: 
National Institute of Allergy and Infectious Diseases (NIAID) 
Division of Microbiology and Infectious Diseases (DMID) 
 
DMID Funding Mechanism: U01-AI143493 
 
 
Principal Investigator: Wilbur H. Chen, MD, MS 
Laboratory Co-Principal Investigator: Eileen M. Barry, PhD 
 
 
 
 
 
 
Version Number: 11.0 
 
06 December  2023 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
NCT04634513

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Statement of Compliance 
The study will be carried out in accordance with Good Clinical Practice (GCP) as required by the 
following: 
• United States 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) 
• The Health Insurance Portability and Accountability Act (HIPAA) Privacy Rule-Final 
Modification (45 CFR Parts 160 and 164) 
• The local laws and regulations, which prevail over the University of Maryland, Baltimore 
• NIH Clinical Terms of Award 
Compliance with these standards provides public assurance that the rights, safety, and well-being 
of study subjects 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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INVESTIGATOR SIGNATURE PAGE 
The signature below constitutes the approval of this protocol and the attachments; it provides the 
necessary assurances that this trial will be conducted according to all stipulations of the protocol, 
including all statements regarding confidentiality, and according to local legal and regulatory 
requirements and applicable US federal regulations and ICH guidelines. 
 
I will provide copies of the protocol and all pertinent information to all individuals responsible to 
me who assist in the conduct of this study. I will discuss this material with them to ensure that 
they are fully informed regarding the product and the conduct of the study. 
I agree that all information pertaining to the study, including protocols, electronic case report 
forms (eCRFs), and verbal and written consent information will be kept strictly confidential. 
Distribution of such information or information on the conduct, progress, or results of the study 
will be restricted to the clinical personnel involved with the conduct of the study, members of the 
Institutional Review Board/Independent Ethics Committee (IRB/IEC), and/or regulatory 
authorities. 
 
 
Principal Investigator: 
 
Name / Title (Print) 
Signature: 
 
Date: 
 

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Table of Contents 
Page 
STATEMENT OF COMPLIANCE ........................................................................................ I 
INVESTIGATOR SIGNATURE PAGE ................................................................................ II 
LIST OF ABBREVIATIONS ................................................................................................. V 
PROTOCOL SUMMARY .................................................................................................... VI 
1 
KEY PERSONNEL ....................................................................................................... 11 
2 
BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE ................... 12 
2.1 
Background Information ..................................................................................... 12 
2.1.1 
Live, Attenuated Shigella vector ......................................................................... 13 
2.1.2 
Pre-clinical studies of Shigella vector expressing ETEC antigens ....................... 13 
2.1.3 
Clinical studies of CVD 1208S-122 .................................................................... 15 
2.2 
Rationale ............................................................................................................... 15 
2.3 
Potential Risks and Benefits ................................................................................ 16 
2.3.1 
Potential Risks ..................................................................................................... 16 
2.3.2 
Known Potential Benefits .................................................................................... 18 
3 
STUDY DESIGN ........................................................................................................... 18 
4 
STUDY POPULATION ................................................................................................ 19 
4.1 
Selection of the Study Population ........................................................................ 19 
4.2 
Inclusion/Exclusion Criteria ................................................................................ 19 
4.3 
Enrollment and Randomization Procedures ...................................................... 22 
4.4 
Withdrawal from Study ....................................................................................... 23 
4.5 
Discontinuation from further vaccination (Cohort 4) ........................................ 24 
5 
STUDY PRODUCT ....................................................................................................... 24 
5.1 
Study Vaccine, CVD 1208S-122 ........................................................................... 24 
5.1.1 
Preparation of Vaccine ........................................................................................ 24 
5.1.2 
Preparation of Placebo ......................................................................................... 25 
5.2 
Study Product Accountability .............................................................................. 25 
5.3 
Blinding of the Study Product ............................................................................. 25 
6 
STUDY PROCEDURES/EVALUATIONS ................................................................. 26 
6.1 
Study Procedures .................................................................................................. 26 
6.2 
Laboratory Evaluations ....................................................................................... 30 
6.2.1 
Laboratory Evaluations/Assays ........................................................................... 30 
6.2.2 
Special Assays or Procedures .............................................................................. 31 
6.2.3 
Specimen Collection, Preparation, Handling and Shipping ................................. 35 
7 
STUDY SCHEDULE .................................................................................................... 35 
7.1 
Screening ............................................................................................................... 35 

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7.2 
For Cohorts 1 – 3, Inpatient Containment Period ............................................. 37 
7.2.1 
Acclimatization (Days -2 and -1, one-two days prior to vaccination,) ................ 37 
7.2.2 
Enrollment/Vaccination Day (Day 1) .................................................................. 38 
7.2.3 
Post-Vaccination Observation Period (Day 1 through discharge) ....................... 38 
7.2.4 
Discharge (~Day 5) ............................................................................................. 38 
7.3 
For Cohort 4, Vaccination Visits for Dose #1 and Dose #2 (Day 1 and Day 
29 ±3) ..................................................................................................................... 39 
7.4 
Outpatient Clinic Visits ........................................................................................ 39 
7.4.1 
For Cohorts 1 – 3, Day 8 (+ 1), Day 29 (± 3), Day 43 (± 3), Day 181 (± 7) ....... 39 
7.4.2 
For Cohort 4, Day 3 (+ 1), Day 8 (+ 1), Day 31 (+ 1), Day 36 (+ 1), Day 57 (± 
3) , Day 210 (± 7) ................................................................................................. 40 
7.5 
Unscheduled Visit(s) or Early Termination Visits, if applicable....................... 40 
7.6 
COVID-19 related considerations ....................................................................... 41 
8 
SAFETY ASSESSMENT AND REPORTING ............................................................ 41 
8.1 
Definition of Adverse Event (AE) ........................................................................ 41 
8.1.1 
Grading of Severity of an AE .............................................................................. 42 
8.2 
Relationship to Study Product ............................................................................. 42 
8.3 
Definition of Serious Adverse Event (SAE) ........................................................ 43 
8.4 
Reporting Procedures........................................................................................... 43 
8.4.1 
SAE Detection and Reporting ............................................................................. 43 
8.4.2 
Regulatory Reporting .......................................................................................... 44 
8.4.3 
Reporting of Pregnancy ....................................................................................... 44 
8.5 
Halting Rules ........................................................................................................ 44 
8.6 
Safety Oversight .................................................................................................... 45 
9 
CLINICAL MONITORING STRUCTURE ............................................................... 45 
9.1 
Site Monitoring Plan ............................................................................................. 46 
10 
STATISTICAL CONSIDERATIONS ......................................................................... 46 
10.1 
Study Outcome Measures .................................................................................... 46 
10.2 
Sample Size Considerations ................................................................................. 46 
10.3 
Statistical Analysis Plan ....................................................................................... 47 
10.4 
General Statistical Principles ............................................................................... 49 
10.5 
Go/No-Go Milestones ........................................................................................... 50 
11 
ACCESS TO SOURCE DATA/DOCUMENTS .......................................................... 50 
12 
QUALITY CONTROL AND QUALITY ASSURANCE ........................................... 51 
13 
ETHICS/PROTECTION OF HUMAN SUBJECTS .................................................. 52 
13.1 
Ethical Standard ................................................................................................... 52 
13.2 
Institutional Review Board .................................................................................. 52 
13.3 
Informed Consent Process ................................................................................... 52 

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13.4 
Exclusion of Women, Minorities, and Children (Special Populations) ............53 
13.5 
Subject Confidentiality .........................................................................................53 
13.6 
Future Use of Stored Specimens ..........................................................................53 
14 
DATA HANDLING AND RECORD KEEPING ........................................................53 
15 
PUBLICATION POLICY ............................................................................................54 
16 
LITERATURE REFERENCES ...................................................................................55 
APPENDIX A: STUDY SCHEDULE OF EVENTS ............................................................58 
APPENDIX B: ACCEPTABLE CLINICAL LABORATORY VALUES FOR 
ELIGIBILITY ...............................................................................................................60 
APPENDIX C: GRADING SCALES FOR CLINICAL SAFETY LABORATORY 
TOXICITY 61 
APPENDIX D: REACTOGENICITY & CLINICAL TOXICITY GRADING .................62 

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List of Abbreviations 
 
AE 
Adverse Event 
AESI 
AE of Special Interest 
ALT 
Alanine aminotransferase 
ALS 
Antibody in Lymphocyte Supernatant 
ANC 
Absolute Neutrophil Count 
ASC 
Antibody Secreting Cell 
BGS 
Buffered Glycerol Saline 
BRC 
Baby Rabbit Complement 
CFA/I 
Colonization Factor Antigen 1 
CFR 
Code of Federal Regulations 
cfu 
Colony Forming Units 
CIOMS 
Council for International Organizations of Medical Sciences 
CRF 
Case Report Form 
CVD 
Center for Vaccine Development and Global Health 
DMID 
Division of Microbiology and Infectious Diseases, NIAID, NIH, DHHS 
DSMB 
Data and Safety Monitoring Board 
ETEC 
Enterotoxigenic Escherichia coli 
FA 
Full Analysis (population) 
FDA 
Food and Drug Administration 
FWA 
Federal-Wide Assurance 
GCP 
Good Clinical Practice 
GN 
Gram-negative 
Hg 
Hemoglobin 
HLA 
Human Leukocyte Antigen 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonization 
IEC 
Independent or Institutional Ethics Committee 
IgA 
Immunoglobulin A 
IgG 
Immunoglobulin G 
IND 
Investigational New Drug 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
JAMA 
Journal of the American Medical Association 
LT 
Heat-Labile Enterotoxin 
LThA2B 
B and A2 subunits of human heat-labile enterotoxin 
MOP 
Manual of Procedures 
N 
Number (typically refers to subjects) 
NEJM 
New England Journal of Medicine 
NIAID 
National Institute of Allergy and Infectious Diseases, NIH, DHHS 
NIH 
National Institutes of Health 
OHRP 
Office for Human Research Protections 
OPKA 
Opsonophagocytic Killing Antibody 

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List of Abbreviations 
 
PBMC 
Peripheral Blood Mononuclear Cell 
PI 
Principal Investigator 
PP 
Per Protocol (population) 
RBC 
Red Blood Cell 
SAE 
Serious Adverse Event 
SBA 
Serum Bactericidal Antibody 
SMC 
Safety Monitoring Committee 
SOP 
Standard Operating Procedure 
ST 
Heat-Stable Enterotoxin 
UMB 
University of Maryland, Baltimore 
UMMC 
University of Maryland Medical Center 
UMSOM 
University of Maryland School of Medicine 
WBC 
White Blood Cell 
WHO 
World Health Organization 

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Protocol Summary 
 
Title: 
Phase 1 Study of the Safety, Tolerability, and Immunogenicity of Oral 
Doses of CVD 1208S-122, a Prototype Attenuated Shigella flexneri 2a 
Live Vector Expressing Enterotoxigenic Escherichia coli antigens 
 
Population: 
A total of 54 healthy adults age 18 – 49 years from the Baltimore- 
Washington metropolitan area will be enrolled 
 
Number of Sites: 
Single site 
 
Study Duration: 
2.5 years 
 
Subject Duration: 
6 – 7 months, not inclusive of the screening period of up to 60 days 
 
Study Products: Vaccine: CVD 1208S-122 is a prototype Shigella - Enterotoxigenic Escherichia 
coli (ETEC) live vector vaccine strain, consisting of an attenuated S. flexneri 
strain CVD 1208S harboring deletion mutations in guaBA, sen and set and 
that has chromosomally integrated genes which express colonization factor 
antigen I (CFA/I) and the B subunit of ETEC heat-labile enterotoxin. 
Placebo: Sodium bicarbonate buffer solution with corn starch USP. 
 
Objectives: 
Primary Objectives: 
1. To evaluate the safety and clinical tolerability of oral doses of CVD 1208S-122, ranging 
from 108 cfu to 1010 cfu organisms, with particular attention to the occurrence of 
objective signs of diarrhea, dysentery or fever 
2. To assess the degree of fecal shedding of CVD 1208S-122 and the genetic stability of the 
excreted live vector isolates by genetic characterization of the vaccine organisms 
recovered in stool cultures 
Secondary Objectives: 
1. To measure the mucosal (α4β7-positive IgA antibody secreting cell [ASC], antibody in 
lymphocyte supernatant [ALS], fecal IgA antibody) and systemic (serum IgG and IgA 
antibody) immune responses to ETEC antigens (CFA/I and LThA2B) and S. flexneri 2a 
antigens (LPS and Ipa) following oral immunization with CVD 1208S-122 
2. To evaluate the ETEC and Shigella functional antibody responses following oral 
immunization with CVD 1208S-122 
3. To assess transmissibility of CVD 1208S-122 by determining whether the vaccine strain 
is acquired (as detected by stool culture) by placebo recipients living in close proximity 
on the Research Isolation Ward, thus approximating household contact conditions (to be 
assessed in Cohorts 1 – 3) 

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Exploratory Objectives: 
1. To measure the circulating B memory cell responses to ETEC antigens (CFA/I and 
LThA2B) and S. flexneri 2a antigens (LPS and Ipa) following oral immunization with 
CVD 1208S-122 
2. To collect, separate and store (at -70°C or colder) peripheral blood mononuclear cells 
(PBMCs) so that subsequently the immune responses to CVD 1208S-122 can be further 
characterized in greater detail including the measurement of T memory and effector cells, 
homing markers and cytokine production 
 
Primary Endpoints: 
To evaluate the safety and clinical tolerability of oral doses of CVD 1208S-122, with 
particular attention to the occurrence of objective signs of diarrhea, dysentery or fever. 
• The number, proportion, and severity of fever, diarrhea, or dysentery (hemoccult testing 
will only be performed during the inpatient days) within 7 days of vaccination, for all 
those receiving vaccine and for each of the vaccine dosages evaluated 
• The number, proportion, and severity of solicited local and systemic adverse reactions 
(diarrhea, dysentery, fever, nausea, vomiting, abdominal discomfort, tenesmus, myalgia, 
arthralgia, and anorexia) within 7 days of vaccination for all those receiving vaccine and 
for each of the vaccine dosages evaluated 
• The number, proportion, severity, and relatedness of non-serious unsolicited adverse 
reactions within 28 days of vaccination for all those receiving vaccine and for each of the 
vaccine dosages evaluated 
• The number, proportion, and severity of clinical safety laboratory adverse events from the 
time of each study vaccination through approximately 7 days after each study 
vaccination. 
• The occurrence of SUSARs in the study 
• The occurrence of all SAEs, regardless of the assessment of relatedness, from the time of 
the first vaccination through approximately 6 months after the last study vaccination 
 
To assess the degree of fecal shedding of CVD 1208S-122 and the genetic stability of the 
excreted live vector isolates by genetic characterization of the vaccine organisms recovered 
in stool cultures, to be determined with inpatient cohorts 1 – 3. 
• The geometric mean number (and interquartile range) of vaccine organisms, per day and 
at the peak of shedding, expressed as cfu/mL for each dosage group 
• The number and proportion of sequential days of fecal shedding of Shigella organisms 
which are documented to contain the genes expressing ETEC antigens for each dosage 
group. Genetically stable organisms will be defined as being PCR positive for Shigella 
(ipaH or virG), CFA/I (cfaB), and LTB (eltB) 
[The endpoints for additional study objectives are described in Section 10.3.] 

