A Double-Blind Placebo Controlled Randomized Phase 2 Trial to Evaluate the Safety, Reactogenicity and Immunogenicity of a Live-Attenuated Shigella Sonnei Vaccine, WRSs2 and Determine Its Efficacy in a Challenge Model of S. Sonnei 53G in Healthy Adults
Phase: PHASE2
Conditions: Immunisation, Shigella Infection
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

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1 
 
A Double-Blind Placebo Controlled, Randomized Phase 2 Trial to 
Evaluate the Safety, Reactogenicity and Immunogenicity of a Live-
Attenuated Shigella sonnei Vaccine, WRSs2 and Determine its 
Efficacy in a Challenge Model of S. sonnei 53G in Healthy Adults 
   
DMID Protocol Number: 17-0112 
DMID Funding Mechanism: HHSN272201300016I/HHSN27200014 
FY.2018.A1B1C1D1.0077 
Pharmaceutical Support Provided by: Department of Defense 
IND Sponsor: NIAID/DMID  
Lead Principal Investigator: Robert W. Frenck, Jr, M.D. 
DMID Clinical Project Manager: Krista Cato, MHA, BSN, RN 
Draft or Version Number: 8.0 
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STATEMENT OF ASSURANCE 
Each Institution will hold a current Federal Wide Assurance (FWA) issued by the Office of 
Human Research Protections (OHRP) for federally-funded human subjects research. Each FWA 
will designate at least one Institutional Review Board (IRB)/Independent Ethics Committee 
(IEC) registered with OHRP, for which the research will be reviewed and approved by the 
IRB/IEC and will be subject to continuing review [45 CFR 46.103(b)]. The IRB/IEC designated 
under an FWA may include an institution’s IRB/IEC, an independent IRB/IEC, or an IRB/IEC of 
another institution after establishing a written agreement with that other institution. 
 

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STATEMENT OF COMPLIANCE 
The trial will be carried out in accordance with Good Clinical Practice (GCP) and as required by 
the following: 
United States Code of Federal Regulations (CFR) 45 CFR Part 46: Protection of 
Human Subjects 
Food and Drug Administration (FDA) Regulations, as applicable: 21 CFR Part 50 
(Protection of Human Subjects), 21 CFR Part 54 (Financial Disclosure by Clinical 
Investigators), 21 CFR Part 56 (Institutional Review Boards), 21 CFR Part 11, 
and 21 CFR Part 312 (Investigational New Drug Application), 21 CFR 812 
(Investigational Device Exemptions) 
International Conference on Harmonisation: Good Clinical Practice (ICH E6); 62 
Federal Register 25691 (1997); and future revisions 
Belmont Report: Ethical Principles and Guidelines for the Protection of Human 
Subjects of Research, Report of the National Commission for the Protection of 
Human Subjects of Biomedical and Behavioral Research 
National Institutes of Health (NIH) Office of Extramural Research, Research 
Involving Human Subjects, as applicable 
National Institute of Allergy and Infectious Diseases (NIAID) Clinical Terms of 
Award, as applicable 
Applicable Federal, State, and Local Regulations and Guidance 
 
 

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SIGNATURE PAGE 
The signature below provides the necessary assurance 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 E6 Good Clinical Practice (GCP) guidelines. 
I agree to conduct the study in compliance with GCP and applicable regulatory requirements. 
I agree to conduct the study in accordance with the current protocol and will not make changes to 
the protocol without obtaining the sponsor’s approval and IRB/IEC approval, except when 
necessary to protect the safety, rights, or welfare of subjects. 
Site Investigator Signature: 
 
Signed:  
Date: 
 
 
Name 
Title 
 
 
 
 

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TABLE OF CONTENTS 
STATEMENT OF ASSURANCE ...................................................................................................2 
STATEMENT OF COMPLIANCE .................................................................................................3 
SIGNATURE PAGE .......................................................................................................................4 
TABLE OF CONTENTS .................................................................................................................5 
LIST OF TABLES .........................................................................................................................10 
LIST OF FIGURES .......................................................................................................................11 
LIST OF ABBREVIATIONS ........................................................................................................12 
PROTOCOL SUMMARY .............................................................................................................16 
1 
KEY ROLES ..............................................................................................................20 
2 
BACKGROUND AND SCIENTIFIC RATIONALE ................................................21 
2.1 
Background .................................................................................................................21 
2.2 
Scientific Rationale .....................................................................................................25 
2.2.1 
Scientific Innovation ...................................................................................................26 
2.2.2 
Purpose of Study .........................................................................................................26 
2.2.3 
Study Population .........................................................................................................26 
2.3 
Potential Risks and Benefits .......................................................................................27 
2.3.1 
Potential Risks ............................................................................................................27 
2.3.2 
Potential Benefits ........................................................................................................31 
3 
STUDY DESIGN, OBJECTIVES AND ENDPOINTS OR OUTCOME 
MEASURES ...............................................................................................................31 
3.1 
Study Design Description ...........................................................................................31 
3.2 
Study Objectives .........................................................................................................37 
3.2.1 
Primary .......................................................................................................................37 
3.2.2 
Secondary ................................................................... Error! Bookmark not defined. 
3.2.3 
Exploratory .................................................................................................................37 
3.2.4 
Secondary Research ....................................................................................................37 
3.3 
Study Endpoints or Outcome Measures .....................................................................38 

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3.3.1 
Primary .......................................................................................................................38 
3.3.2 
Secondary ...................................................................................................................38 
3.3.3 
Exploratory .................................................................................................................39 
3.3.4 
Secondary Research ....................................................................................................40 
4 
INTERVENTION/INVESTIGATIONAL PRODUCT ..............................................40 
4.1 
Study Product Description ..........................................................................................40 
4.1.1 
Product Storage and Stability .....................................................................................42 
4.2 
Acquisition/Distribution .............................................................................................43 
4.3 
Protocol-Specified Medications/Treatments other than Study Products ....................45 
4.4 
Dosage/Regimen, Preparation, Dispensing and Administration of Study 
Intervention/Investigational Product ..........................................................................45 
4.5 
Accountability Procedures for the Study Intervention/Investigational 
Product(s) ....................................................................................................................47 
5 
SELECTION OF SUBJECTS AND STUDY ENROLLMENT AND 
WITHDRAWAL ........................................................................................................48 
5.1 
Eligibility Criteria .......................................................................................................48 
5.1.1 
Subject Inclusion Criteria ...........................................................................................48 
5.1.2 
Subject Exclusion Criteria ..........................................................................................49 
5.2 
Withdrawal from the Study, Discontinuation of Study Product, or Study 
Termination .................................................................................................................51 
5.2.1 
Withdrawal from the Study or Discontinuation of the Study Product ........................51 
5.2.2 
Handling of Withdrawals ............................................................................................52 
5.2.3 
Study Termination ......................................................................................................52 
5.2.4 
Study Discontinuation ................................................................................................53 
6 
STUDY PROCEDURES ............................................................................................53 
6.1 
Screening ....................................................................................................................53 
6.2 
Enrollment ..................................................................................................................55 
6.3 
Planned Study Visits ...................................................................................................55 
6.3.1 
Follow-up ....................................................................................................................68 
6.3.2 
Final Study Visit .........................................................................................................70 

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6.3.3 
Early Termination Visit ..............................................................................................70 
6.4 
Unscheduled Study Visits ...........................................................................................71 
6.5 
Protocol Deviations ....................................................................................................71 
7 
DESCRIPTION OF CLINICAL AND LABORATORY EVALUATIONS .............72 
7.1 
Clinical Evaluations ....................................................................................................72 
7.1.1 
Assessment of Concomitant Medications/Treatments other than Study 
Product ........................................................................................................................73 
7.1.2 
Non-Research Standard of Care .................................................................................74 
7.2 
Laboratory Evaluations ...............................................................................................74 
7.2.1 
Clinical Laboratory Evaluations .................................................................................74 
7.2.2 
Secondary Research Assays .......................................................................................76 
7.2.3 
Laboratory Specimen Preparation, Handling, and Storage ........................................76 
7.2.4 
Laboratory Specimen Shipping ..................................................................................77 
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ASSESSMENT OF SAFETY .....................................................................................77 
8.1 
Assessing and Recording Safety Parameters ..............................................................77 
8.1.1 
Adverse Events (AEs) .................................................................................................78 
8.1.2 
AE Grading .................................................................................................................78 
8.1.3 
Reactogenicity ............................................................................................................80 
8.1.4 
Serious Adverse Events (SAEs) .................................................................................80 
8.2 
Specification of Safety Parameters .............................................................................81 
8.2.1 
Solicited Events ..........................................................................................................81 
8.2.2 
Unsolicited Events ......................................................................................................81 
8.3 
Reporting Procedures ..................................................................................................81 
8.3.1 
Reporting SAEs ..........................................................................................................81 
8.3.2 
Regulatory Reporting for Studies Conducted Under DMID-Sponsored IND ............82 
8.3.3 
Reporting of Pregnancy ..............................................................................................83 
8.4 
Type and Duration of Follow-up of Subjects after AEs .............................................83 
8.5 
Procedures to be Followed in the Event of Abnormal Laboratory Test Values 
or Abnormal Clinical Findings ...................................................................................83 