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Brief Description of Study Design: 
This will be a phase 1, double-blind, placebo-controlled, dose-escalating, single-center study, 
involving three vaccine dosage escalation cohorts (108, 109, and 1010 cfu vaccine organisms). 
Each of the three dose-escalation cohorts will consist of 8 study participants who will be 
randomly allocated to receive either vaccine (n=6) or placebo (n=2), as a single, oral dose. A 
Safety Monitoring Committee (SMC) will review the available safety data for Cohorts 1 – 3 
through 7 days post-vaccination before proceeding to the enrollment of the next cohort. The 
fourth cohort will be an adaptive design cohort consisting of 30 study participants to be 
randomly allocated to receive either two doses of vaccine (n=12), one dose of vaccine (n=12), or 
two doses of placebo (n=6), with the dosage selection based on the highest well-tolerated dose in 
the dose-escalation cohorts (1 – 3), as determined by the SMC. 
Each of the dose-escalation cohorts (1 – 3) will receive the oral dose of blinded study product 
while in the inpatient setting. During the following subsequent 96 hours (4 days), participants 
will remain on the inpatient research isolation ward to be closely monitored, and each stool will 
be collected by study staff. The evaluation and monitoring of participants enrolled in the fourth 
cohort will be conducted entirely in the outpatient setting. (Table 1) 
Table 1: Overview of the Study Cohorts 
 
Cohort 
Setting 
Dose (cfu) 
No. Subjects 
Dose #1 
Dose #2 
Vaccination 
Days 
1 
Inpatient 
108 
6 
Vaccine 
- 
1 
- 
2 
Placebo 
- 
2 
Inpatient 
109 
6 
Vaccine 
- 
1 
- 
2 
Placebo 
- 
3 
Inpatient 
1010 
6 
Vaccine 
- 
1 
- 
2 
Placebo 
- 
4 
Outpatient 
108 
12 
Vaccine 
Vaccine 
1 & 29* 
108 
12 
Vaccine 
Placebo 
1 & (29)* 
- 
6 
Placebo 
Placebo 
(1) & (29)* 
(highest, well- 
tolerated dose 
among cohorts 1 – 3, 
determined by SMC) 
Total 
N = 54 
* In Cohort D, the vaccine will be administered as a single dose (Day 1) or as a two-dose (Days 1 and 29) regimen. In order to maintain 
the blinding of study products, a placebo will be administered when indicated by the parentheses. 
SMC, Safety Monitoring Committee 

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1 
KEY PERSONNEL 
Principal Investigator 
Wilbur H. Chen, MD, MS 
Professor of Medicine 
Center for Vaccine Development and Global Health 
Phone: 410-706-5328 
Email: wchen@som.umaryland.edu 
 
Laboratory Co-Principal Investigator 
Eileen M. Barry, PhD 
Professor of Medicine and Microbiology & Immunology 
Center for Vaccine Development and Global Health 
Phone: 410-706-3702 
Email: embarry@som.umaryland.edu 
 
Immunology Co-Investigators 
Marcela F. Pasetti, PhD 
Professor of Pediatrics and Microbiology & Immunology 
Center for Vaccine Development and Global Health 
Phone: 410-706-2341 
Email: mpasetti@som.umaryland.edu 
 
Marcelo B. Sztein, MD 
Professor of Pediatrics, Medicine, and Microbiology & Immunology 
Center for Vaccine Development and Global Health 
Phone: 410-706-2345 
Email: msztein@som.umaryland.edu 
 
Microbiology Co-Investigator 
Sharon M. Tennant, PhD 
Associate Professor of Medicine and Microbiology & Immunology 
Center for Vaccine Development and Global Health 
Phone: 410-706-5336 
Email: stennant@som.umaryland.edu 
 
Research Institution 
Center for Vaccine Development and Global Health 
University of Maryland School of Medicine 
685 W. Baltimore Street, Suite 480 
Baltimore, MD 21201, U.S.A. 

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2 
BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE 
 
2.1 
Background Information 
Diarrheal disease is a significant cause of morbidity and mortality worldwide; it is the second 
leading cause of death in children under the age of 51, with approximately 800,000 deaths annually. 
In 1999, the global burden of Shigella was estimated to include 163 million cases and >600,000 
deaths each year with the greatest burden in children under five years of age in developing 
countries.2 The Global Enteric Multicenter Study (GEMS), which investigated the cause of 
moderate-to-severe diarrhea (MSD) in children under 5 years of age in 7 sites in Africa and Asia 
identified Shigella among the top 4 pathogens.1 GEMS follow-on studies using molecular 
diagnostic methods revealed an increased attributable burden due to Shigella and confirmed that it 
was the number one pathogen associated with MSD in the two older age strata (12 – 23 and 24 – 
59 months old) and fourth most important pathogen in the youngest age group (0 – 11 months) 
across all sites.3,4 Moreover, Shigella was disproportionately associated with severe outcomes. 
In industrialized countries shigellosis, mostly caused by the S. sonnei species, persists both as 
sporadic cases,5 and as a problem in certain sub-populations, such as children in daycare and other 
settings with suboptimal personal hygiene.6 Shigella also causes significant disease in travelers to 
endemic regions including military personnel.7,8 The increasing resistance of Shigella isolates to 
multiple antibiotics reduces therapeutic options.6,9 The World Health Organization (WHO) has 
named Shigella a priority for vaccine development and implementation,10 but despite intense 
efforts, no licensed vaccine to prevent Shigella diarrheal illness is currently available. 
The GEMS also identified ETEC as an important diarrheal pathogen in young children of 
developing countries. Similarly, ETEC is known as the leading cause of diarrhea in travelers to 
endemic regions, including military populations.11,12 Following ingestion of contaminated food or 
water, ETEC attach to the small intestine by fimbriae (also named colonization factor antigens or 
coli surface antigens, CFAs or CSs), organelles emanating from the bacterial surface. Thereupon, 
the bacteria elaborate heat stable toxin (ST) and/or heat labile toxin (LT), which are responsible 
for secretion of fluid into the intestinal lumen and subsequent watery diarrhea. 
Earlier vaccine studies support the protective capacity of immune responses targeting fimbriae (to 
block colonization) and LT toxins (toxin-neutralizing antibodies) from ETEC.13 ST consists of 
small peptides (18 or 19 amino acids). Neutralizing anti-ST has not been observed in nature.14 In 
order to induce neutralizing anti-LT antibodies, the B subunit must form a pentamer by inclusion 
of the LTA2 subunit.15 LThA2B allows for the expression of B subunits of human LT (as opposed 
to porcine LT), which can form pentamers when stabilized by the A2 domain and which lack the 
enzymatically-active and toxigenic A1 subunit domain. The most common fimbriae expressed on 
clinically important isolates include CFA/I, CS1, CS2, CS3, CS4, CS5, and CS6.16,17 
There are currently no licensed vaccines for ETEC. Both Shigella and ETEC are classified as 
category B agents of biodefense concern, making them priorities for development of therapeutics 
and vaccines. A multitude of strategies have been pursued to develop vaccines against Shigella 
including live attenuated, killed whole cell, subunit, and conjugates.18-20 Decades of literature 

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including field trials and volunteer challenge studies support the protective efficacy of live 
attenuated vaccines, which present the entire antigenic repertoire of the pathogen and which are 
delivered via the most immunologically relevant route, i.e., oral.21-23 Protective immunity against 
Shigella is directed against the LPS O-antigen and is serogroup-specific and often serotype- 
specific. 
 
2.1.1 Live, Attenuated Shigella vector 
From wild-type S. flexneri 2a strain 2457T, a prototype vaccine candidate, designated strain CVD 
1208, containing deletion mutations in guaBA, set, and sen was constructed. The guaBA operon 
encodes two enzymes involved in the synthesis of guanine nucleotides—thereby deletion of this 
operon creates a guanine auxotroph incapable of survival without a source of exogenous guanine. 
The mutations in the set and sen genes, encoding Shigella enterotoxins ShET1 and ShET2 
respectively, are further attenuations intended to render the organism incapable of diarrheal 
reactogenicity. A phase 1 trial of oral doses of 107, 108, and 109 cfu with the attenuated strain CVD 
1208 demonstrated good tolerability and strong immunogenicity.24 
Strain CVD 1208 was subsequently reconstructed on animal-free media (peptone soy medium, Hy 
soy) to address general regulatory concerns about the possible contamination of vaccine products 
constructed on animal-containing media with prions, causing Bovine Spongiform Encephalopathy 
(BSE). This new construct was designated strain CVD 1208S. A phase 1 clinical trial confirmed 
that oral doses of 108 and 109 cfu of strain CVD 1208S was similarly well-tolerated and 
immunogenic compared to CVD 1208.25 A subsequent phase 1 clinical trial of oral doses of 107 
and 108 cfu of strain CVD 1208S with a current Good Manufacturing Practice (cGMP) pilot lot of 
vaccine (Shigella 28000) grown in Hy soy broth (manufactured at the Walter Reed Army Institute 
of Research Pilot Bioproduction Facility [WRAIR PBF]) also demonstrated minimal 
reactogenicity and good immunogenicity26, thus encouraging continued clinical development of 
this prototype strain as a viable oral attenuated vaccine. 
In order to construct a rational prototype Shigella-ETEC vaccine strain, two ETEC genes were 
added into the chromosome of the parent vaccine strain CVD 1208S. The integration of genes 
encoding CFA/I fimbriae and LT antigen, LThA2B, into the chromosome resulted in strain CVD 
1208S::PmLpp-CFA/I-LTA2B (renamed strain CVD 1208S-122). Western blot analysis 
demonstrated high level expression of CfaB, the structural subunit of CFA/I. However, electron 
microscopy analysis of cGMP lot material of CVD 1208S-122 did not show full-length CFA/I 
fimbriae on the surface of the bacteria. Instead, the CfaB subunit monomers are expressed at high 
levels in the periplasm and also on the bacterial surface but not in the native morphology. 
Additional information can be found in IND section 1.11.3. 
 
2.1.2 Pre-clinical studies of Shigella vector expressing ETEC antigens 
Immunization of guinea pigs with two doses of CVD 1208S-122 induced robust responses to the 
Shigella live vector, as well as CFA/I, and to a lesser extent the LTB antigen (Figure 1). The lead 
chromosomally-encoded gene construct, CVD 1208S-122, was compared to the multi-copy 
plasmid-bearing construct, strain CVD 1208S(pCFA/I-LTB), that was previously tested in 

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volunteers. All vaccinated animals responded with robust and comparable serum and mucosal anti- 
S. flexneri 2a LPS IgG and IgA titers following two doses; there was no significant difference in 
the geometric mean titers between any vaccine groups. Consistent with this strong response, all 
vaccinated animals were protected against keratoconjunctivitis following Sereny test challenge 
with wild-type S. flexneri 2a strain 2457T two weeks after the second immunization. All five 
unimmunized control animals succumbed to infection, reaching a clinical score of 4 by 48 – 72 h 
post-inoculation. All of the vaccinated animals responded with strong serum and mucosal 
antibodies to CFA/I. Responses to the LTB antigen were variable; 75% (3 of 4) animals immunized 
with the plasmid-bearing construct responded to LTB and 50% (4 of 8) animals immunized with 
CVD 1208S-122 responded with anti-LTB IgG titers above pre-immune levels.27 
 
 
 
Figure 1. Mucosal IgA (left) and serum IgG (right) responses to S. 
flexneri LPS, CFA/I and LTB in immunized guinea pigs. Blood and 
tears were collected from animals immunized with CVD 1208S-122 
(red) or CVD 1208S(pCFA/LTB) (blue) prior to immunization (open 
symbols) and 2 weeks following the final dose (closed symbols). 
Endpoint titers for individual animals are shown for each antigen. 
 
The functional activity of elicited antibodies was assessed with Shigella serum bactericidal assays 
(SBA), ETEC hemagglutination inhibition, and cell binding inhibition assays for ETEC.28 All 
animals vaccinated with CVD 1208S-122 had SBA titers >1200 (an endpoint titer that exhibits 
50% killing) against wild-type Shigella (Figure 2A). These serum samples were also able to inhibit 
hemagglutination (HAI) of human type A red blood cells by wild-type ETEC strain H10407 (which 
expresses CFA/I). This measurement is considered a proxy for inhibition of ETEC binding to the 
human host.29,30 Additional studies confirmed the capacity of immune sera from immunized 
animals to inhibit wild-type ETEC binding to human intestinal cell lines in vitro (Figure 2B). 

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Figure 2. Functional activity of immune sera. A) Mean bactericidal activities against WT Shigella at 
increasing dilutions. B) cfu of attached WT ETEC strain H10407 following incubation with no sera, rabbit 
polyclonal anti-CFA/I, pre-immune guinea pig sera, or pooled sera from animals immunized with CVD 
2108S-122 (immune sera). ***p<0.001, **p<0.01 2-way ANOVA with Sidak and Tukey’s tests for multiple 
comparisons. 
 
 
 
2.1.3 Clinical studies of CVD 1208S-122 
No human clinical studies have evaluated CVD 1208S-122. 
 
2.2 
Rationale 
This will be a phase 1, randomized, double-blind, placebo-controlled, dose-escalating, single- 
center study of CVD 1208S-122. We hypothesize that the chromosomal integration of foreign 
genes for CFA/I and LThA2B into the parent attenuated vector (CVD-1208S) will be stable and 
not introduce further reactogenicity. It is possible that the metabolic burden of the expression of 
these foreign genes could further attenuate the live organism and diminish stability or 
immunogenicity. The proposed starting dose of 108 cfu of CVD 1208S-122 will be evaluated in 
the first dose-escalation cohort (Cohort 1). Cohort 2 will evaluate a target dose of 109 cfu of 
CVD 1208S-122. Cohort 3 will evaluate a target dose of 1010 cfu of CVD 1208S-122. Each of 
these three dose-escalation cohorts will consist of a total of 8 participants who will be randomly 
allocated to receive a single oral dose of a blinded study product such that six participants will 
receive vaccine and two participants will receive placebo. 
A fourth cohort will be included to further evaluate the highest well-tolerated dose of the vaccine 
and will evaluate the safety and immunogenicity of one or two doses of vaccine. The dose range 
was selected because the safety and immunogenicity at these doses was demonstrated with 107 - 
109 cfu of the parent strain (CVD 1208S) and 107 – 1010 cfu of the plasmid-expression-based 
Shigella-ETEC strain (CVD 1208S(pCFA/I-LThA2B)) in prior clinical studies.25,26,31 
In order to characterize the fecal shedding and genetic stability of the ingested organism, Cohorts 
1 – 3 will be administered the assigned dose of blinded study product while in the inpatient 
setting at the Research Isolation Ward. Reactogenicity will be assessed for 96 hours (4 days) 
following administration of the product. Participants will be discharged after 96 hours of 
observation if they are free from objective signs of adverse reactions, including fever, diarrhea, 

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and dysentery for at least 12 hours. Twice daily stool cultures will be performed (one in the AM 
and one in the PM) to characterize the pattern of fecal shedding, assess the genetic stability of the 
excreted live vector vaccine organisms, determine whether the vaccine is transmitted to placebo 
recipients living in close proximity on the ward, and document the duration of vaccine excretion. 
Blood and stool specimens will also be collected to measure the immune response to the oral 
doses of vaccine. Since the assumption will be for the fourth cohort to be selected based on the 
elicitation of minimal reactogenicity in the dose-escalation cohorts, this cohort of the study will 
be conducted on an outpatient basis. Nonetheless, to ensure the assessment of safety, 
reactogenicity information will be collected from the participants during the first week post- 
vaccination. 
 
2.3 
Potential Risks and Benefits 
 
2.3.1 Potential Risks 
Shigellosis and Diarrheal Disease. Although engineered to be attenuated, since CVD- 
1208S-122 is a live vaccine, subjects could develop shigellosis with symptoms such as 
fever, headache, diarrhea, dysentery, abdominal cramps, nausea, and vomiting. If a 
subject develops unacceptable reactogenicity (as described above), is uncomfortable, 
and/or does not appear to be recovering spontaneously, he/she will be offered early 
antibiotic therapy. The potential exists for virulent S. flexneri strains to cause dehydrating 
diarrheal disease. Under the controlled management in this study, even the dehydrating 
effects of heavy purging can be prevented by early recognition of dehydration, by giving 
oral rehydration solutions preventively after each loose stool (Grade 3 – 5), and by 
appropriate therapy with oral and/or intravenous fluids. If intravenous fluids are required, 
blood chemistries will be monitored. As a precaution, subjects who have poor venous 
access will be excluded from participating in the trial. 
Reactive Arthritis. Reactive inflammatory arthritis, alone32 or as part of a constellation of 
arthritis, conjunctivitis or iritis (uveitis), and urethritis33 is a rare post-infectious 
complication that occurs mostly in adults who have had an infection with a wild-type S. 
flexneri serotype. Arthritis usually begins acutely, 2 to 4 weeks after the intestinal illness. 
Joint symptoms become chronic in about 10% of cases, ranging from mild arthralgia to 
severe polyarthritis. Persons with the human leukocyte antigen type B27 (HLA-B27) 
histocompatibility antigen are predisposed, accounting for approximately half of the 
cases.34 The risk to the remaining 92 – 99% of the HLA B27-negative population is thus 
extremely low. As such, subjects who carry HLA-B27 will be excluded from 
participating in the study. Uveitis can be a component of the reactive arthritis syndrome 
(which has many causes), but to our knowledge has never been associated with Shigella 
human challenge infections. Uveitis in association with reactive inflammatory arthritis 
can cause pain, eye redness, and visual disturbance, but often completely resolves within 
3 months. 