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8.6 
Study Halting Rules ....................................................................................................84 
8.6.1 
Individual Halting Criteria ..........................................................................................84 
8.7 
Safety Oversight (DSMB, as applicable)....................................................................84 
8.7.1 
Data and Safety Monitoring Board (DSMB) ..............................................................84 
9 
HUMAN SUBJECTS PROTECTION .......................................................................86 
9.1 
Institutional Review Board/Independent Ethics Committee ......................................86 
9.2 
Informed Consent Process ..........................................................................................86 
9.3 
Consent for Secondary Research of  Residual Specimens and Data ..........................88 
9.4 
Exclusion of Women, Minorities, and Children (Special Populations) ......................89 
9.5 
Subject Confidentiality ...............................................................................................89 
9.6 
Certificate of Confidentiality ......................................................................................90 
9.7 
Costs, Subject Compensation, and Research Related Injuries ...................................91 
10 
STATISTICAL CONSIDERATIONS .......................................................................91 
10.1 
Study Hypotheses .......................................................................................................91 
10.2 
Sample Size Considerations .......................................................................................91 
10.3 
Treatment Assignment Procedures .............................................................................92 
10.3.1 
Randomization Procedures .........................................................................................92 
10.3.2 
Masking Procedures ....................................................................................................93 
10.4 
Planned Interim Analyses ...........................................................................................93 
10.4.1 
Interim Safety Review ................................................................................................93 
10.4.2 
Interim Immunogenicity or Efficacy Review .............................................................94 
10.5 
Final Analysis Plan .....................................................................................................94 
10.5.1 
Endpoint Review Committee ......................................................................................94 
10.5.2 
Analysis Populations ..................................................................................................94 
10.5.3 
Primary Efficacy Analysis ..........................................................................................95 
10.5.4 
Safety Data ..................................................................................................................95 
10.5.5 
Immunogenicity Data .................................................................................................96 
10.5.6 
Missing Values and Outliers .......................................................................................96 

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SOURCE DOCUMENTS AND ACCESS TO SOURCE 
DATA/DOCUMENTS ...............................................................................................97 
12 
QUALITY CONTROL AND QUALITY ASSURANCE .........................................97 
13 
DATA HANDLING AND RECORD KEEPING ......................................................98 
13.1 
Data Management Responsibilities ............................................................................98 
13.2 
Data Coordinating Center/Biostatistician Responsibilities ........................................98 
13.3 
Data Capture Methods ................................................................................................99 
13.4 
Types of Data ..............................................................................................................99 
13.5 
Study Records Retention ............................................................................................99 
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CLINICAL MONITORING .......................................................................................99 
15 
PUBLICATION POLICY ........................................................................................100 
16 
LITERATURE REFERENCES................................................................................101 
17 
APPENDICES ..........................................................................................................104 
Appendix A: Schedule of Events .................................................................................................105 
Appendix B: Toxicity Table/Laboratory AE Grading Scale .......................................................113 
Appendix C: Vital Signs ADVERSE EVENT Grading Scale .....................................................114 
Appendix D: Diarrhea Classification ...........................................................................................115 
Appendix E: Reactogenicity Grading Scale ................................................................................116 
 
 

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LIST OF TABLES 
Table 1: S. sonnei strain 53G Controlled Human Infection Clinical Outcome .............................25 
Table 2: Maximum Severity of Solicited Adverse Events in Subjects Receiving WRSs2 ...........28 
Table 3: Vaccine Administration, Challenge and Follow-up ........................................................32 
Table 4: Primary Endpoint .............................................................................................................38 
 
 

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LIST OF FIGURES 
Figure 1: 
Schematic of Study Design .........................................................................................19 
 
 

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LIST OF ABBREVIATIONS 
AE 
Adverse Event 
ALS 
Antibody Lymphocyte Supernatant 
ALT 
Alanine Aminotransferase 
ANC 
Absolute Neutrophil Count 
ASC   
Antibody Secreting Cell 
AST 
Aspartate Aminotransferase 
BPM 
Beats Per Minute 
BUN 
Blood Urea Nitrogen  
°C 
Degrees Celsius 
CCHMC 
Cincinnati Children’s Hospital Medical Center 
CMS 
Clinical Materials Services 
CFR 
Code of Federal Regulations 
CFU 
Colony Forming Units 
COVID-19 
Coronavirus Disease 2019 
CRF 
Case Report Form 
CVD 
Center for Vaccine Development 
DCC 
Data Coordinating Center 
DSMB 
Data and Safety Monitoring Board 