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Bacteremia and/or Localized Infection. Available information suggests that even during 
infection with virulent Shigella spp., bacteremia and localized infection are rare in 
persons of good nutritional state; i.e., typically only observed in developing country 
settings and in association with poor nutritional status. Among adults of developing 
countries, Shigella bacteremia has only been documented among older persons with 
underlying chronic medical conditions or among the immunocompromised, therefore, 
this theoretic risk is prevented through our eligibility criteria and not specifically 
discussed in the consent form. 
Transmission and Secondary Spread of the Live Organism. Wild-type Shigella are 
capable of efficient person-to-person transmission in settings where there is lack of 
sanitation, safe water, and hygiene. This risk has been minimized for several reasons: 
a) Previous studies with either wild-type or vaccine strains of S. flexneri at the CVD 
have not detected symptomatic transmission events. We acknowledge there was an 
observation of two recipients of an early attenuated Shigella strain (strain CVD 
1204) at 107 cfu and one placebo recipient which demonstrated positive stool 
cultures in enrichment broth while on ciprofloxacin therapy; this is believed to be 
the result of a laboratory error and not nosocomial transmission of the vaccine 
strain. In this study, there will be the performance of daily stool cultures to 
systematically identify whether any transmission events occur while inpatient. 
b) The vaccine strain has a stringent growth requirement (guanine auxotrophy) 
rendering it capable of only limited survival in the environment. This inability to 
persist in the environment further minimizes the risk of transmission of the 
organism. 
c) A lapse in observing hygiene is the ultimate culprit for transmission events in 
industrialized country settings. Therefore, study participants will be repeatedly 
educated during the course of the study about the precautions needed to prevent 
spread of the vaccine strain to close contacts. Hand washing will be emphasized to 
all the participants. Stools will be treated with bleach for five minutes prior to 
flushing. In Cohort 4 (outpatients), training on proper toileting and handing of 
waste will be provided. Those who have close contact with vulnerable persons who 
can develop more severe or even fatal infections with wild-type Shigella strains 
(children, immunocompromised, or older adults), healthcare workers, or who are 
occupational food-handlers will not be eligible to participate. 
Pregnancy. This Shigella-ETEC vaccine has not been evaluated in pregnancy, so 
pregnant women may not participate in this first in human study. Women of child-bearing 
potential must agree to use effective methods of birth control for at least 4 weeks after 
vaccination. 
Special Circumstance. 
COVID-19. At the time of this writing, there is a pandemic caused by the SARS-CoV-2 
virus and causing COVID-19 illness. As long as the threat of COVID-19 continues to 
represent a U.S. Public Health Emergency, a number of steps will be undertaken to 

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protect research staff and study participants from COVID-19. The eligibility criteria will 
include a question regarding the presence of symptomatic COVID-19-like illness, 
wherein a positive response regarding symptomatic illness will exclude the participant 
from eligibility to be vaccinated. Also, the incorporation of SARS-CoV-2 testing will be 
a required element of the procedures to be performed prior to inpatient admission 
(Cohorts 1-3), wherein a negative molecular diagnostic test result will be required for 
final eligibility for vaccination. This testing is intended to further reduce the risk of 
transmission of COVID-19 on the inpatient setting from an asymptomatic or pre- 
symptomatic infected participant. 
Furthermore, at all points during the conduct of the study, a number of non- 
pharmacologic interventions will be required. The observance of universal masking, 
physical distancing, hand hygiene, administrative changes (e.g., decreasing the density of 
study personnel), and other control or mitigation measures will be implemented by 
research staff. All study participants will be required to wear a face mask and instructed 
to perform hand washing or hand sanitation during the inpatient period (Cohorts 1-3) and 
for all scheduled outpatient visits. Lastly, COVID-19 vaccination must have been 
completed at least 14 days prior to scheduled first vaccination. A valid COVID-19 
vaccine is one that has been authorized by the U.S. FDA or has received WHO pre- 
qualification. 
When the COVID-19 pandemic resolves and the U.S. Public Health Emergency has 
ended, the study team will re-evaluate the local conditions for consideration of removing 
these additional precautionary measures. 
 
2.3.2 Known Potential Benefits 
This is a healthy volunteer study which does not provide any guarantee of benefit. The 
benefit is largely the scientific knowledge to be gained from the study. 
 
3 
STUDY DESIGN 
This will be a phase 1, double-blind, placebo-controlled, dose-escalating, first-in-human, single- 
center study involving healthy adults (age 18 – 49 years). This study will involve three vaccine 
dose-escalation cohorts and a fourth adaptive design cohort. Participants will be screened for 
eligibility up to 60 days prior to enrollment. Randomization will be performed by the assigned 
unblinded biostatistician and will occur upon enrollment, after a final confirmation of eligibility. 
Study product administration will be performed by prospectively assigned unblinded study staff 
members, who will not have any role in the subsequent follow-up assessments of the 
participants. Following completion of each dose-escalation cohort (7 days post-vaccination), 
there will be an independent Safety Monitoring Committee (SMC) which will review the 
cumulative safety data and will provide their recommendation for whether the study should 
proceed to the subsequent cohort. 

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The three dose-escalation cohorts will evaluate doses of 108, 109, and 1010 cfu vaccine 
organisms. Each of the three dose-escalation cohorts will be randomly allocated to receive either 
vaccine (n=6) or placebo (n=2). In Cohorts 1 – 3, eligible participants will be administered a 
single oral dose of blinded study product while residing in a Research Isolation Ward (inpatient 
setting). All participants will be observed for at least 4 days (96 hours) following product 
administration while inpatient. During the inpatient observation period, protocol-specified 
clinical evaluation procedures will be implemented, including management of diarrhea, 
procedures for fluid replacement, and criteria for antibiotic therapy. 
Subjects will be discharged after 96 hours of observation if they are free of fever, diarrhea, and 
dysentery for at least 12 hours—i.e., subjects have an oral temperature <100.4°F, 12 hours have 
passed since the last loose stool meeting the definition of diarrhea, and 12 hours have passed 
since the last loose stool containing gross blood. After discharge and through 7-days following 
vaccination, the subject will record onto a standardized memory aid the occurrence of solicited 
symptoms, graded by severity, oral temperature, record the character of all stools passed as either 
formed or loose (defined as stools which take the shape of the container) or containing visible 
blood. 
Subsequent scheduled outpatient follow-up visits will be completed for the continued collection 
of safety information and blood and stool specimens. Subjects will return to the CVD outpatient 
clinic on Day 8 and research staff will collect and review the information in the memory aid. 
Additional scheduled outpatient clinic visits will also occur on Days 29, 43, and 181. 
The adaptive design cohort (Cohort 4) will consist of 30 participants that will receive either two 
doses of vaccine, one dose of vaccine, or placebo; the dosage selection for Cohort 4 was based 
on the highest well-tolerated dose in the dose-escalation cohorts. Cohort 4 is planned to be 
conducted entirely in the outpatient setting with several in-clinic follow-up visits. Blinded oral 
doses of study product will be administered on Days 1 and 29. A stool specimen and safety 
information will be collected 2 days following each vaccination (Days 3 and 31). Another stool 
specimen and a blood sample will be collected, and a complete review of the memory aid will be 
performed 1 week following vaccination (Days 8 and 36). Additional scheduled outpatient 
follow-up visits will also occur on Days 57 and 210. A complete schedule of events in provided 
in Appendix A: Study Schedule of Events. 
 
4 
STUDY POPULATION 
 
4.1 
Selection of the Study Population 
Healthy subjects, ages 18 – 49 years will be recruited on the basis of satisfying eligibility criteria 
during screening. Up to 54 subjects will be enrolled, meaning randomized and receiving the first 
dose of blinded study product. 
 
4.2 
Inclusion/Exclusion Criteria 
Inclusion Criteria: 

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1. Male or female, 18 – 49 years of age 
2. Written informed consent provided 
3. Determined to be in good health* based on medical history and review of concomitant 
medications 
*Good health as defined by an absence of an active chronic medical condition which requires daily prescription 
medication(s). Participants may be eligible if the medical condition only requires infrequent as needed (PRN) 
medication and if the investigator determines that the condition does not pose a risk to participant safety or the 
assessment of reactogenicity and immunogenicity. Any chronic medical condition which does not require a daily 
prescription medication but might pose a risk to a participant with rapid dehydration (i.e., rapid intravascular volume 
changes) would be ineligible to participate. 
4. Documented acceptable results from screening laboratory work (defined in Appendix B), 
including: 
• Complete blood count (CBC) with differential for total white blood cell count 
(WBC), absolute neutrophil count (ANC), hemoglobin (Hg), platelet count 
• Creatinine, alanine aminotransferase (ALT), total bilirubin 
• Serum Immunoglobulin A (IgA) level 
• Human immunodeficiency virus (HIV) antibody, Hepatitis B surface antigen 
(HBsAg), Hepatitis C virus antibody (HCV) 
• HLA-B27 histocompatibility testing 
• Serum Beta human chorionic gonadotropin (β-HCG) test, if the participant is a 
woman of child-bearing potential 
5. A passing score (≥70%) on a Comprehension Assessment Tool 
6. Agrees not to participate in another interventional clinical trial during the study period 
(participation in non-interventional studies, such as surveys, is acceptable) 
7. Females of child-bearing potential† agree to use an acceptable form of birth control‡ from 
enrollment and through at least 4 weeks after vaccination 
†Females of child-bearing potential, defined as having not been sterilized via tubal ligation, bilateral oophorectomy, 
salpingectomy, hysterectomy, or successful Essure® placement (permanent, non-surgical, non-hormonal sterilization) 
with documented radiological confirmation test at least 90 days after the procedure, and still menstruating or <1 year 
has passed since the last menses, if menopausal. 
‡Acceptable birth control includes barrier methods such as condoms or diaphragms/cervical cap with spermicide; 
effective intrauterine devices; NuvaRing®; and licensed hormonal methods such as implants, injectables or oral 
contraceptives (“the pill”) or alternatively, monogamous relationship with a vasectomized partner who has been 
vasectomized for 180 days or more prior to the subject receiving the study vaccination, abstinence from sexual 
intercourse with a male partner, or sexual relationships with non-male partners. 
8. Available for up to a 6-day inpatient stay 
9. For Participants of Cohorts 1-3 during the time when a U.S. Public Health Emergency for 
COVID-19 exists, SARS-CoV-2 testing must be performed prior to admission to the 
inpatient ward and must document a negative test result prior to vaccination. 

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Exclusion Criteria: 
1. 
A positive pregnancy test at screening or within 24 h prior to study product dosing 
2. 
A female who is breastfeeding 
3. 
Poor venous access, as defined by inability to obtain venous blood, for screening labs, 
after 3 venipuncture attempts 
4. 
Abnormal vital signs, defined as: 
• Systolic BP >150 mmHg or Diastolic BP >90 mmHg 
• Resting heart rate >100 bpm 
• Oral temperature ≥38.0°C 
5. Having received prior vaccines for or have had prior infection with ETEC, LT, cholera, 
or Shigella, within the past 3 years 
6. History of diarrhea during travel to a developing country within the past 3 years 
7. History of chronic gastrointestinal illness, including severe dyspepsia, lactose intolerance, 
or another significant gastrointestinal tract disease (e.g., irritable bowel syndrome, 
inflammatory bowel syndrome, gastric ulcer disease) 
8. Regular use (≥once weekly) of laxatives, anti-diarrheal agents, anti-constipation agents, 
or antacid therapies 
9. History of major gastrointestinal surgery (uncomplicated laparoscopic appendectomy or 
cholecystectomy >1-year prior is permitted) 
10. Abnormal bowel habits, as defined by <3 stools per week or >2 stools per day in the past 
6 months 
11. Use of systemic antibacterials§ within the past 2 weeks 
§use of topical (skin), otic, or ophthalmic antibiotics is acceptable, if those doses are not expected to result in 
significant systemic absorption levels 
12. Use of oral, parenteral or high-dose inhaled steroids within 30 days 
(high-dose oral steroids is defined as prednisone ≥ 20 mg total daily dose, or equivalent dose of other glucocorticoids; 
high-dose inhaled steroids is defined as >800 µg/day of beclomethasone dipropionate or equivalent) 
13. Use of any medication which might affect immune function# within 30 days⁑ 
#examples include anti-cancer drugs, immunomodulating monoclonal antibody therapeutics, and rheumatologic 
therapies 
⁑Due to the prolonged half-life of some immunosuppressive medications (e.g., monoclonal antibody biologic response 
modifiers), the restricted use period may need to be extended to 3 or 6 months, based on the anticipated duration of 
activity with longer half-lives. This is to be interpreted by the investigator. 
14. Diagnosis of schizophrenia or other major psychiatric disease 
15. Alcohol or drug abuse within last 5 years 
16. Presence of immunosuppression, which could be due to active neoplastic disease or a 

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history of any hematologic malignancy (excluding resolved non-melanoma skin cancers), 
radiation therapy, or primary or secondary immunodeficiencies 
17. History of allergy to quinolone (e.g., ciprofloxacin), sulfa drugs (e.g., trimethoprim- 
sulfamethoxazole), or soy or corn products 
18. Known history of seizure disorder (remote history of a childhood seizure disorder which 
has completely resolved is acceptable) 
19. Occupation involving the handling of ETEC, cholera, or Shigella bacteria 
20. Occupation as a healthcare worker, in food handling industry or care of very young 
children (<2 years old), elderly (≥70 years), or immunocompromised; this exclusion also 
applies to individuals that have close contact with young children (<2 years old), elderly 
(≥70 years), or immunocompromised, for example by virtue of living in the same 
household with such individuals 
21. During the time when a U.S. Public Health Emergency for COVID-19 exists, within 14 
days prior to the time of vaccination, the presence of 2 or more of any of the following 
symptoms: fever (≥38°C, chills, myalgia, headache, sore throat, or new olfactory and 
taste disorder 
22. During the time when a U.S. Public Health Emergency for COVID-19 exists, within 14 
days prior to the time of vaccination, the presence of 1 or more of any of the following 
respiratory symptoms: cough which cannot otherwise be explained, shortness of breath, 
or difficulty breathing 
23. During the time when a U.S. Public Health Emergency for COVID-19 exists, has not 
completed a COVID-19 vaccine within 14 days prior to the time of vaccination 
24. Any other criteria which, in the investigator’s opinion, would compromise the safety of 
the study, the ability of a subject to participate, or the results of the study 
 
4.3 
Enrollment and Randomization Procedures 
Per International Conference on Harmonisation (ICH) guideline E6: Good Clinical Practice 
(GCP), screening records will be kept at the clinical site to document the reason why an 
individual was screened, but failed trial entry criteria. The reasons why individuals failed 
screening will be recorded in the data system. 
Once consented to and upon confirmation of eligibility for this trial, the subjects will be enrolled 
and randomized. Subjects will be randomized at a 3:1 ratio (vaccine to placebo) for Cohorts 1 – 
3. In Cohort 4, randomization will be a 2:2:1 ratio for 2-dose vaccine vs. 1-dose vaccine vs. 
placebo. The randomization code will be prepared by a statistician at CVD. A designated 
individual at the site will be provided with a code list for emergency unblinding purposes, which 
will be kept in a secure place. 
Subjects who sign the informed consent form and are enrolled and randomized but have not 
received study vaccine may be replaced by back-up eligible participants. Subjects who sign the 

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informed consent form, are randomized and vaccinated, and subsequently withdraw, are 
withdrawn, terminated from this trial, or are lost to follow-up will not be replaced. 
 
4.4 
Withdrawal from Study 
Reasons for Withdrawal or Termination. Participation in the study is strictly voluntary. 
Participants have the right to withdraw from the study at any time and for any reason, without 
penalty. The Principal Investigator (PI) and/or designee may, at his discretion, withdraw a 
subject from continuing in the study if it is considered to be in the participant’s best interest, or if 
the participant is not willing or able to comply with the study requirements. The reason for 
withdrawal will be documented. 
Handling of Participant Withdrawals or Termination. Every effort will be made to undertake 
protocol-specified safety follow-up procedures to capture adverse effects (AEs), serious adverse 
events (SAEs), and unanticipated problems (UPs). In the event of withdrawal from the study, 
reasonable efforts should be made to conduct the following procedures: 
• A review of the diary card/memory aid, if still in use prior to withdrawal 
• Update any ongoing AE/SAEs that remain ongoing at time of subject’s last visit prior to 
withdrawal 
• Query about AEs, SAEs and concomitant medications, if the interval between the 
subject’s last visit and the time of withdrawal is within the protocol-defined reporting 
period 
• Physical examination 
• Blood specimen collection for safety testing, if withdrawal occurs before Visit 28 
• Update contact information 
Premature Termination or Suspension of Study. This study may be temporarily suspended or 
prematurely terminated if there is sufficient reasonable cause. Written notification, documenting 
the reason for study suspension or termination, will be provided by the suspending or terminating 
party to the investigator. If the study is prematurely terminated or suspended, the PI will 
promptly inform the IRB and will provide the reason(s) for the termination or suspension. 
Circumstances that may warrant termination or suspension include, but are not limited to: 
• Determination of unexpected, significant, or unacceptable risk to participants 
• Demonstration of insufficient immunogenicity that would warrant halting the study 
• Insufficient compliance to protocol requirements 
• Data that are not sufficiently complete and/or evaluable 
• Determination of futility 
Study may resume once concerns about safety, protocol compliance, data quality is addressed 
and satisfy the sponsor, IRB and/or FDA. 

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4.5 
Discontinuation from further vaccination (Cohort 4) 
Participants that are planned to receive two sequential doses of blinded study product (Cohort 4) 
may be discontinued from further vaccination to avoid exposure of subjects to unreasonable risk. 
If a participant experiences a grade 3 or higher AE which is deemed to be related to study 
product or an SAE that is deemed to be related to study product, then the second dose of study 
product will be discontinued. Also, antimicrobials will be administered immediately if a subject 
meets the criteria for discontinuation from further vaccination. Antimicrobials will consist of a 3- 
day course of ciprofloxacin (primary) or trimethoprim-sulfamethoxazole (secondary). The 
investigator may also discontinue a participant from second dose administration for other 
significant and unexpected reasons; whereupon the investigator may also promptly treat the 
participant with a 3-day course of antimicrobial therapy. The reason for discontinuation must be 
clearly documented by the investigator. Any participant that is discontinued from further 
vaccinations will be asked to remain in the study to complete all immunologic and safety follow 
up visits, except the participant will not complete the 7-day memory aid related to the second 
dose of vaccine. 
 