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eCRF 
Electronic Case Report Form 
ELISA 
Enzyme-Linked Immunosorbent Assay 
ERC 
Endpoint Review Committee 
°F 
Degrees Fahrenheit 
FDA 
Food and Drug Administration 
FWA 
Federal Wide Assurance 
GCP 
Good Clinical Practice 
HEENT 
Head, Eyes, Ears, Nose, Throat  
Hgb 
Hemoglobin  
HIV 
Human Immunodeficiency Virus 
IBD 
Inflammatory Bowel Disease 
IBS 
Irritable Bowel Syndrome 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation 
IDES 
Internet Data Entry System 
IEC 
Independent or Institutional Ethics Committee 
IgA 
Immunoglobulin A 
IgG 
Immunoglobulin G 
IND 
Investigational New Drug Application 

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IpaB 
Invasion plasmid antigen B 
IpaC 
Invasion plasmid antigen C 
IRB 
Institutional Review Board 
KI 
Killing Index 
MAAE 
Medically Attended Adverse Event 
MOP 
Manual of Procedures 
N 
Number (typically refers to subjects) 
NDA 
New Drug Application 
NPO 
Nothing by mouth 
OHRP 
Office for Human Research Protections 
ORS 
Oral Rehydration Solution 
PBF 
Pilot Bioproduction Facility 
PBMC 
Peripheral Blood Mononuclear Cell 
PBS 
Phosphate Buffered Saline 
PE 
Physical Exam 
QA 
Quality Assurance 
QC 
Quality Control 
PI-IBS 
Post-Infectious Irritable Bowel Syndrome 
qPCR 
quantitative Polymerase Chain Reaction 

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RPR 
Rapid Plasma Reagin 
SAE 
Serious Adverse Event 
SARS-CoV-2  
Severe Acute Respiratory Syndrome Coronavirus 2 
SBA 
Serum Bactericidal Assay 
SDCC 
Statistical Data Coordinating Center 
SOP 
Standard Operating Procedure 
SOC 
System Organ Class 
SWI 
Sterile Water for Injection, USP 
UC 
Ulcerative Colitis 
ULN 
Upper Limit of Normal 
WBC 
White Blood Cell 
WRAIR 
Walter Reed Army Institute of Research 
 
 
 
 

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PROTOCOL SUMMARY 
 
Title: 
A Double-Blind Placebo Controlled, Randomized Phase 2 Trial to 
Evaluate the Safety, Reactogenicity and Immunogenicity of a Live-
Attenuated Shigella sonnei Vaccine, WRSs2 and Determine its Efficacy in 
a Challenge Model of S. sonnei 53G in Healthy Adults 
Design of the 
Study: 
This is a double-blind, placebo controlled, randomized study to test the 
safety, reactogenicity, immunogenicity and efficacy of up to a 106 cfu dose 
of an oral live-attenuated Shigella sonnei vaccine (WRSs2) to protect 
against shigellosis after a targeted oral challenge with S. sonnei 53G, a 
virulent strain of S sonnei. Up to 120 subjects will be enrolled and 
randomized in this study. Using the 106 CFU dose, subjects were 
randomized 1:1:1 into one of three vaccination arms to receive 2 doses of 
study agent 28 days apart. Arm 1 received 2 doses of WRSs2, Arm 2 
received placebo followed by WRSs2 and Arm 3 received 2 doses of 
placebo. Twenty-eight (+2) days after the second dose of study agent, 
subjects were admitted to the inpatient unit and given an oral challenge of 
approximately 1500 cfu of 53G. The goal was to have 90 subjects (30 per 
arm) receive a challenge dose of 53G.  
After the initiation of the study, two participants had Grade 3 diarrhea 
and/or vomiting in the days following vaccination. This triggered a halting 
rule. To meet the recommendations of the DSMB and evaluate the safety 
and efficacy signal of a lower dose, several changes to the protocol were 
made. The vaccination dose was reduced to 5x105, enrollment was changed 
to 2 arms and randomized 2:1 (vaccine: placebo). Furthermore, participants 
with morbid obesity were excluded and weight loss medications 
prohibited. No changes in sample collection or challenge phase were made. 
Study Phase: 
 Phase 2 
Study Population: 
Up to 120 healthy male and non-pregnant females, 18-49 years inclusive at 
the time of first vaccination, recruited from the general population at the 
participating VTEU site(s) 

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Number of Sites: 
Up to 3 sites 
Description of 
Study Product or 
Intervention: 
Up to a 106 cfu oral dose of live attenuated S. sonnei vaccine WRSs2, 0.9% 
sterile normal saline as placebo and targeted oral challenge of 
approximately 1.5x103 cfu of wild type S. sonnei 53G.  
For this amendment, the remaining subjects to be randomized will receive 
5x105 cfu vaccine dose or placebo.   
 
Study Objectives: 
 
 
Primary: 
Estimate combined vaccine efficacy of 2 doses of WRSs2 (106 cfu 
or 5x105 cfu) in preventing shigellosis, as defined in Table 4, 
following challenge with S. sonnei strain 53G 
Secondary: 
Estimate vaccine efficacy of 1 dose of 106 cfu, 2 doses of 106 cfu, 
and 2 doses of 5x105 cfu of WRSs2 in preventing shigellosis 
following challenge with S. sonnei strain 53G 
Safety evaluations of WRSs2   
Evaluate immune responses following vaccination 
(immunogenicity) with WRSs2 and after challenge with S. sonnei 
strain 53G by serum anti-LPS and anti-Invaplex IgG and IgA by 
ELISA  
Determine fecal shedding of S. sonnei after WRSs2 vaccination and 
53G challenge by qualitative stool culture   
Exploratory:  
Evaluate fecal shedding of S. sonnei post vaccination and post 
challenge by colony immunoblot and PCR 

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Evaluate S. sonnei antigen (LPS and Invaplex) specific IgG and 
IgA-ASC following vaccination and challenge 
Evaluate fecal IgA following vaccination and challenge 
Secondary Research: 
Blood and stool samples will be collected and stored to characterize 
in depth innate and acquired immune response to WRSs2 and 
Shigella infection.  
 
 
Duration of 
Individual Subject 
Participation: 
 
Approximately 8 months  
Estimated Time to 
Last Subject/Last 
Day: 
Approximately 24 months 

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Figure 2: 
Change in Schematic of Study Design 
Following changes will be implemented in the schematic design of the study as described in 
Figure 1. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Screening will occur from day -60 to -3 (-2). Prospective subjects will be screened 
until 90 (approximately 30 per arm) are eligible for inpatient challenge. After 
informed consent is obtained, screening will occur including inclusion/exclusion 
criteria, medical history, safety labs and physical exam  
Randomize 2:1 Vaccine: Placebo 
Once participants are randomized, the vaccination procedure, follow-up 
and sample collection will remain the same as described in Figure 1.  
Placebo/Placebo  
WRSs2/ WRSs2 
5x105 

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1 KEY ROLES 
Lead Principal 
Investigator: 
 
                                    
 
 
 
DMID Clinical Project 
Manager: 
 
Robert W. Frenck, Jr., MD 
Director, Gamble Vaccine Research Center 
Cincinnati Children’s Hospital Medical Center 
3333 Burnet Ave, Cincinnati, OH, 45229  
Ph: 
 
robert.frenck@cchmc.org 
 
Krista Cato, MHA, BSN, RN  
5601 Fishers Lane Room 8F56 Bethesda, MD 20892-9825 
Ph:
  
Krista.cato@nih.gov 
 
Statistical and Data 
Coordinating Center 
The Emmes Company, LLC 
401 N. Washington St., Suite 700 
Rockville, MD 20850 
Phone: 
 
Email: enterics@emmes.com 
 
2 BACKGROUND AND SCIENTIFIC RATIONALE 
2.1 Background  
Diarrheal diseases continue to be major public health problems throughout the world. According 
to the 2015 Global Burden of Disease (GBD) study, diarrhea was estimated to be a leading cause 
of death (1.31 million deaths) among all ages as well as a leading cause of disability adjusted life 
years (DALYs) (71.59 million DALYs)1. After rotavirus, Shigella was the second leading cause 