5 
STUDY PRODUCT 
 
5.1 
Study Vaccine, CVD 1208S-122 
This prototype monovalent, live, attenuated, vaccine strain consists of S. flexneri 2a into which 
deletion mutations have been introduced into the guaBA, set, and sen genes. The guaBA operon 
encodes two enzymes involved in the biosynthesis of guanine nucleotides. The set and sen genes 
encode Shigella enterotoxins ShET1 and ShET2, respectively. The CFA/I operon of ETEC, with 
the genes encoding the A2 and B subunits of LTh toxin (also of ETEC), were inserted into the 
chromosome of this live, attenuated vector. Thus, the resulting strain CVD 1208S-122 bears the 
O-antigen of S. flexneri and expresses both CFA/I and LT of ETEC. 
 
5.1.1 Preparation of Vaccine 
Vials of the study product, CVD 1208S-122, will be removed from the freezer and 
allowed to thaw at room temperature in a biosafety cabinet (BSC). After thawing, while 
within the BSC, the vial contents will be diluted with sterile Phosphate Buffered Saline 
(PBS) to the desired concentration. Dilutions of vaccine will be kept on wet ice, until just 
prior to administration. Once the vial has been thawed, the contents should be 
administered to subjects within 2 hours of thawing. A bicarbonate buffer solution will be 
prepared with each vaccine dose (see below). Subjects will be required to fast for at least 
90 minutes prior to vaccination and for 90 minutes after dosing. Approximately 1 – 2 
minutes prior to vaccination, subjects will drink 120 mL of the bicarbonate solution. 
Then, the designated dosage of vaccine will be administered orally to the subject, 
delivered in 30 mL of bicarbonate buffer. If a subject vomits after receiving the vaccine, 

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the subject will not be re-dosed, but will remain in the inpatient unit for observation as if 
they had received the intended dose. 
To prepare the sodium bicarbonate buffer solution, combine 2 g USP-grade white 
crystalline powder sodium bicarbonate (NaHCO3) to 150 mL sterile non-bacteriostatic 
water. 
 
5.1.2 Preparation of Placebo 
The placebo will have the appearance of, and be administered in a similar manner to, that 
of the active vaccine. The placebo will consist of corn starch USP mixed in 30 mL of 
sodium bicarbonate buffer, as described above. Subjects will be required to fast for at 
least 90 minutes prior to vaccination and for 90 minutes after dosing. The bicarbonate 
buffer solution will be prepared as described. Approximately 1 – 2 minutes prior to 
placebo vaccination, subjects will drink 120 mL of the bicarbonate solution. Then, the 30 
mL volume of bicarbonate buffer with corn starch USP will be administered orally. If a 
subject vomits after receiving the vaccine, the subject will not be re-dosed, but will 
remain in the inpatient unit for observation. 
 
5.2 
Study Product Accountability 
The study product will be stored in the University of Maryland Medical Center (UMMC) 
Investigational Drug Service (IDS) pharmacy or the CVD Clinical Microbiology Lab. The FDA 
requires accounting for the disposition of all investigational products. The Investigator is 
responsible for ensuring that an accurate record of product disposition is maintained, and product 
is dispensed only by authorized personnel, as required by applicable regulations and guidelines. 
Records of product disposition, as required by federal law, consist of the date received, date 
administered, quantity administered, and the subject number to whom the study product was 
administered. The investigational pharmacist will be responsible for maintaining accurate 
records of the shipment and accountability of the study product. The dispensing of the study 
product (thawing, dilutions, and dosing of volunteers) will be performed by CVD research staff; 
the process and procedures are documented and maintained at CVD. The pharmacy records 
(either IDS pharmacy and/or CVD) will be made available for inspection by external monitors 
and by the relevant regulatory agencies (e.g., FDA) at any time. 
 
5.3 
Blinding of the Study Product 
A prospectively assigned vaccinator and checker will be designated as unblinded personnel and 
will not have any role in the management of illness or assessment of AEs in study participants. 
Unblinded personnel will be responsible for the preparation of the study products and for the 
blinded labeling of the study products. The designated vaccinator will be responsible for 
maintaining the randomization key and will secure these study documents in a locked cabinet. 

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6 
STUDY PROCEDURES/EVALUATIONS 
 
6.1 
Study Procedures 
Informed Consent. All aspects of the protocol will be reviewed with prospective subjects. 
Subjects who satisfy all inclusion/exclusion criteria will provide informed, written consent to 
participate in the trial. To evaluate comprehension of the study and to document that informed 
consent has been elicited, all subjects must pass a written examination before vaccination 
(minimum passing grade is 70%). Staff will review incorrect answers with the subject. A subject 
who scores below 70% may take the test a second time but will be excluded if she/he scores 
<70% again. 
 
Medical Screening. After signing the consent form, receipt of Notification of Privacy Practices 
(NPP) will be verified and HIPAA authorization forms will be completed. Subjects will undergo 
a clinical evaluation to ensure that they are in good mental and physical health, which will 
include medical history, vital signs, and a brief physical examination (auscultation of heart and 
lungs and oral cavity, abdominal, and lymph node examinations). This screening will also 
include a review of concomitant medications, relevant travel history, normal stool characteristics, 
and other aspects needed to document eligibility. 
Blood will be drawn for selected laboratory measures including a CBC with differential (WBC, 
ANC, hemoglobin, platelets), blood chemistries (creatinine, ALT, total bilirubin), serum IgA, 
serological tests for HIV, hepatitis C, hepatitis B surface antigen, and HLA-B27 typing. Serum 
pregnancy test in females must be negative during screening and a urine pregnancy test must be 
negative within 24 h prior to vaccination. 
Subjects who are excluded based on screening results will be counseled by the clinical staff and 
referred for medical care, as appropriate. If elevated blood pressures are detected after 
enrollment, the investigator will make a determination as to whether continued participation 
would be unsafe or would interfere with the study evaluations. 
 
Acclimatization (Cohorts 1 – 3). Study participants will begin their inpatient stay one day prior 
to vaccination for acclimatization, when each participant will be educated and familiarized with 
the protocol-required procedures (e.g., stool handling), hygiene practices, and the Policies and 
Procedures to be followed while on the inpatient unit. During the acclimatization period, person- 
to-person interactions, mood, and other behaviors will be monitored to assess for any conduct or 
attitudes which might not be appropriate for an inpatient study (i.e., combativeness, anti-social 
behavior, anger outbursts, destruction of property, etc.). Evidence that a subject has 
demonstrated behavior which might pose a safety risk to themselves, other subjects, or staff 
could be cause for ineligibility for vaccination and the remainder of the inpatient stay. Refusal to 
comply with protocol-required procedures, adherence to hygiene practices, or repetitively 
breaking the Policies and Procedures may also constitute ineligibility. Any subject who is 
deemed ineligible will be discharged, prior to vaccination. 

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During the time when a U.S. Public Health Emergency for COVID-19 exists, a SARS-CoV-2 
molecular diagnostic test will be performed on each participant prior to admission to the 
inpatient unit. A negative test result and the absence of COVID-19 symptoms will be required 
for final eligibility for vaccination. 
 
Blinded Vaccination. The vaccine and placebo will be administered by the oral route for 
ingestion. Two grams of NaHCO3 (sodium bicarbonate) will be dissolved in 150 mL of sterile 
water, to make sodium bicarbonate buffer solution. 120-mL of the bicarbonate buffer solution 
will be aliquoted for the participant to drink, to neutralize gastric acidity, prior to drinking the 
vaccine. One to two minutes later, the participants will ingest 1 mL of the appropriate assigned 
vaccine dose, suspended in the remaining 30 mL of the buffer solution, or 1 mL of placebo 
suspended in the remaining 30 mL of buffer solution to which corn starch USP has been added to 
match the turbidity of the vaccine inoculum. Subjects will not eat, drink, or smoke for 90 
minutes pre- and post-inoculation. 
To maintain the blind, a member of the investigative team who will not be involved in the 
clinical evaluation of the subjects will be responsible for preparing and labeling the vaccine and 
placebo with a randomized number. An unblinded checker will be present to verify that the 
correct randomization scheme has been followed. 
 
Inpatient Observation (Cohorts 1 – 3). Subsequent to vaccination, participants will remain on the 
ward for at least 4 days (96 h). Vital signs and oral temperature will be measured at least every 8 
± 1 h by staff nurses who remain on the ward 24 h/day. Participants will be interviewed daily by 
a study physician to determine the occurrence of illness signs and symptoms (e.g., anorexia, 
malaise, abdominal cramps, headache); these data will be recorded and graded for severity. A 
focused physical examination may be performed at the discretion of the physician according to 
the nature of a participant’s complaint. All stools will be collected by research staff and there 
will be a requirement for at least two stool samples per day, one every morning and one every 
evening. If a participant is unable to produce a stool within the 12-hour period, a rectal swab 
will be performed. 
 
Measurement of Diarrhea and Dysentery (Cohorts 1 – 3). Since diarrhea and/or dysentery are 
key measures of the safety of the vaccine, all participants will be expected to collect every stool 
that is passed from the time of vaccination until discharge. Participants will be instructed to use a 
plastic stool collection basin, commonly called a “hat.” All stools will be graded for consistency 
by the study staff. The grading of stool consistency is as follows: 
• 
Grade 1 – well formed (normal stool, does not take the shape of the container) 
• 
Grade 2 – soft (normal stool, does not take the shape of the container) 
• 
Grade 3 – thick liquid (diarrhea, readily takes the shape of the container) 
• 
Grade 4 – opaque, watery liquid (diarrhea 
• 
Grade 5 – rice water (clear, watery diarrhea) 
Any Grade 3 or higher (looser) stool is considered a stool having diarrheal consistency and must 
be weighed to estimate the volume of fluid loss (assume ~1 g diarrheal stool = 1 mL of fluid 

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lost). Gross blood or suspicion of blood in any stools must be evaluated with a hemoccult test, 
for confirmation of dysentery. 
Similarly, any episodes of vomiting should be collected in a provided stool “hat” or plastic 
“kidney” basin. If a vomiting or diarrhea episode is not able to be collected in a basin (e.g., the 
subject has an “accident” while sleeping or before they are able to reach a toilet), then the 
volume of the output will be estimated. 
 
Management of Fluid Losses (Cohorts 1 – 3). Subjects who develop diarrheal stools (Grade 3 or 
higher) during the inpatient observation will be required to ingest standard World Health 
Organization (WHO) Oral Rehydration Solution (ORS) at 1.5 times the stool volume. Vomitus 
will be replaced with ORS in equal amounts, 1:1 ratio. At the discretion of the investigator, 
additional ORS may be administered. If a subject develops severe watery diarrhea or persistent 
vomiting and cannot maintain full hydration by oral means, IV fluid replacement will be 
administered. 
For the duration of diarrhea, the subject will be requested to provide a urine specimen from every 
void that they experience; the specimen will be tested for specific gravity. In the event that IV 
fluids are required, serum electrolytes (Sodium, Potassium, Chloride, Bicarbonate), BUN, and 
creatinine will be measured. 
A physician investigator shall always be available by telephone or beeper. Nurses will notify the 
on-call physician if any of the following occurs in a subject who is experiencing diarrhea and/or 
vomiting: 
• Syncope 
• Complaint of dizziness or light-headedness or established orthostatic hypotension 
• Urine specific gravity >1.025 
• >500 mL behind in ORS replacement 
• Vomiting ≥500 mL once or total volume within the past 4 hours 
• High fever ≥39C (≥102.2F) 
• Severe headache, severe malaise, or severe abdominal pain 
• Subject has a complaint for which he/she requests treatment 
• Any other clinical situation that concerns the nurse 
 
Frequency of Vital Signs Assessment (Cohorts 1 – 3). Vital signs (blood pressure, pulse, and oral 
temperature) will be measured approximately every 8 h, unless more frequent monitoring is 
needed. Once a subject has passed a diarrheal stool (Grade 3 or higher), vital signs will be 
measured every 4 h, until either the subject passes a Grade 1 or 2 stool, or 24 h have passed since 
the last grade 3 – 5 stools, whichever comes first. Vital signs will also be measured every 4 h 
when a subject has a fever ≥39C (≥102.2F), until the subject has 2 consecutive temperatures of 
≤38C (100.4F). Any subject that complains of dizziness or light-headedness upon standing will 
have orthostatic blood pressures assessed. Orthostatic hypotension is defined as a drop in systolic 
BP >20 mmHg or in diastolic BP >10 mmHg. 

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Fluid Therapy/Hydration (Cohorts 1 – 3). ORS (Jianas Bros ORS) will be offered as the primary 
means of hydration and will be prepared according to the manufacturer’s package insert. Unused 
ORS should be discarded 24 h after preparation. 
Intravenous fluids (Lactated Ringers solution) will be administered to subjects with diarrhea who 
meet any of the following criteria: 
• Orthostatic hypotension 
• Urine specific gravity >1.025, or determined as necessary by the Investigator 
• No urine output for ≥8 h 
• >1000 mL deficit in ORS replacement 
• At the investigator’s discretion, based on clinical evaluation or on a subject’s 
difficulty in keeping up with ongoing diarrheal losses by oral rehydration alone 
Intravenous therapy will continue until the above criteria are no longer satisfied, the subject is 
able to take fluid by mouth, and a study physician determines that it is no longer required. 
 
Indications for Antibiotics. Antimicrobials will be administered immediately if a subject meets 
the criteria for moderate diarrhea or dysentery (defined in Appendix D). Antimicrobials will 
consist of a 3-day course of ciprofloxacin (primary) or trimethoprim-sulfamethoxazole 
(secondary). In rare and unexpected instances, the study investigator will be allowed to 
administer antimicrobials for alternate reasons—e.g., if in his/her judgment it is necessary to 
ensure the safety of the subject. Otherwise, antibiotic therapy will be initiated for any individual 
that demonstrates continued shedding of vaccine organisms on the Day 8 visit (one-week post- 
vaccination), to be started as soon as the participant is able to return back to clinic for this 
unscheduled visit. 
Indications for Other Concomitant Medications. Any concomitant prescription or over-the- 
counter medications will be evaluated for continuation during the inpatient setting. Any such 
medication will need to be discussed with and approved by the Investigator prior to admission. 
The supply of medication will be the responsibility of the subject and will be handed over to the 
research staff upon admission; daily administration will be recorded. Any prescription or over- 
the-counter medications that were not declared and are discovered during the inpatient stay will 
constitute a violation of the ward Policies and Procedures. 
Other medications may be administered during the study period as follows: 
• Smoking is not allowed on the ward, but subjects will be able to request a nicotine patch. 
• Antipyretics and analgesics (i.e., ibuprofen, acetaminophen, aspirin, or similar non- 
steroidal agents) may be prescribed for severe headaches, other pains, or fevers (e.g., 
sustained temperatures of ≥39C). 
• At the investigator’s discretion (e.g., upon review of serum electrolyte results during 
severe diarrhea), oral potassium may be administered for repletion of electrolyte losses. 
• Other medications, which are deemed necessary for the safety and welfare of the subject 
Any medications prescribed must be ordered and signed by the Investigator and each 
administration must be recorded. 

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Stool Microbiology. For Cohorts 1 – 3, stool cultures will be performed to characterize the 
pattern of fecal shedding, to determine whether the vaccine is transmitted to placebo recipients 
living in close proximity on the ward, to assess the genetic stability of the excreted live vector 
vaccine organisms, and to document the duration of vaccine excretion. All stools will be 
collected, graded, and weighed while participants are on the Research Isolation Ward; 
quantitative cultures will be performed on the first stool sample of each 12-h period; qualitative 
cultures will be performed on all additional stool samples. Qualitative cultures will be performed 
on rectal swabs. A stool specimen or rectal swab will also be collected on the Day 8 clinic visit. 
For Cohort 4, a stool specimen will be collected for qualitative culture on the day of (prior to) 
vaccination (Days 1 and 29), 2 days after vaccination (Days 3 and 32), and 7 days after 
vaccination (Days 8 and 36). In addition, quantitative stool culture will be performed on the 
stools collected on Days 3 and 32. 
Shed Shigella organisms will be genetically characterized to ensure that the vaccine organism 
remains stable. 
Memory Aids. During the 7 days after each dose of vaccination, the documentation of 
reactogenicity will be completed. For Cohorts 1 – 3, the PI (or designee) will complete the 
reactogenicity case report form for those days the participant is on the inpatient unit; this is 
typically performed during the daily physician rounds. For Cohorts 1 – 3, upon discharge and 
until the Day 8 clinic visit, the participant will complete a paper memory aid, to document 
reactogenicity. For Cohort 4, The participant will complete a paper memory aid for the 7-day 
post-vaccination period. On the Day 8 clinic visit (for Cohort 4, both the Day 8 and Day 36 clinic 
visits), the memory aid document will be reviewed by study staff and the reactogenicity case 
report form will be completed by the study staff, based on the information on the memory aid 
and a discussion with the participant regarding the responses on the memory aid. 
 
6.2 
Laboratory Evaluations 
 
6.2.1 Laboratory Evaluations/Assays 
 
Screening Laboratory. The following clinical laboratory assessments will be performed 
as part of the screening for eligibility (Appendix B): 
• CBC with differential for WBC, ANC, Hg, platelet count 
• Creatinine, ALT, total bilirubin 
• HBsAg, HCV, HIV 
• Serum β-HCG (if woman of child-bearing potential) 
• Serum IgA level 
• HLA-B27 histocompatibility testing 
 
During the time when a U.S. Public Health Emergency for COVID-19 exists, a SARS- 
CoV-2 molecular diagnostic test will be performed for Cohort 1-3 participants. 

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Safety Laboratory. The following clinical laboratory assessments will be performed at 
baseline (Day 1, prior to vaccination) and at the Day 8, 29, and 36 visits (see Appendix C 
for Toxicity): 
• CBC with differential for WBC, ANC, Hg, platelet count 
• Creatinine, ALT, total bilirubin 
• Sodium, potassium, aspartate aminotransferase (AST), and alkaline phosphatase 
may be added clinical laboratory measures if indicated by the Investigator’s 
judgement, in order to further investigate reactogenicity or adverse effects 
 
The following tubes are be used for the evaluation of clinical laboratories: 
• 4 mL EDTA for CBC with differential 
• 8.5 mL tiger top for chemistries, serum IgA, HBsAg, HCV, and β-HCG 
• 4 mL EDTA for HLA-B27 test (Monday - Thursday shipments only) 
• 3.5 mL tiger top for HIV test 
 
6.2.2 Special Assays or Procedures 
 
Serum ELISA. Serum antibody responses will be measured against the Shigella live vector 
(anti-S. flexneri 2a LPS and T3SS component IpaB) and to each ETEC antigen (CFA/I and 
LTB). Standardized ELISAs for immunoglobulin G (IgG) and IgA will be used to quantify 
antibody responses to all antigens.24,25 ELISA titers will be calculated as the inverse serum 
dilution that produces an absorbance value (450 nm) of 0.2 above background. 
 
Shigella Serum Bactericidal Antibody (SBA) and Opsonophagocytic Killing Antibody 
(OPKA). The functional activity of immune sera will be analyzed using the serum 
bactericidal antibody (SBA) assay and opsonophagocytic killing antibody (OPKA) assay.28 
For the SBA assay, heat-inactivated serum are serially diluted 2-fold with S. flexneri 2a 
strain 2457T and baby rabbit complement (BRC). For the OPKA assay, heat-inactivated 
serum samples are serially diluted 2-fold with S. flexneri 2a strain 2457T, BRC, and 
dimethylformamide-differentiated HL-60 cells (ATCC CCL-240). After incubation for 1 h 
(for SBA) or 45 min (for OPKA) at 37°C, viable colony counts are determined by plating 
10 µL of reaction mixture on Trypticase Soy Agar (TSA) plates. Negative-control wells 
containing only bacteria (no serum), bacteria plus BRC, bacteria plus HL-60 cells (for 
OPKA) and a positive-control serum with known high bactericidal activity will be included 
in each assay. SBA and OPKA titers are calculated from the reciprocal of the serum dilution 
that produces 50% bacterial killing. 
 
ETEC Mannose-resistant Hemagglutination Assay of Human Group A Erythrocytes. The 
functional ability of immune serum to inhibit hemagglutination of human type A red blood 
cells (RBC) by wild-type ETEC strain H10407 will be tested.37,38 Serial dilutions of 
immune serum (100 µL) are mixed with a standardized suspension of ETEC strain H10407 
and incubated at 37°C for 1 h. The bacteria-serum mixtures are then added to U-bottom 96-
well plates. Equal volumes of 0.1 M D-mannose solution and standardized human type 

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A RBC solution are added to each well. The plates are incubated for 2 h at 4°C. 
Hemagglutination inhibition titer is calculated from the highest dilution which results in 
the loss of the pelleting of RBC at the bottom of the well. Each serum sample is tested in 
duplicate. 
 
Serum Inhibition of ETEC Adhesion to HT-29 cells. The immune serum will be tested for 
the ability to inhibit wild-type ETEC H10407 binding to HT-29 cells.39 Bacteria are grown 
overnight in 2 mL of LB, diluted 1:100 into fresh medium on the morning of the 
experiment, and grown for an additional 90 min to mid-logarithmic growth phase. Serial 
dilutions of immune serum (100 µL) are mixed with a standardized suspension of ETEC 
strain H10407 and incubated at 37°C for 1 h. The bacteria-serum mixtures are then added 
to triplicate wells of a 48-well tissue culture plate containing confluent monolayers of HT- 
29 cells. After 1 h, the monolayers are washed three times with tissue culture medium and 
then treated with 0.1% Triton X-100 in PBS for 5 min. Triton X-100 lysates containing 
total cell-associated bacteria are then diluted 1:10 in PBS and plated onto agar plates to 
enumerate attached bacteria. Inhibition of bacterial adherence (IC50) is defined as the 
highest dilution of immune serum needed to inhibit 50% recovery of organisms, compared 
to an irrelevant control antibody. 
 
LT Neutralizing Antibody Assays. Serum will be used to determine toxin neutralization 
using an in vitro neutralization assay employing Y-1 adrenal cells as substrate, following 
neutral red uptake.40 Titers (ED50s) will be calculated as the reciprocal of the serum dilution 
which resulted in a 50% reduction in toxin activity. 
Alternatively, LT neutralization can be measured by inhibition of intracellular cAMP 
accumulation in CaCo2 or T84 cells. 
Antibody-Secreting Cells (ASC). Plasmablasts recognizing S. flexneri LPS and IpaB and 
ETEC CFA/I and LT will be measured using ELISPOT. In brief, purified peripheral blood 
mononuclear cells (PBMCs) resuspended in complete medium (2 x105 cells/well in 
quadruplicate) will be incubated in flat-bottom, 96-well nitrocellulose plates (MAHA 
S4510, Millipore) previously coated with target antigens, overnight at 37°C, 5% CO2. 
Optimal coating concentrations have been determined in preliminary experiments. 
Antibodies produced by antigen-specific plasmablasts will be detected by horse radish 
peroxidase-labeled anti-human IgG and IgA antibodies (HRP, Jackson Immunoresearch, 
West Grove, PA), followed by 3-amino-9-ethylcarbazole substrate (AEC, Calbiochem). 
The frequency of spot-forming cells (sfc) from 4 wells will be determined and reported as 
ASC/106 PBMC. A positive response will be defined as ≥8 ASC/106 PBMC. 
Antibodies in Lymphocyte Supernatants (ALS). To measure the antibodies in lymphocyte 
supernatants (ALS), isolated PBMCs at 1x107 viable lymphocytes per mL, will be re- 
suspended and incubated at 37°C, 5% CO2 for 72 h without antigenic stimulation. 
Supernatant fluid will be collected and tested in an ELISA assay to measure antigen- 
specific IgA and IgG antibodies released by the PBMCs. Briefly, ALS samples will be 
added to flat-bottom 96-well ELISA plates that have been previously coated with S. 

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flexneri LPS and IpaB and ETEC CFA/I and LT. Antigen-specific antibodies will be 
revealed by adding goat anti-human IgA conjugated to HRP, followed by AEC substrate. 
Endpoint titers will be calculated as the reciprocal dilution giving rise to an absorbance 
value of 0.4 above the background at 450 nm. A positive response will be defined as a ≥4- 
fold increase in ALS titer over baseline. 
ASC Homing Studies (only for Cohort 4). To measure the homing potential of these 
plasmablasts, PBMC will enumerated by flow cytometry based on their expression of 
molecules that are expressed in naïve (Bn) and memory B (BM) cells (e.g., CD19, CD27) 
and that direct these cells to home to distinct sites (e.g., integrin α4β7, CD62L).41 Several 
of these populations (e.g., Bn [CD19+ CD27-], BM [CD19+ CD27+] expressing integrin 
α4β7, but not CD62L, etc.) will be sorted and their ability to secrete specific anti-S. flexneri 
LPS and IpaB and ETEC CFA/I and LT antibodies will be determined by ELISPOT as 
described above. Sorting will be performed using a MoFlow Astrios EQ flow 
cytometer/cell sorter state-of-the-art system available in the CVD Flow Cytometry Core 
Laboratory. 
B Memory Cells (BM). BM cells are responsible for mounting rapid anamnestic antibody 
responses (recall responses) upon re-exposure to microbial antigens and thus are 
considered an indicator of long-term protection induced by vaccines or natural infection. 
BM responses will be measured using a dual color ELISPOT assay method, allowing for 
the simultaneous measurement of IgA and IgG sfc.42-45 Briefly, PBMC will be seeded in 
duplicate wells of plates coated with Shigella or ETEC antigens or total IgG or IgA. Plates 
will then be incubated with anti-human IgG-alkaline phosphatase and IgA-horseradish 
peroxidase (Jackson ImmunoResearch). IgA sfc are visualized with nitro-blue tetrazolium 
chloride (NBT) substrate (Vector laboratories) as blue spots, washed, and incubated with 
AEC substrate to visualize IgG sfc as red spots. Spots (sfc) will be enumerated using an 
automated ELISPOT reader (Immunospot 3B, Cellular Technologies Ltd) with aid of the 
Immunospot software version 5.0 (Cellular Technologies Ltd). Total and antigen-specific 
BM SFC are calculated as sfc/106 cells. Isotype-specific frequencies of BM against specific 
antigens are expressed as the percentages of specific BM per total IgA or IgG (specific BM 
sfc per 106/total Ig sfc per 106 × 100). 
Qualitative/Quantitative Stool Culture. For each stool culture specimen, a sterile cotton 
swab will be inserted into the stool (sampling nearest the most loose or liquid parts of stool 
or areas with gross blood) and then placed into a tube containing buffered glycerol saline 
(BGS) transport medium; short-term storage at 4°C (2°C-8°C). If a rectal swab is obtained, 
then the swab will be placed into a tube containing Gram-negative enrichment broth (GN, 
Fisher) with short-term storage at 4°C (2°C-8°C). For all stool specimens collected during 
the inpatient period and one specimen per outpatient day that requires stool culture, the 
CVD Microbiology Lab will plate the stool specimen onto Salmonella-Shigella agar (SS, 
Fisher) and MacConkey agar (MAC, Fisher) plates and GN broth for incubation overnight 
at 37°C. Hemoccult testing is to be repeated from stools that appear to contain gross blood; 
this procedure will have already been performed by the clinical research team on the 
inpatient unit and is a confirmation test. After overnight incubation, the GN broth tubes 

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will be sub-cultured onto SS agar and incubated overnight at 37°C. After incubation, SS 
and MAC plates are examined for single pure lactose-negative colonies (colorless and 
translucent) and are picked onto Triple Sugar Iron (TSI, Remel) agar slants and Motility 
Indole Ornithine medium (MIO, BD-Difco) for overnight incubation at 37°C. Those 
colonies that are non-motile and produce typical TSI reactions for Shigella (alkaline slant 
over acid but without gas) may be identified by commercial API 20E and must be 
confirmed by agglutination. Positive agglutination of a Shigella colony with one or more 
specific antisera (Denka Seiken available from Hardy Diagnostics as MastAssure) will 
identify a S. flexneri 2a strain. 
To quantify the level of excretion of the strain (quantitative counts), 10-fold dilutions will 
be made from ~1 g of each stool; from each dilution, 100 µL will be spread on MAC agar 
and incubated overnight at 37°C. From the dilution plates, lactose negative colonies are 
counted; plates containing 30 – 300 colonies yield the most accurate counts. Up to 10 
suspicious colonies will be agglutinated with antisera to calculate the quantitative counts 
of the Shigella strain. The quantitative count is calculated by multiplying the number of 
suspicious colonies by the ratio of confirmed positive isolates out of the 10 isolates tested. 
Genetic Stability of Vaccine Organisms. In selected recovered isolates, the confirmation of 
the presence of the genes encoding both CFA/I and LThA2B will be performed in the 
laboratory of Dr. Eileen Barry. Colonies identified as Shigella will be tested by PCR for 
the presence of the genes encoding CFA/I and LTB. Briefly, 10 – 20 Shigella-positive 
colonies from each volunteer at each time point post-vaccination will be tested. Direct 
colony PCR or extracted DNA will be used as the template for PCR using primer pairs 
specific for Shigella (targeting the ipaH or virG genes), CFA/I (targeting cfaB gene) or 
LTB (targeting eltB gene). These data are expected to confirm the proportion of Shigella 
isolates shed that maintain ETEC antigen-encoding genes over time in the human host. A 
subset of colonies from different volunteers will be selected for expansion, genomic DNA 
isolation, and sequencing of the genomic region encoding the CFA/I and LTB antigens. 
These studies will confirm the chromosomal integrity and location of transgene insertion, 
which will be an exploratory endpoint. 
An additional subset of colonies from multiple volunteers will be expanded to confirm 
maintenance of Shigella and ETEC antigen-specific phenotypes. Isolates will be tested for 
expression of CFA/I by western blot and positive hemagglutination of human type A RBC. 
LTB expression will be confirmed by western blot analysis of periplasmic fractions. The 
invasion capacity of Shigella in intestinal HT-29 cells will be quantified and lack of 
intracellular replication confirmed. 
Fecal IgA. Fecal supernatants will be tested for total and antigen-specific (Shigella LPS, 
IpA, CFA/I, and LT) IgA ELISA as previously described.46 Total and antigen-specific IgA 
concentrations are calculated by interpolation of the regression-corrected absorbance 
values produced by serially diluted stool samples and extrapolation from a standard curve 
of known concentrations of human IgA (Calbiochem). In order to adjust for individual 
variations in the IgA content of stools, the ratio of specific IgA to total IgA antibody are 

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calculated, and a positive response is defined as a ≥4-fold increase of the ratio over the 
baseline ratio. The threshold of detection for each assay will be determined for each antigen 
and any baseline values below this level of detection will be designated the value of that 
limit of detection for the calculation of fold increases post-vaccination. 
 
6.2.3 Specimen Collection, Preparation, Handling and Shipping 
Clinical laboratory specimens will be shipped to the contract CLIA-approved clinical 
laboratory for analysis. Research study specimens are intended to be stored for potential 
future-use research at CVD’s long-term storage facility. Instructions for research specimen 
preparation, handling, and storage are described in the Manual of Procedures (MOP). At 
this time, there is no planned shipment of research study specimens. 
 
7 
STUDY SCHEDULE 
Complete study schedule details listed by type of visit are described below. Refer also to 
Appendix A: Study Schedule of Events. 
 
7.1 
Screening 
Potential participants may be screened for eligibility up to 60 days prior to enrollment. Interested 
potential participants, responding to advertisements, campus flyers, or notification through 
existing databases, will be instructed to call CVD to receive further information about the study. 
CVD research staff will receive these inquiries and read through an IRB-approved telephone 
script which describes basic information on the study topic, duration and number of visits, 
procedures, and eligibility criteria. No personal health information is solicited and afterwards 
interested potential participants may be given an appointment to attend an Orientation Session 
for formal screening. 
At the Orientation Session, the consent process continues. CVD research staff will provide a 
detailed description of all aspects of the study, including the rationale and background, the public 
health significance, the procedures and schedule of visits, and a detailed discussion of the risks 
and the presence or absence of benefit to them, as appropriate. This Orientation Session is 
commonly one hour long. The prospective subject is given a copy the study consent form 
(without the signature page) and is encouraged to discuss his/her participation with family 
members or advisors before agreeing to sign the informed consent form and proceeding with any 
screening procedures. 
After the informed consent form is signed, the subject is interviewed one-on-one by a member of 
the study team to discuss the study. A brief written examination is administered to assess the 
volunteer’s comprehension of the study (i.e., Comprehension Assessment Tool). If this quiz is 
passed (≥70% correct answers), the research staff complete the medical history and concomitant 
medication forms and draw blood for eligibility testing. Study subjects are also asked to sign a 
verification of Notification of Privacy Practices receipt and Health Insurance Portability and 
Accountability Act (HIPAA) authorization form. The subject also meets the PI, or designee, and 

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has a physical examination to complete the eligibility screening. If the subject has passed all the 
eligibility criteria, he/she is then invited to proceed in the study and is given an appointment for 
the next visit. The screening procedures may be completed in a single visit, but it is acceptable to 
complete all the screening procedures on multiple visits, as long as the data of initiation of 
screening does not exceed 60 days prior to vaccination. 
It is recognized that volunteer studies must be carried out in an environment where no coercion is 
applied, and where volunteers can be adequately informed of the purpose, nature, procedures, 
risks and hazards of the study. To assess and document comprehension of the material presented, 
each subject must pass a written quiz that covers aspects of the study including the purpose, 
procedures, risks, benefits and pertinent microbiology with a score 70% or higher. Incorrect 
answers will be reviewed with the subject. The quiz may be retaken once, after a review of the 
consent form. The Comprehension Assessment Tool is dated and signed by the subject and by a 
research staff member and made part of the permanent record. 
Another important feature of our consent process is the repeated demonstration of both initiative 
and reliability by the prospective subject. There are multiple opportunities for the subject to 
decline to proceed further in the process. This deliberate education and screening process 
contributes to the informed nature of the subject’s consent. The process also increases the 
likelihood that the subject will be committed to completing the entire inpatient containment 
portion of this study. Prospective subjects will be carefully screened to ensure that they are in 
good physical and mental health. 
The screening criteria also are intended to rule out those with histories of major gastrointestinal 
surgery, functional gut problems, and persons with IgA deficiencies. 
The screening procedures include: 
• Signed informed consent 
• Administration of the study Comprehension Assessment Tool 
• Obtaining vital signs (oral temperature, blood pressure, pulse, height, and weight) 
• Collection of medical history 
• Collection of concomitant medication history 
• Collection of a detailed travel history, including any episodes of diarrhea 
• Perform physical examination, to be performed by a study clinician 
• Obtain the following screening studies (~16 mL blood); the acceptable values for the 
screening tests are defined in Appendix B: 
o Complete Blood Count (CBC) with differential and platelet count for the 
evaluation of WBC, ANC, Hemoglobin, and Platelets 
o Creatinine, ALT, and Total Bilirubin 
o Serum IgA 
o HLA-B27 histocompatibility 
o Pregnancy test (if female; serum β-HCG) 
o HIV antibody 
o Hepatitis B surface antigen 
o Hepatitis C antibody 

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Note: Since intravenous fluids are a part of the planned management of severe diarrhea, if more 
than 3 attempts at venipuncture are required for obtaining screening labs, then we will consider 
that person ineligible on the basis of poor venous access. 
 