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of diarrheal deaths (~164,300)1. Additionally, diarrhea caused by Shigella continues to be a 
common cause of mortality and morbidity in children <5 years of age in developing countries 
where malnutrition and unsafe water and sanitation constituted leading risk factors for diarrheal 
deaths and morbidity2. More recent estimates of mortality rates are lower, however 
epidemiological information on incidence rates of Shigella-mediated diarrhea from much of Asia 
and Africa is missing3. 
Shigella is comprised of four species (S. dysenteriae, S. flexneri, S. boydii and S. sonnei) with S. 
sonnei being the most common species of Shigella-associated diarrhea in developed countries 
and S. flexneri being the most common species of Shigella-associated diarrhea in the developing 
world4.  
Shigella is a low-inoculum infection transmitted by the fecal-oral route with as few as 10 colony 
forming units (cfu) sufficient to cause disease5. Due to efficiency of transmission, multiple 
secondary cases from an initial case are common5. Infections caused by Shigella can range from 
mild watery diarrhea to severe disease characterized by fever, headaches, abdominal cramps, and 
dysentery (frequent small volume stools containing blood and mucus). Although Shigella-
associated diarrhea typically is self-limiting, infection frequently is debilitating for 5-7 days if 
untreated3, 6. 
Currently, the mainstays against Shigella are prevention and treatment of infections. Improved 
sanitation and education about improved hygienic practices are the principal means of prevention 
while treatment typically requires administration of antimicrobial therapy7, 8. Improving 
sanitation is difficult, particularly in resource-limited areas thus decreasing the effectiveness of 
prevention. Additionally, the emergence of multidrug-resistant Shigella strains has complicated 
the treatment of shigellosis9-11. Thus, methods to prevent infection, such as vaccines, are 
critically needed.  
A live-attenuated S. sonnei vaccine candidate, WRSS1 was derived from a virulent strain of S. 
sonnei called Moseley. The principal attenuating feature of WRSS1 is the loss of the virulence-
plasmid encoded protein VirG (IcsA). Shigella lacking VirG (IcsA) have decreased intracellular 
motility and movement from one cell to another which inhibits bacterial spread within the host 
tissue. Loss of VirG (IcsA) results in a significantly attenuated bacterial strain12-16.  
WRSS1 underwent Phase 1 trials in the US (inpatient) and in Israeli adults (outpatient). The 
vaccine was highly immunogenic when orally administered at a dose of 103 to 104 cfu16, 17. 
However, of the subjects who received a 1x104 cfu dose of WRSS1, 15% to 20% developed 
diarrhea and fever, albeit mild and transient16, 17. The safety and efficacy of WRSS1 also was 
tested among Thai adults18. Thirteen adults were given a 1.6 x104 cfu dose of WRSS1 and then, 
60 days after vaccination, administered a 1.67x103 cfu dose of S. sonnei 53G to evaluate efficacy 

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of WRSS1 in protecting against dysentery and shigellosis. Similar to US and Israeli studies, 4 of 
the 13 (31%) Thai subjects immunized with WRSS1 developed fever or diarrhea, although 
symptoms were described as mild and not affecting the daily routine of the subjects18. 
Although WRSS1 has been immunogenic, the vaccine was unacceptably reactogenic at doses 
higher than 104 cfu leading to the development of more attenuated vaccine candidates; WRSs2 
and WRSs3. The goal for these VirG (IcsA)-based second generation candidates was to maintain 
immunogenicity while having a better safety profile. In addition to the loss of the virG (icsA) 
gene, WRSs2 has two additional deletions in the genes senA and senB, that are present on the 
virulence plasmid19, 20. The gene senA (also known as shet2-1 and ospD3) encodes the 
enterotoxin SenA (ShET2-1 and OspD3), which has been shown to cause fluid accumulation in 
rabbit ileal loops21, 22. The gene senB (also known as shet2-2 and ospD2) encodes SenB (or ShET 
2-2 and OspD2) a similarly sized protein as SenA with 40% identity with SenA at the amino acid 
level. A recent study using cell culture assays has indicated that SenB may inhibit epithelial cell 
death, implicating that one of its functions is to facilitate bacterial colonization23. The enterotoxic 
activity of SenB remains to be demonstrated19, 24. WRSs3 includes the added deletion of the 
virulence plasmid-based msbB2 gene. Lack of the msbB2 gene product has previously been 
shown to produce a less toxic lipopolysaccharide (LPS) molecule, which may reduce the 
proportion of vaccinated subjects with fever19, 24. Animal safety, immunogenicity and efficacy 
studies have shown that WRSs2 and WRSs3 compare favorably with WRSS1.  
A Phase 1 trial evaluating the safety and immunogenicity of WRSs2 and WRSs3 was recently 
completed25. Healthy adults 18-45 years of age, assigned to 5 cohorts of 18 subjects each 
(WRSs2 (n=8), WRSs3 (n=8) or placebo (n=2) were housed in an inpatient facility and 
administered a single oral dose of study agent 5 minutes after ingestion of oral bicarbonate. 
Ascending dosages of vaccine (from 103 cfu to 107 cfu) were evaluated. On day 8, treatment with 
ciprofloxacin (500 mg BID for 3 days) was initiated and subjects were discharged home 2 days 
after completing antibiotics. Subjects returned for outpatient visits on day 14, 28 and 56 post-
vaccination for monitoring and collection of stool and blood samples.  
Both WRSs2 and WRSs3 were generally well-tolerated and safe over the entire dose range. 
Among the 80 vaccinees, 11 subjects developed diarrhea, 8 of which were mild and did not 
affect daily activities. At the 107 cfu dose, moderate diarrhea occurred in one subject receiving 
WRSs2 while at the same dose of WRSs3, two subjects had moderate to severe diarrhea (see 
Risk Section for more detailed description of AEs associated with WRSs2). As predicted, doses 
of WRSs2 and WRSs3 that were 2 logs higher than what was overly reactogenic for WRSS1 
were well-tolerated by subjects. Additionally, the attenuation of both WRSs2 and WRSs3 was 
demonstrated by the fact that despite close housing for 9 days with subjects who received and 