7.2 
For Cohorts 1 – 3, Inpatient Containment Period 
Study participants that are determined to be eligible will be scheduled for admission to the 
inpatient containment unit (research isolation ward). The anticipated duration of the inpatient 
stay is 6 days. 
 
7.2.1 SARS-CoV-2 PCR test (within 72 hours before admission to the inpatient unit) 
During the time when a U.S. Public Health Emergency for COVID-19 exists, study 
participants will be instructed to come to the research clinic to perform a SARS-CoV-2 
PCR test. This is a procedure that is consistent with current hospital policy (UMMC 
PolicyStat 11947187). It is anticipated that should the testing policy change, the protocol 
would be revised to be consistent with the hospital policy. A nasal swab is to be 
performed for the PCR test; no additional procedures are planned to be performed at this 
visit. This test result must be negative for eligibility to enter the inpatient unit. 
 
7.2.2 Acclimatization (Day -1, one-day prior to vaccination,) 
Study participants will start their inpatient stay with 1 day for acclimatization, during 
which we educate and familiarize each subject with the protocol-required procedures 
(e.g., stool handling), hygiene practices, and the “Rules and Procedures” to be followed 
while on the inpatient unit. In addition, during the acclimatization period, we monitor 
behavior, person-to-person interactions, mood, etc. to assess each study participant for 
any behavior or attitudes which might not be appropriate for an inpatient containment 
study (i.e., combativeness, anti-social behavior, anger outbursts, destruction of property, 
etc.). Any evidence that a subject may demonstrate behavior which might pose a safety 
risk to themselves, other subjects, or staff could be cause for ineligibility for vaccination 
and the remainder of the inpatient stay. Refusal to comply with protocol-required 
procedures, adherence to hygiene practices, or repetitive breaking of the “Rules and 
Procedures” could also constitute ineligibility. This observation during the 
acclimatization period may be considered an imperfect and rather subjective method, but 
we have not identified any other good alternate objective measures that substitute for this 
direct observation procedure. Any subject who is deemed ineligible will be discharged, 
prior to vaccination, and eligible back-up participants will be used. All back-up study 
participants that are not vaccinated will be discharged from the inpatient ward upon the 
completion of the target number of vaccinations. 

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7.2.3 Enrollment/Vaccination Day (Day 1) 
On the morning of vaccination, the enrollment and randomization process will be 
completed prior to oral ingestion of the blinded study product and a final eligibility 
confirmation will be completed. Additionally, the following lab tests will be completed 
prior to vaccination: 
• Baseline vitals (oral temperature, pulse, and blood pressure) will be recorded 
• Baseline Clinical Safety Laboratory tests (a 12.5 mL blood sample, collected prior 
to vaccination) which will include: 
o CBC with differential and platelet count for the evaluation of WBC, ANC, 
Hemoglobin, and Platelets 
o Creatinine, ALT, and total bilirubin 
• Baseline Research Blood tests (approximately 125 mL blood volume will be 
obtained) 
After fasting for a minimum of 90 minutes, participants will drink 120 mL of sodium 
bicarbonate solution (~1.3% NaHCO3); approximately 1 – 2 minutes later, subjects will 
ingest the blinded study product. Subjects will have nothing by mouth, except water, for 
90 minutes before and after ingestion of the blinded study product. 
 
7.2.4 Post-Vaccination Observation Period (Day 1 through discharge) 
For the 96 hours (4 days) following vaccination, study participants will be observed on 
the Research Isolation Ward and the following procedures will be performed: 
• Vital signs will be measured at least 3 times daily (approximately every 8 h) 
• All stools will be graded for consistency (Grade 1 – 5) and any diarrheal stool 
(grade 3 or higher) will be weighed; a 1:1 weight per volume ratio will be assumed 
for conversion of diarrheal stools 
• All vomitus will be weighed 
• For each day post-vaccination, the maximum temperature, total diarrheal stool 
volume, number of diarrheal stools, total vomitus volume, and number of vomiting 
episodes will be calculated, as part of the reactogenicity assessment. 
• For each day, the maximum of an adverse symptom over that day will be recorded. 
The documentation of the previous day’s maximum adverse symptom (prior 24 h) 
will include an attribution—either to the study drug or an alternate etiology 
(unrelated to study). The anticipated subjective adverse symptoms include 
abdominal discomfort (pain/cramping), nausea, myalgia/arthralgia, anorexia, 
malaise, and headache. These adverse symptoms will be graded according to the 
scales shown in Appendix D. 
 
7.2.5 Discharge (~Day 5) 
Participant must feel in good (baseline) health and have no fever or diarrhea for at least 
12 h prior to discharge. Should there be excessive reactogenicity (e.g., severe diarrhea or 

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ongoing diarrhea within 12 h of discharge), then the participant will be initiated on a 3- 
day course of antibiotic therapy. Subjects may be discharged during their 3-day course of 
antibiotics as long as they are symptom-free for at least 12 h, unless the investigator 
deems discharge not to be in the best interest of the participant’s welfare. 
Prior to discharge, participants will be provided a digital thermometer for the assessment 
of daily oral temperatures; participants will be encouraged to take their temperature at the 
same time each day. Participants will also be provided a Memory Aid document and will 
be instructed on how to record the daily temperature and adverse events onto the 
document. 
 
7.3 
For Cohort 4, Vaccination Visits for Dose #1 and Dose #2 (Day 1 and Day 29 ±3) 
On the morning of vaccination, subjects will report to the clinic and baseline vitals will be 
recorded (oral temperature, pulse, and blood pressure) and a final eligibility confirmation will be 
completed prior to oral ingestion of the first dose of blinded study product. The enrollment and 
randomization process will be completed, prior to vaccination. 
Baseline Clinical Safety Laboratory tests (12.5 mL) will be obtained prior to vaccination to 
include: 
• CBC with differential and platelet count for the evaluation of WBC, ANC, Hemoglobin, and 
Platelets 
• Creatinine, ALT, and total bilirubin 
Baseline Research Blood tests (approximately 125 mL) will be obtained prior to vaccination. 
Baseline Stool will be collected prior to vaccination. A stool collection kit and instructions will 
have been provided prior to the scheduled visit. 
After confirmation of at least 90 minutes of fasting, eligible subjects will drink 120 mL of 
sodium bicarbonate solution (~1.3% NaHCO3); approximately 1 – 2 minutes later, subjects will 
ingest the blinded study product. Subjects will have nothing by mouth, except water, for 90 
minutes before and after ingestion of the blinded study product. 
Prior to the completion of the first vaccination day visit, participants will be provided a digital 
thermometer for the assessment of daily oral temperatures; participants will be encouraged to 
take their temperature at the same time each day. Prior to the completion of both vaccination day 
visits, participants will be provided a Memory Aid document and will be instructed on how to 
record the daily temperature and adverse events onto the document. 
 
7.4 
Outpatient Clinic Visits 
 
7.4.1 For Cohorts 1 – 3, Day 8 (+ 1), Day 29 (± 3), Day 43 (± 3), Day 181 (± 7) 
Participants will be scheduled for outpatient visits at approximately 1 week, 4 weeks, 6 
weeks and 6 months after vaccination. On visit Day 8, the Memory Aid document will be 

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collected and the information with be reviewed with the participant. During the visits on 
Day 8 and Day 29, the emergence of adverse events and any changes to medical history 
and concomitant medications will be assessed. The occurrence of any serious adverse 
events will be assessed through Day 181. Blood will be drawn for safety labs (12.5 mL) 
on Day 8 and for research (approximately 37.5 – 100 mL) on Days 8, 29, 43, and 181 
(target volumes according to Appendix A). Stool specimens are to be collected on each 
scheduled outpatient clinic visit. A stool collection kit and instructions will have been 
provided prior to the scheduled visit. 
 
7.4.2 For Cohort 4, Day 3 (+ 1), Day 8 (+ 1), Day 31 (+ 1), Day 36 (+ 1), Day 57 (± 3), Day 
210 (± 7) 
 
Outpatient follow-up visits will be scheduled 2 days and one-week post-vaccination for 
each dose. (The timing of Days 31 and 36, and the allowable windows associated with 
those visits, are intended to be relative to the timing of dose #2.) Additional scheduled 
clinic visits are to be completed at 4 weeks and 6 months after second dose of 
vaccination. On visits for Day 8 and Day 36, the Memory Aid document will be 
collected, and the information will be reviewed with the participant. 
During all visits through Day 57 (or 28 days following the last dose of vaccine), the 
emergence of adverse events (AEs) and any changes to medical history and concomitant 
medications will be assessed. The occurrence of any serious adverse events (SAEs) will 
be assessed through Day 181. Blood will be drawn for safety labs (12.5 mL) on Days 8 
and 36 and for research (approximately 50 – 100 mL) on Days 8, 36, 57, and 210 (target 
volumes according to Appendix A). Stool specimens are to be collected on each 
scheduled outpatient clinic visit. A stool collection kit and instructions will have been 
provided prior to the scheduled visit. 
 
7.5 
Unscheduled Visit(s) or Early Termination Visits, if applicable 
Subjects who experience any serious or severe adverse effects or who experience an event of 
concern can be scheduled for an outpatient visit for further evaluation. If an unscheduled visit 
occurs, a member of the clinical study team (PI, sub-investigator, nurse coordinator, or clinical 
nurse) will interview and evaluate the subject to determine the cause of the visit and provide care 
as needed. 
Symptomatic participants who demonstrate shedding of vaccine organisms on the Day 8 visit 
will be contacted for the initiation of antibiotic therapy and the collection of a stool specimen; 
this unscheduled visit will be arranged for the earliest convenient time for that participant. 
Asymptomatic participants who demonstrate shedding of vaccine organisms on the Day 8 visit 
will not be initiated on antibiotic therapy but will have an unscheduled visit arranged for the 
earliest convenient time for that participant for the collection of a stool specimen. Additional 
outpatient clinic visits, scheduled every other day, will be continued for the collection of stool 
specimens until there are two negative stool culture days in a row. 

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7.6 
COVID-19 related considerations 
The investigators will monitor the situation related to the COVID-19 pandemic to ensure that 
potential risks to study participants and staff are mitigated. The following strategies will be 
implemented: 
• The conduct of the study will be in accordance with state and local travel 
limitations/restrictions. 
• Study staff will take appropriate precautions to protect study participants 
• Safety assessments may be performed by phone call, when appropriate 
• If travel restrictions or COVID-19 related illness impacts the conduct of the study, 
specific measures will be taken to mitigate risk to study staff and participants and monitor 
protocol deviations due to COVID-19 illness and/or COVID-19 control measures. 
• The investigators will attempt to comply with the FDA Guidance on Conduct of Clinical 
Trials of Medical Products during COVID-19 Public Health Emergency. 
https://www.fda.gov/media/136238/download 
• compliance with hospital (UMMC) policies, when applicable 
 
8 
SAFETY ASSESSMENT AND REPORTING 
 
8.1 
Definition of Adverse Event (AE) 
An AE is any untoward medical occurrence in a participant after administration of the blinded 
study product regardless of its causal relationship to the study treatment. An AE can therefore be 
any unfavorable and unintended sign (including abnormal laboratory findings), symptoms, 
physical examinations, or disease temporally associated with the use of the study product, 
whether or not related to the study product. This definition includes exacerbations of pre-existing 
conditions. Stable, pre-existing conditions which do not change in nature or severity during the 
study are not considered AEs; however, these should be reported as part of the medical history. 
Solicited AEs are pre-specified AEs that could potentially be in association with the blinded 
study product. Typically, a solicited AE is assumed to be associated with the study product, but 
investigators may assign causality to an alternate etiology, if clearly present. 
• The solicited AEs are diarrhea, dysentery*, fever, nausea, vomiting, abdominal 
discomfort, tenesmus (a feeling on inadequate defecation), myalgia (muscle aches), 
arthralgia (joint aches), and anorexia (decreased appetite) (see Appendix D). 
*Dysentery can only be reliably assessed during the inpatient stay (Cohorts 1 – 3), whereupon every stool will be 
evaluated with hemoccult testing. 
Unsolicited AEs are any AEs reported spontaneously by the participant, observed by the study 
personnel during study visits or those identified during review of medical records or source 
documents. Investigators may attempt to assign causality of unsolicited AEs to either the study 
product or an alternate etiology. 

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Adverse events of special interest (AESI) which may occur will be recorded and given special 
attention for review of ascertainment of relationship with vaccination. Some example AESIs 
include any occurrence of reactive arthritis, uveitis, urethritis, bacteremia, and new diagnoses of 
auto-immune or immune-mediated conditions post-vaccination. 
 
8.1.1 Grading of Severity of an AE 
All AEs will be assessed by the clinician using a protocol-defined grading system 
(Appendix D). For events not included in the protocol-defined grading system or in the 
DAIDS toxicity table or the FDA toxicity table, the following guidelines will be used to 
quantify severity: 
• 
Mild: events which require minimal or no treatment and do not interfere with the 
participant’s daily activities. 
• 
Moderate: events which result in a low level of inconvenience or concern with the 
therapeutic measures. Moderate events may cause some interference with 
functioning. 
• 
Severe: events which interrupt a participant’s usual daily activity and may require 
systemic drug therapy or other treatment. Severe events are usually incapacitating. 
• 
Life-threatening: any adverse drug experience that, in the view of the investigator, 
places the participant at immediate risk of death as a result of the reaction as it 
occurred; this does not include a reaction that, had it occurred in a more severe 
form, might have caused death. 
Changes in the severity of an AE should be documented to allow an assessment of the 
duration of the event at each level of intensity to be performed. AEs characterized as 
intermittent require documentation of onset and duration of each episode. 
 
8.2 
Relationship to Study Product 
Relationship (causality or attribution) of all AEs to the study product or to an alternate 
etiology (unrelated to the study) is part of the documentation process, but it is not a factor 
in determining what is (or is not) reported in the study. If there is any doubt as to whether 
a clinical observation is an AE, the event should be reported. All AEs must have their 
relationship assessed using one of two terms: “related to study drug” or “not related.” To 
help assess whether an AE is related to a study product or not, the following guidelines 
are to be used: 
• Related to study product – There is a reasonable possibility that the study drug 
caused the adverse event (perhaps due to the timing of onset of the symptoms). 
“Reasonable possibility” means that there is evidence to suggest a causal 
relationship between the study product and the AE and no reasonable alternate 
etiology can be determined. 
• Not Related – The event is not related to the study product because of a reasonable 
alternate etiology. That is, an alternate etiology can be identified and is likely and/or 
feasible. 

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8.3 
Definition of Serious Adverse Event (SAE) 
An SAE is any AE that results in any of the following outcomes: 
1. Death 
2. A life-threatening event. Life-threatening events mean that the study participant was, in 
the opinion of the site PI or Sponsor, at risk of death at the time of the event; it does not 
refer to an event which hypothetically might have caused death if it were more severe. 
3. Requires inpatient hospitalization or prolongation of existing hospitalization 
4. Results in persistent or significant incapacity or substantial disruption of the ability to 
conduct normal life functions 
5. Congenital abnormality or birth defect 
6. An important medical event that may not result in one of the above outcomes but may 
jeopardize the health of the study participant and/or requires medical or surgical 
intervention to prevent one of the outcomes listed in the above definition of SAE. 
Examples of such medical events include allergic bronchospasm requiring intensive 
treatment in an emergency room or at home, blood dyscrasias or convulsions that do not 
result in inpatient hospitalization, or the development of drug dependency or drug abuse. 
 
8.4 
Reporting Procedures 
The assessment of the safety of the blinded study product will be through the detection and 
documentation of AEs, both solicited AEs (reactogenicity) and unsolicited AEs, and/or clinically 
significant laboratory abnormalities, through 28 days after the last dose of vaccination. The 
occurrence of SAEs will be reported for the interval between the last day of vaccination and 180 
days after (Days 29-181 for Cohorts 1 – 3; Days 57-210 for Cohort 4). 
 