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excreted either WRSs2 or WRSs3, none of the 9 placebo subjects shed the vaccine candidate nor 
did placebo recipients develop any symptoms during the inpatient stay. 
Humans are the only natural host for Shigella making it more difficult to study the prevention 
and/or treatment of infections with the organism. While non-human primate models have been 
developed, inoculums that are multiple logs higher than needed to cause an infection in humans, 
are required to reproducibly induce infection in monkeys. In a rhesus monkey model, 2 x109 cfu 
of Shigella dysenteriae 1 were required to reproducibly induce disease26. Similarly, a dose of 
1x1011 cfu of Shigella flexneri 2a was needed to induce infection in an Aotus nancymaae monkey 
model27. The limitations of the currently available animal models reinforce the need for a safe 
and reproducible human challenge model.  
While human challenge studies of Shigella have produced important data, many concerns have 
been raised about these data including; lack of standard inoculum, lack of reproducibility, small 
sample size of study subjects, varying clinical outcome definitions, and minimal understanding 
of the immune response to infection28. Additionally, the method of preparation of the Shigella 
inoculum from a bacterial suspension of freshly harvested cells from agar plates with colony 
count estimated from a fixed optical density has been cited as a barrier to standardization and 
reproducibility28.  
A Phase 1 study recently completed, established a controlled human infection model (CHIM) for 
S. sonnei using a lyophilized, standardized inoculum of strain 53G  to address some of the 
concerns raised about earlier studies29. The lot of 53G used in the study (
) was grown, 
harvested and lyophilized at the WRAIR Pilot Bioproduction Facility under conditions of cGMP. 
The study was designed as an ascending dose study with the goal to identify the dose of 53G that 
would induce shigellosis in approximately 60% of subjects. The study outcome is summarized 
below in Table 1. 
 
 

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Table 1: S. sonnei strain 53G Controlled Human Infection Clinical Outcome 
 
Dose of 53G (n) 
Symptom (%) 
500 CFU (10)  800 CFU (10)  900 CFU (10)  1100 CFU (16) 1760 CFU (10) 
Diarrhea (any) 
20 
60 
70 
75 
70 
Severe Diarrhea 
0 
40 
30 
44 
60 
Dysentery 
0 
60 
40 
38 
50 
Fever 
0 
30 
10 
19 
40 
Abd pain/cramps 
10 
90 
70 
56 
70 
Headache 
50 
70 
80 
63 
70 
Severe GI Sxs. 
0 
20 
0 
6 
20 
Overall, of subjects receiving the 1.1-1.7 x103 cfu of 53G, approximately 63% developed one of 
the primary endpoints of shedding Shigella and either having mod-severe diarrhea and fever or 
mod-severe diarrhea and dysentery with more than one constitutional symptom of at least 
moderate severity. Therefore, a targeted approximately 1.5 x103 cfu dose of S. sonnei 53G was 
determined to be the optimal dose to be used in future CHIM studies.  
Based on the safety and immunological profile of WRSs2 and the availability of a robust 
challenge model, we originally planned to evaluate the safety, immunogenicity and protective 
efficacy of 1 and 2 doses of WRSs2 (106 cfu) in a human challenge model in protecting subjects 
against shigellosis (see Table 4 for definitions) after challenge with a targeted approximately 1.5 
x103 cfu dose of S. sonnei 53G (
). After the initiation of the study using 1x106 (see 
version 7.0 for prior study design), two participants had Grade 3 diarrhea and/or vomiting in the 
days following receipt of study product. This triggered a stopping rule. To meet the 
recommendations of the DSMB and evaluate the safety and efficacy signal of a lower dose, 
several changes to the protocol were made. The vaccination dose was reduced to 5x105, 
enrollment was changed to only two arms and randomized 2:1 (vaccine: placebo). Furthermore, 
participants with morbid obesity were excluded and weight loss medications prohibited.  
2.2 Scientific Rationale 
Shigella are exclusively human pathogens. While improved sanitation and education on safe 
eating practices could lessen the frequency of Shigella-associated diarrhea, these methods have 
been difficult to achieve. Thus, alternative methods to prevent infection, such as vaccines, are 