8.4.1 SAE Detection and Reporting 
All SAEs will be: 
• Recorded in the research record 
• Reported to the local IRB, per local IRB guidelines 
• Reviewed and evaluated by a study clinician and the PI 
• Followed through resolution by a study clinician 
All deaths and immediately life-threatening events, whether related or unrelated, will be 
reported to the study sponsor within 24 h of site awareness. Other SAEs regardless of 
relationship, will be reported to the study sponsor within 24 h of site awareness 
All SAEs will also be reported to DMID within 24 hours of awareness. The DMID 
Phamacovigilance Group (PVG) will forward the SAE report to the designated DMID 
Medical Monitor for review. The DMID Medical Monitor may contact the PI for any 
questions or concerns regarding the event. The PI holds the responsibility of the 
assessment of the SAE and reporting requirements to the FDA and DMID’s role is 
consultative. 

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DMID Pharmacovigilance Group 
Clinical Research Operations and Management Support (CROMS) 
6500 Rock Spring Dr., Suite 650 
Bethesda, MD 20817 
SAE Hot Line: 1-800-537-9979 (US) 
SAE Fax: 800-275-7619 (US) 
SAE Email: PVG@dmidcroms.com 
 
8.4.2 Regulatory Reporting 
A suspected unexpected serious adverse reaction (SUSAR) is a suspected adverse 
reaction that is both serious and unexpected. As specified in 21 CFR 312.32, SUSARs 
will be reported by the Sponsor of the Investigational New Drug Application (IND) to the 
FDA and to all participating PIs in an IND safety report as soon as possible, no later than 
15 calendar days after the Sponsor becomes aware of the suspected adverse reaction (21 
CFR 312.32(c)(1)). 
Unexpected fatal or life-threatening suspected adverse reactions represent especially 
important safety information and will be reported to FDA as soon as possible but no later 
than 7 calendar days following the sponsor’s initial receipt of the information. 
 
8.4.3 Reporting of Pregnancy 
Pregnancies occurring in study subjects will be reported on the Pregnancy Report form. 
No further study vaccinations will be administered to pregnant subjects, but with the 
subject’s permission all protocol-required venous blood samples will be obtained and the 
subject will continue to be followed for safety for the duration of this trial. Efforts will be 
made to follow all pregnancies reported over the course of this trial through pregnancy 
outcome, pending the subject’s permission. 
 
8.5 
Halting Rules 
Further enrollments and study vaccinations will be halted for an ad hoc Safety Monitoring 
Committee (SMC) review if any of the following events occur: 
• One or more participants experience an SAE related to the product 
• Two or more participants experience the same or similar Grade 3 AE considered related to 
the study product 
• Three or more participants experience the same moderate (Grade 2) safety laboratory AE. 
The study will not continue until the SMC has made the determination that the halt may be lifted. 
The lifting of a halt may require changes to the protocol and/or Informed Consent Form and is 
upon the advisement of the SMC. 

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Should any subject be experiencing a higher than grade 2 AE at the time of the scheduled second 
dose of blinded vaccine, then the second dose will be deferred or cancelled. This decision can be 
made upon investigator judgement and with discussion with the study participant. 
 
8.6 
Safety Oversight 
An independent SMC will perform the oversight of safety for this study. The SMC voting 
members will consist of 4 scientists that are not involved in the conduct of the study. The 
primary responsibility of the SMC is to monitor participant safety. The SMC will take study- 
specific data, as well as relevant background information about the disease, test agent, and target 
population under study into consideration. The SMC will review the cumulative safety data 
shortly after completion of each inpatient phase, prior to initiation of enrollment for the 
subsequent study cohort and will approve or reject advancement to the next cohort. The SMC 
will be authorized to suspend the study, recommend amendments to the protocol, and/or request 
further information for their review. One of the non-voting members of the SMC will be 
designated as the Independent Safety Monitor (ISM); this person will be local to CVD but will 
not be a part of the study team. The ISM will be responsible for conducting a comprehensive 
review, should there be a halt in the study or at the request of the SMC. 
Should there be a halt in the study, no further study product dosing will be performed until the 
halt is lifted; all enrolled participants will continue to be followed for safety. For participants that 
are in the Research Isolation Ward at the time of a halt, the management of diarrhea or other 
symptoms will continue, until resolution. An SMC Charter will be reviewed and approved by the 
SMC members prior to the initiation of the trial and will include the scheduled frequency/timing 
of SMC meetings, types of data for review, halting rules, and roles/responsibilities. 
The SMC will also recommend the dose to be evaluated in the fourth cohort. The intent will be 
for the fourth cohort to evaluate the highest dose that is well-tolerated, that is, the highest dose 
for which there is an absence of significant reactogenicity. Nonetheless, the SMC may also 
consider data from immunological outcome measures as part of the target dose selection for the 
fourth cohort. The immunological outcome data, which may be available at the time of SMC 
review, may consist of serum IgG and IgA ELISA, ASC, and ALS responses. Functional 
antibody assay data and cellular-mediated immune responses may not be available at the time of 
the SMC review. 
 
9 
CLINICAL MONITORING STRUCTURE 
Site monitoring is conducted to ensure that the rights and well-being of human subjects are 
protected, that the reported trial data are accurate, complete, and verifiable, and that the conduct 
of the trial is in compliance with the currently approved protocol/amendment(s), with GCP, and 
with applicable regulatory requirement(s). External monitoring will be performed at standard 
intervals to oversee the conduct of and reporting of data from this study. The site monitoring will 
include ensuring appropriate clinical investigator supervision of study site staff and third-party 
contractors. 

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9.1 
Site Monitoring Plan 
Independent internal audits will be conducted, as described in the CVD Quality Management 
Plan. All clinical trials conducted at CVD are monitored for quality assurance and quality control 
under the CVD-Wide Quality Management (QMP) Plan. The results of the internal audit are 
reviewed with the PI, coordinator, and Clinical Research Manager; Corrective Action Plans are 
formulated in response to any issue. The CVD’s 19-page QMP (Version 10.0, 04 February 2022) 
has been reviewed against the minimum requirements set forth in current standards, such as the 
DMID CQMP Policy (Version 6.0, 13 July 2016). 
 
10 STATISTICAL CONSIDERATIONS 
 
10.1 
Study Outcome Measures 
The safety of CVD 1208S-122 will be measured by reactogenicity and AEs, within the time 
periods described. The immune response to CVD 1208S-122 will be summarized according to 
the outcome measures described. The genetic stability of the attenuated organism will be 
evaluated by carefully documenting shed organisms recovered from stool cultures and examining 
these isolates for the genes that have been inserted into the chromosome. 
 
10.2 
Sample Size Considerations 
This is a phase 1 first-in-human study, so the primary purpose of statistical comparisons is to 
screen out potential solicited reactogenicity events that need further clinical evaluation. 
Therefore, the statistical analyses are not considered formal statistical hypothesis tests and it is 
acknowledged that there will be inflated Type I errors (i.e., inflated false statistical significances) 
from performing multiple unadjusted comparisons. No formal sample size calculations were 
performed to guide the sample size of this phase 1 study. Based on the result of the observed 
highest tolerated dose (Table 2), a total of 18 to 30 participants will receive active vaccine and 
this study design allows for at least 87% chance of observing an AE that has an 11% chance of 
occurrence. This study is not designed to evaluate for rare AEs. 
Table 2: Sample size 
 
Highest 
tolerated 
dose 
Number of 
participants 
receiving active 
vaccine 
Number of 
participants 
receiving placebo 
Among the participants who receive 
active vaccine, probability of 
observing at least one participant 
with an adverse effect that has an 
11% chance of occurrence 
108 cfu 
6+12= 18 
2+4=6 
0.877 
109 cfu 
6+6+12 = 24 
2+2+4=8 
0.939 
1010 cfu 
6+6+6+12 = 30 
2+2+2+4=10 
0.970 

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10.3 
Statistical Analysis Plan 
Primary Objective 1. To evaluate the safety and clinical tolerability of oral doses of CVD 
1208S-122, ranging from 108 cfu to 1010 cfu organisms, with particular attention to the 
occurrence of objective signs of diarrhea, dysentery or fever. 
Endpoints: 
• The number, proportion, and severity of fever, diarrhea, or dysentery (hemoccult testing 
will only be performed during the inpatient days) within 7 days of vaccination, for all 
those receiving vaccine and for each of the vaccine dosages evaluated 
• The number, proportion, and severity of solicited local and systemic adverse reactions 
within 7 days of vaccination for all those receiving vaccine and for each of the vaccine 
dosages evaluated 
• The number, proportion, severity, and relatedness of non-serious unsolicited adverse 
reactions within 28 days of vaccination for all those receiving vaccine and for each of the 
vaccine dosages evaluated 
• The number, proportion, and severity of clinical safety laboratory adverse events from the 
time of each study vaccination through approximately 7 days after each study 
vaccination. 
• The occurrence of SUSARs in the study 
• The occurrence of all SAEs, regardless of the assessment of relatedness, from the time of 
the first vaccination through approximately 6 months after the last study vaccination 
 
Primary Objective 2. To assess the degree of fecal shedding of CVD 1208S-122 and the genetic 
stability of the excreted live vector isolates by genetic characterization of the vaccine organisms 
recovered in stool cultures, to be determined with inpatient Cohorts 1 – 3. 
Endpoints: 
• The geometric mean number (and interquartile range) of vaccine organisms expressed as 
cfu/mL for each dosage group, per day, and at the peak of shedding 
• The number and proportion of sequential days of fecal shedding of Shigella organisms 
which are documented to contain the genes expressing ETEC antigens for each dosage 
group. Genetically stable organisms will be defined as being PCR positive for Shigella 
(ipaH or virG), CFA/I (cfaB), and LTB (eltB) 
 
Secondary Objective 1. To measure the mucosal (α4β7-positive IgA antibody secreting cell 
[ASC], antibody in lymphocyte supernatant [ALS], fecal IgA antibody) and systemic (serum IgG 
and IgA antibody) immune responses to ETEC antigens (CFA/I and LThA2B) and Shigella 
flexneri 2a antigens (LPS and Ipa) following oral immunization with CVD 1208S-122. 
Endpoints: 
• The number and proportion of 4-fold rises; GMT; mean fold-rises (compared to 
baseline); median titer; and peak post-vaccination responses in serum anti-LPS, anti-Ipa, 

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anti-CFA/I, and anti-LT IgA, IgG, or both antibodies post-vaccination, for each dosage 
group 
• The number and proportion of participants with a positive response (≥8 sfc/106 cells); 
mean (and geometric mean) count in anti-LPS, anti-Ipa, anti-CFA/I, and anti-LT ASC 
IgA and IgG, for each dosage group 
• The number and proportion of participants with a 4-fold rise; mean (and geometric 
mean) titers in anti-LPS, anti-Ipa, anti-CFA/I, and anti-LT ALS IgA and IgG, for each 
dosage group 
• The number and proportion of 4-fold rises; GMT; mean fold-rises (compared to 
baseline); median titer; and peak post-vaccination responses in adjusted antigen-specific 
fecal IgA ratios, for each dosage group 
• The number and proportion of participants with anti-LPS, anti-Ipa, anti-CFA/I, and anti- 
LT α4β7-positive IgA ASC, for each dosage group (ASC mucosal homing studies will 
only be performed on participants of Cohort 4) 
 
Secondary Objective 2. To evaluate the ETEC and Shigella functional antibody responses 
following oral immunization CVD 1208S-122. 
Endpoints: 
• The number and proportion of 4-fold rises; GMT; mean fold-rises (compared to 
baseline); median titer; and peak post-vaccination responses in Shigella SBA and OPKA 
titer, for each dosage group 
• The number and proportion of 4-fold rises; GMT; mean fold-rises (compared to 
baseline); median titer; and peak post-vaccination responses in ETEC mannose-resistant 
Hemagglutination Assay (MRHA) titer, for each dosage group 
• The number and proportion of 4-fold rises; GMT; mean fold-rises (compared to 
baseline); median titer; and peak post-vaccination responses in ETEC IC50 titer (for 
adhesion to HT-29 cells), for each dosage group 
• The number and proportion of 4-fold rises; GMT; mean fold-rises (compared to 
baseline); median titer; and peak post-vaccination responses in LT neutralizing titer, for 
each dosage group and timepoint 
 
Secondary Objective 3. To assess transmissibility of CVD 1208S-122 by determining whether 
the vaccine strain is acquired (as detected by stool culture) by placebo recipients living in close 
proximity on the Research Isolation Ward, thus approximating household contact conditions (to 
be assessed in Cohorts 1 – 3). 
Endpoint: 
• The number and proportion of transmission events, defined as any time during the 
inpatient period a placebo recipient has a detectable vaccine strain in a stool sample (as 
long as it is not considered a contaminant during handling or in the lab), for each dosage 
group 

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Exploratory Objective 1. To measure the circulating B memory cell responses to ETEC 
antigens (CFA/I and LThA2B) and Shigella flexneri 2a antigens (LPS and Ipa) following oral 
immunization with one dose of CVD 1208S-122 
Endpoint: 
• The number and proportion of participants with a positive response (≥8 sfc/106 cells); 
mean (and geometric mean) count in anti-LPS, anti-Ipa, anti-CFA/I, and anti-LT IgA and 
IgG memory B cells, for each dosage group 
 
Exploratory Objective 2. To collect, separate and store peripheral blood mononuclear cells 
(PBMCs) so that subsequently the immune responses to CVD 1208S-122 can be further 
characterized in greater detail including the measurement of T memory and effector cells, 
homing markers and cytokine production 
Endpoint: 
• Not applicable 
 
10.4 
General Statistical Principles 
The distributions of all measures will be examined and described in terms of numbers, means, 
standard deviations, median, interquartile ranges, minima and maxima for continuous variables 
(such as geometric mean titers); counts and proportions for categorical variables (such as the 
presence of the genes encoding CFA/I and LTB, antibody response [Y/N]), will be determined 
for each group separately. Continuous variables of interest that are not normally distributed will 
be transformed if needed. To compare each vaccine dose group with the placebo group, t-test or 
Mann-Whitney U test will be used for continuous variables as appropriate; a Chi-squared test or 
Fisher’s exact will be used for categorical variables as appropriate. All analyses will be 
performed using Stata/SE version 15. 
All participants in the enrolled population who are randomized and receive a blinded vaccination 
will be included in the Full Analysis (FA) Population. All safety, fecal shedding, and genetic 
stability analysis will be performed using this FA population. Treatment groups for the safety 
analysis will be assigned according to the actual treatment received at enrollment. 
All participants in the FA population and that have no major protocol violations that are 
determined to potentially interfere with the immunogenicity assessment of the vaccine and who 
have at least one immunogenicity assessment will be included in the Per Protocol (PP) 
Population. The PP population will serve as the primary analysis population for the 
immunogenicity endpoints. The specific criteria for exclusion of participants from the PP 
population will be established before breaking the blind and will be based on the blinded review 
of protocol violations. 
In general, all missing data will be treated as missing completely at random and no imputation 
will be performed except for the safety endpoints, as described below. If some safety data are 
available for a participant in the FA population, but respective secondary endpoint-related data 

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are missing, then the participant will be included in the safety analysis and data will be treated as 
follows for immediate AEs, unsolicited AEs and SAEs. 
▪ If Severity is missing for any AE, then it will be considered as an AE of maximum 
severity (Grade 3) "Severe", unless it is captured as an SAE 
▪ If "Relationship" is missing, then it will be considered as "Related" to the vaccine 
administered 
▪ If, for Start date, the day of event/condition is missing due to any adverse event, then it 
will be imputed as the date of last visit 
▪ If the Stop date of an AE is missing, then it will be treated as ongoing 
For solicited AEs, the following assumptions will be made: 
▪ If dates are missing, but symptoms are reported, then the best estimated day post- 
vaccination will be used to calculate the date of the symptom 
▪ If a symptom is not reported at any time through Day 7, then no imputation for missing 
data will be performed; the data will be summarized as “not reported” on the tables 
 
10.5 
Go/No-Go Milestones 
 
A set of criteria are to be defined for the safety and tolerability of each of the dose-escalation 
cohorts, in order to proceed to the next higher dosage cohort and/or to decide on the target dose 
in Cohort 4. These criteria are to be reviewed and accepted by the independent SMC as described 
in Section 7.6. 
 
Any dose of vaccine which results in more than one episode of a Grade 2 or higher diarrhea or 
dysentery may be considered too reactogenic for dose-escalation. Any dose of vaccine which 
results in more than one episode of a Grade 2 or higher solicited AE of the same type may also 
be considered too reactogenic for dose-escalation, unless the SMC recommends otherwise. 
 
11 ACCESS TO SOURCE DATA/DOCUMENTS 
Source data are all information, original records of clinical findings, observations, or other 
activities in a clinical trial necessary for validation of the clinical data. Examples of these 
original documents and data records include, but are not limited to, hospital records, clinical and 
office charts, laboratory notes, memoranda, subjects’ memory aid or evaluation checklists, 
pharmacy dispensing records, recorded data from automated instruments, copies or transcriptions 
certified after verification as being accurate and complete, microfiches, photographic negatives, 
microfilm or magnetic media, x-rays, and subject files and records kept at the pharmacy, at the 
laboratories, and medico-technical departments involved in the clinical trial. All information on 
the Case Report Form (CRF) will be traceable to these source documents, which are generally 
maintained in the subject’s study file. The source documents will include a copy of the signed 
Informed Consent/HIPAA authorization. The source document data collection forms for 

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Screening, Inpatient Pre-Challenge to Day of Challenge, Inpatient Post-Challenge, Flow Sheet of 
Stool and Emesis Record, Outpatient Visits and AEs will also serve as Case Report Form (CRF) 
data collection instruments. 
The Investigator is responsible for ensuring the accuracy, completeness, legibility, and timeliness 
of the data reported. Source documents are maintained for recording data for each subject 
enrolled in this clinical study. Study subjects’ data collected on the CRF during the trial will only 
be identified by subject number. If, as an exception, it is necessary for safety or regulatory 
reasons to identify the patient, both the Sponsor and the Investigator are bound to keep this 
information confidential. 
All the information required by the protocol should be provided; any omissions require 
explanation. Each source document and corresponding CRF should be completed and available 
for monitoring and/or collection within a timely manner so that the monitor may check the 
entries for completeness, accuracy and legibility, ensure the CRF is signed by the Investigator, 
and transmit the data to the Sponsor. 
All source documents and CRFs should be completed in a neat, legible manner to ensure 
accurate interpretation of data. Black or blue ink is required to ensure clarity of reproduced 
copies. When making a change or correction, cross out the original entry with a single line and 
initial and date the change. DO NOT ERASE, OVERWRITE OR USE CORRECTION FLUID 
OR TAPE ON THE ORIGINAL. 
The Investigator or designee must enter the information required by the protocol onto the CRF 
provided. The Sponsor’s clinical site monitor will review the CRF for completeness and 
accuracy and instruct site personnel to make any required corrections or additions. 
 
12 QUALITY CONTROL AND QUALITY ASSURANCE 
All clinical trials conducted by CVD are internally monitored for quality assurance and control 
under the CVD-Wide Quality Management (QMP) Plan. The purpose of the QMP is to describe 
Quality Control (QC) and Quality Assurance (QA) procedures designed to ensure that the site 
and Investigator comply with applicable regulations, adhere to the IRB-approved protocol, 
generate quality data, protect data integrity, and safeguard the safety and well-being of study 
participants. The plan has the authority to enforce and correct deficiencies in clinical and 
laboratory conduct of a trial. A Protocol-Specific Quality Assurance Plan is also developed for 
each study. This plan, composed of QC and QA, is developed in collaboration with the PI, 
clinical study coordinator, Regulatory Affairs Specialist, and the QM Coordinator. The QC 
process involves day-to-day logic and edits checks of source documents, case report forms, and 
laboratory documents. The QA process involves periodic retrospective audits of study records 
and prospective reviews of clinical operations and assurance that all research required training 
has been completed as applicable (e.g. GCP, HIPAA, CITI, Lab Safety, Biological 
Specimen/Shipping). These audits also involve the review of the regulatory file, consent 
forms/process, eligibility, vaccine accountability and storage, specimen collection, processing, 

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storage, and shipping. All observations are reviewed with the PI, coordinator, and Clinical 
Research Manager; Corrective Action Plans are formulated in response to any issue. 
 
13 ETHICS/PROTECTION OF HUMAN SUBJECTS 
 
13.1 
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 and/or the 
ICH E6; 62 Federal Regulations 25691 (1997). 
 
13.2 
Institutional Review Board 
The local institution must provide for the review and approval of this protocol and the associated 
informed consent documents by an appropriate ethics review committee or Institutional Review 
Board (IRB). Any amendments to the protocol or consent materials must also be approved before 
they are placed into use unless it is in the best interest of the subjects’ safety to implement 
changes prior to approval. In both the United States and in other countries, only institutions 
holding a current US Federal-Wide Assurance, issued by the Office for Human Research 
Protections (OHRP), may participate. 
Refer to: http://www.hhs.gov/ohrp/assurances/ 
Prior to enrollment of subjects into this clinical study, the protocol and the informed consent 
form(s) will be reviewed and approved by the appropriate IRB. Any amendments to the protocol 
or consent materials will also be reviewed and approved by the appropriate IRB. The responsible 
official for the IRB will sign the IRB letter of approval of the protocol prior to the start of this 
clinical study. Should amendments to the protocol be required, the amendments will be 
submitted to the IRB; an IRB letter of approval of the amendment must be obtained prior to 
acting upon the amendment in the protocol. 
 
13.3 
Informed Consent Process 
Informed consent is a process that is initiated prior to an individual agreeing to participate in the 
study and continues throughout the individual’s participation in the study. Extensive discussion 
of risks and possible benefits of this therapy will be provided to the participant. Consent forms 
describing, in detail, the study, study procedures and risks are given to the potential participant 
and written documentation of informed consent is required prior to starting any study procedure. 
Consent forms will be IRB-approved. Participants will be asked to read and review the 
document. Upon reviewing the document, the Investigator will explain the research study to the 
participant and answer any questions that may arise. The participants will sign the informed 
consent document prior to the start of any procedures specific to the study. The participant 

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should have the opportunity to discuss the study with their surrogates or think about it prior to 
agreeing to participate. The participant may withdraw consent at any time throughout the course 
of the trial. A copy of the informed consent document will be given to the participant for their 
records. The rights and welfare of the participant 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. 
 
13.4 
Exclusion of Women, Minorities, and Children (Special Populations) 
This clinical study will include women, children of 18 years of age and older, and all minorities 
who meet the inclusion/exclusion criteria, regardless of religion, sex, or ethnic background. 
 
13.5 
Subject Confidentiality 
Participant confidentiality is strictly held in trust by the investigators, their staff, and the 
Sponsor. This confidentiality is extended to cover testing of biological samples and other testing, 
in addition to clinical information relating to the participant. 
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. 
The study monitor or other representatives authorized by the sponsor 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 
clinical study site will permit access to such records. 
Participant identity data will be contained in paper study records which will be kept in a locked 
file cabinet and in a secure electronic database, accessible only to authorized users at each 
clinical site. The study database will be user-restricted and password-protected. Participants will 
not be identified by name. The study database will identify participants by a coded study 
Volunteer ID number assigned by clinical site personnel. 
 
13.6 
Future Use of Stored Specimens 
It is intended that any remaining specimens at the closure of the study will be stored at CVD 
indefinitely. Participants must provide a written request to the PI to have their specimens 
destroyed. This is described in the consent form. 
 
14 DATA HANDLING AND RECORD KEEPING 
The site’s PI is responsible to ensure the accuracy, completeness, legibility, and timeliness of the 
data reported. 
Source data may be collected on data collection forms that will be generated by the site or 
entered directly into the case report forms by study personnel or received by direct data transfer. 

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Source data collected on data collection forms will be entered into the REDCap data 
management system, managed by the Clinical and Translational Research Informatics Center 
(CTRIC), University of Maryland School of Medicine. REDCap is a secure web-based 
application for building an electronic database that provides a 21 CFR Part 11, FISMA, and 
HIPAA-compliant environment. Quality control audits of all key safety, laboratory, and clinical 
data in the database will be made after data entry has been completed. Coexisting medical 
conditions, AEs and other medical events will be coded using MedDRA dictionary. Concomitant 
medications will be coded using the WHO-DD dictionary. When the database has been declared 
to be complete and accurate, the database will be locked. Any changes to the database after that 
time will only be by joint written agreement of the study team. 
All data collection forms will be completed in a neat, legible manner to ensure accurate 
interpretation of data. Black or blue ink is required to ensure clarity of reproduced copies. When 
making a change or correction, research staff members will cross out the original entry with a 
single line and initial and date the change. They will not erase, overwrite, or use correction fluid 
or tape on the original. 
Data reported in REDCap are derived from the data collection forms and should be consistent 
with the data collection forms or the discrepancies should be explained. 
The sponsor and/or its designee will provide guidance to the site PI and other study personnel on 
making corrections to the data collection forms and eCRF. 
 
15 PUBLICATION POLICY 
All investigators funded by the NIH must submit, or have submitted for them, an electronic 
version of their final, peer-reviewed manuscripts, upon acceptance for publication, to the 
National Library of Medicine’s PubMed Central (http://www.ncbi.nlm.nih.gov/pmc/). 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 the official date of publication. 
Refer to: 
• 
NIH Public Access Policy, http://publicaccess.nih.gov/ 
• 
NIH Office of Extramural Research (OER) Grants and Funding, 
http://grants.nih.gov/grants/oer.htm 
Following completion of this clinical trial, the PI will publish the results of this research in a 
scientific journal. 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 
(http://clinicaltrials.gov/), which is sponsored by the National Library of Medicine. Other 
biomedical journals are considering adopting similar policies. 

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APPENDIX A: STUDY SCHEDULE OF EVENTS 
 
Schedule of Events, Cohorts 1 – 3 
Study Period 
Screen 
Inpatient Period 
Outpatient Follow- 
Up 
Study Day 
-60 to -2 -3* -1 1 
2 
3 
4 
5 
8 29 43 181 ET 
Informed Consent 
X 
Screening Procedures1 
X 
* 
Admission & Acclimatization 
X 
Pregnancy Test (females only) 
X 
Vaccination 
X 
Clinical Safety Labs2 
12.5 
12.5 
Reactogenicity Assessments 
X X X X X 
Adverse Event Monitoring 
X X X X X X X X X X 
Research Blood for Serology (mL) 
(x) 10 
10 
10 
10 
10 
Research Blood for ASC/ALS 
Assay (mL) 
(x) 25 
25 
Research Blood for PBMC (mL) 
(x) 90 
90 
90 
90 
90 
Stool Grading (while inpatient 
only) 
X X X X X 
Stool Culture, for fecal shedding 
X X X X X X 
X 
Stool IgA 
X 
X X X X 
Total daily blood volume (mL) 
16 
137.5 
37.5 100 
100 
100 
Cumulative blood volume (mL) 
16 
153.5 
191 
291 
391 
491 
 
Schedule of Events, Cohort 4 
Study Period 
Screen 
Outpatient 
Study Day 
-60 to - 
2 
1 
3 
8 29 31 36 57 210 ET 
Informed Consent 
X 
Screening Procedures1 
X 
Pregnancy Test (females only) 
X 
X 
Vaccination 
X 
X 
Clinical Safety Labs2 
12.5 
12.5 12.5 
12.5 
Memory Aid (disperse or collect) 
d 
c 
d 
c 
Adverse Event Monitoring 
X X X X X X X X X 
Research Blood for Serology (mL) 
10 
10 
10 
10 10 
Research Blood for ASC/ALS Assay plus 
homing (mL) 
25 
50 
25 
50 
Research Blood for PBMC (mL) 
90 
50 
90 
90 90 
Stool for Culture 
X X X X X X 
Stool IgA 
X 
X 
X X X 
Total daily blood volume (mL) 
16 
137.5 
62.5 
97.5 
62.5 100 
100 
Cumulative blood volume (mL) 
16 
153.5 
216 
313. 
5 
376 
476 
576 

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ET = early termination 
(x) = research blood for serology, ASC/ALS, and PBMC may be collected on Day -1 or Day 1, as long as it is prior to 
vaccination 
1 - Screening includes clinical laboratory testing: 
• Serology: HIV Ab, HBsAg, HCV Ab 
• Other labs: HLA B27, hematology (WBC, ANC, hemoglobin, platelet count), chemistry (creatinine, ALT, total 
bilirubin), serum IgA, and β-HCG for women of Childbearing Potential (CBP) 
2 - Clinical Safety Labs include: hematology (WBC, ANC, hemoglobin, platelet count), and chemistry (creatinine, ALT, 
total bilirubin) 
* A nasal swab for a SARS-CoV-2 test is to be performed within 72 hours prior to admission to the inpatient unit, for 
Cohorts 1-3 only, and as per current hospital policy 
Note: all research blood volumes indicated are intended to be approximate target volumes; therefore, the collection of 
volumes which are not exactly equal to the volumes indicated in the protocol do not necessarily result in protocol 
deviations 

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APPENDIX B: ACCEPTABLE CLINICAL LABORATORY VALUES FOR 
ELIGIBILITY 
 
Analyte 
Unit 
Reference 
Range* 
Acceptable Values for 
Screening Labs: 
White Blood Cell count 
(WBC) 
103/µL 
3.5 – 11.5 
3.15 – 12.6 
(2.8 – 12.6 for African 
Americans) 
Absolute Neutrophil Count 
(ANC) 
103/µL 
1560 – 8100 
1400 – 8910 
(1200 – 8910 for African 
Americans) 
Hemoglobin (Females) 
g/dL 
12.0 – 16.0 
10.8 – 17.6 
Hemoglobin (Males) 
g/dL 
13.5 – 17.5 
12.2 – 19.2 
Platelet count 
103/µL 
140 – 400 
126 – 440 
Creatinine (Females) 
mg/dL 
0.6 – 1.2 
≤1.3 
Creatinine (Males) 
mg/dL 
0.7 – 1.3 
≤1.4 
Alanine aminotransferase 
(ALT) 
U/L 
7 - 45 
≤54 
Bilirubin, total 
mg/dL 
0.2 – 1.2 
<1.5 
Serum IgA (Females) 
mg/dL 
87 - 352 
≥87 
Serum IgA (Males) 
mg/dL 
–90 - 386 
≥90 
Urine pregnancy test 
n/a 
n/a 
Negative 
Serum β-HCG (Females) 
n/a 
n/a 
Negative 
HLA-B27 
n/a 
n/a 
Negative 
Hepatitis B surface antigen 
n/a 
n/a 
Negative 
Hepatitis C virus ELISA 
n/a 
n/a 
Negative 
HIV 
n/a 
n/a 
Negative 
SARS-CoV-2# 
n/a 
n/a 
Negative 
*The reference ranges provided are from Garcia Labs, published 10 October 2022. 
#A SARS-CoV-2 molecular diagnostic test 

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APPENDIX C: GRADING SCALES FOR CLINICAL SAFETY LABORATORY 
TOXICITY 
 
Test 
Mild 
Grade 1 
Moderate 
Grade 2 
Severe 
Grade 3 
WBC, increase (xULN) 
>1.1 – 1.5 
1.6 – 2.0 
≥2.1 
WBC, decrease* (xLLN) 
<1.1 – 1.5 
1.6 – 2.0 
≥2.1 
ANC, increase (xULN) 
>1.1 – 1.5 
1.6 – 2.0 
≥2.1 
ANC decrease* (xLLN) 
<1.1 – 1.5 
1.6 – 2.0 
≥2.1 
Hemoglobin, increase 
(xULN) 
>1.1 – 1.5 
1.6 – 2.0 
≥2.1 
Hemoglobin, decrease* 
(xLLN) 
<1.1 – 1.5 
1.6 – 2.0 
≥2.1 
Platelet, increase (xULN) 
>1.1 – 1.5 
1.6 – 2.0 
≥2.1 
Platelet, decrease* (xLLN) 
<1.1 – 1.5 
1.6 – 2.0 
≥2.1 
Creatinine* (xULN) 
>1.1 – 1.5 
1.6 – 2.0 
≥2.1 
ALT (xULN) 
>1.2 – 2.0 
2.1 – 4.0 
≥4.1 
Total Bilirubin* (xULN) 
>1.2 – 1.5 
1.6 – 2.0 
≥2.1 
*if the baseline value is outside of the reference range, then the relative changes 
to the baseline will be evaluated for toxicity 
ULN, upper limit of normal 
LLN, lower limit of normal 

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APPENDIX D: REACTOGENICITY & CLINICAL TOXICITY GRADING 
 
Objective 
Reactogenicity 
Mild 
Moderate 
Severe 
Potentially 
Life- 
Threatening 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Diarrhea 
(Number of loose stools 
with Grade 3 – 5 
consistency, as defined 
below) 
2 or more loose 
stools of ≥200 
mL within 48 h 
or 
a single loose 
stool of ≥300 mL 
Cumulative 
loose stools of ≥ 
1 L over 96 h or 
loose stools of 
≥400 mL in 24 h 
or 4 – 5 loose 
stools in 24 h 
Cumulative 
loose stools of 
≥1 L in 48 h or 
loose stools of 
≥800 mL in 24 h 
or >5 loose 
stools in 24 h 
Requires 
hospitalization 
Dysentery (Number of 
heme-positive loose 
stools) 
1 
2 – 3 
≥4 
Vomiting (Number of 
episodes/24 h) 
1 
2 – 3 
≥4 
Fever (°C (°F)) 
38.1 – 38.4 
(100.5 – 101.1) 
38.5 – 38.9 
(101.2 – 102.1) 
≥39 
(≥102.2) 
Tachycardia (bpm) 
101 – 115 
116 – 125 
>125 
Systolic Hypotension 
(mmHg) 
85 – 94 
80 – 84 
<80 
Diastolic Hypotension 
(mmHg) 
45-59 
35-44 
<35 
Subjective 
Reactogenicity and 
Unsolicited AEs, 
including abdominal 
pain/cramping, tenesmus, 
malaise, nausea, 
anorexia, arthralgia, and 
myalgia 
No interference 
with activity 
Some 
interference with 
activity 
Prevents daily 
activity 
Requires 
hospitalization 
 
 
Stool Consistency Grading Scale 
Normal stool 
Loose or diarrheal stool 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Grade 5 
Well formed; 
Soft; 
Thick liquid stool; 
Clear, watery or 
“rice water” 
diarrheal stool 
does not take the 
does not easily 
easily takes the 
Opaque, watery 
shape of the 
take the shape of 
shape of the 
diarrheal stool 
container 
the container 
container