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critically needed. As animal models of Shigella do not closely mimic infections in humans; to 
assess the effectiveness of a vaccine against Shigella, a human model of infection is required.  
2.2.1 Scientific Innovation 
Currently, no licensed vaccine exists to prevent infection with Shigella. In a dose escalation 
study, a live attenuated S. sonnei vaccine candidate, WRSs2, was shown to be both safe and 
immunogenic. The next step will be to evaluate the efficacy of WRSs2 using a human challenge 
infection model (CHIM). In contrast to previous CHIM studies where the challenge strain 
typically has been obtained by harvesting confluent bacterial lawns from agar plates that were 
grown from thawed glycerol stocks or starter cultures from one or more earlier passages; in the 
current project a lyophilized preparation of S. sonnei strain 53G (
) will be used for the 
challenge. The use of a lyophilized product provides many advantages over the plating-and-
harvesting the bacterial lawn method including a) consistency and reproducibility between the 
inoculum used for challenge, b) no need to plate-harvest the inoculum for challenge, and c) 
minimizing the error related to handling viscous and incompletely homogenized bacterial 
suspensions.   
S. sonnei vaccine candidate WRSs2 (
) and challenge strain 53G (
) have been 
successfully produced and lyophilized by WRAIR and demonstrated highly reproducible colony 
counts in a recently completed series of S. sonnei vaccine trials at Cincinnati Children’s Hospital 
Medical Center (CCHMC)25, 29.  
Another critical area to explore is the “systems biology” to an infection with Shigella. In contrast 
to previous “reductionist” methods of study, systems biology is a field of study that focuses on 
complex interactions within biological systems, using a holistic approach. By studying the 
“signature of infections”, it may be possible to predict people immune to a specific 
microorganism as well as who may be at increased risk for developing severe infection. This 
approach may allow us to better understand the immune response to natural infections which 
could result in vaccines targeted to stimulate those portions of the immune system.  
2.2.2 Purpose of Study 
The study is designed to evaluate the safety, reactogenicity, immunogenicity, and efficacy of 
WRSs2, a live oral S. sonnei vaccine candidate to protect against shigellosis. Efficacy will be 
determined by challenging WRSs2-vaccinated and control subjects with a dose of S. sonnei 53G.    
2.2.3 Study Population 
Healthy males and non-pregnant females, 18-49 years inclusive at the time of first vaccination, 
regardless of religion, sex, or ethnic background who meet all of the inclusion and none of the 

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exclusion criteria. Subjects will be recruited from the general population of the participating 
VTEU sites.  
2.3 Potential Risks and Benefits 
2.3.1 Potential Risks 
Blood Collection/Intravenous Catheter Placement 
Drawing blood may cause transient discomfort and may cause people to feel faint. Bruising at 
the blood draw site may occur but can be prevented or lessened by applying pressure for several 
minutes. Infection at the site of blood collection is a rare but possible event. If required for 
intravenous fluid hydration, placement of an intravenous catheter may be associated with mild 
pain, redness and bruising which may take up to 14 days to resolve. Rarely, infection or a blood 
clot may develop. Risks of blood collection and intravenous catheter placement will be 
minimized by using sterile technique and having experienced personnel perform the blood 
collection. 
Study Agent (Live, Attenuated WRSs2 Oral S. sonnei Vaccine)   
WRSs2 is an attenuated strain, no cases of shigellosis are expected from the vaccine when given 
at the planned dose of up to 106 cfu. Nausea, vomiting, abdominal pain/cramping, headache, 
anorexia/loss of appetite, chills, muscle and/or joint aches and malaise may occur.  
As WRSs2 is derived from a virulent S. sonnei strain Moseley, subjects potentially could be at 
risk for developing the signs and symptoms associated with a Shigella infection. However, our 
experience with the vaccine has demonstrated that, at the dose proposed for the current study, 
AEs were generally uncommon, of mild severity and resolved spontaneously without treatment.   
In a recently completed Phase 1 study, forty subjects (8 subjects per dose) received a single dose 
of WRSs2 ranging from 103 – 107 cfu25. The vaccine was generally safe and well-tolerated. No 
subject developed shigellosis, dysentery or dehydration and there were no vaccine-related SAEs. 
Except for the symptom of moderate diarrhea at the highest vaccine dose of 107 cfu, vaccine-
associated AEs at lower doses were mild, short-lived and resolved without treatment. Of the six 
subjects who developed diarrhea after the doses of WRSs2, 5 were mild, characterized by 2-3 
loose stools of small volume (average weight 108 g) over one 24-hour period that did not affect 
the daily routine (Table 2). At the highest WRSs2 dose, one subject had 4 loose stools over a 24-
hour period with a total weight of 178 grams with one of the stools being 0.2 grams in weight. 
On the same day, this subject also experienced a single episode of mild fever (38.4°C) for which 
he was given a single 200 mg dose of ibuprofen. The fever resolved within 5 hours and never 
recurred.   

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Table 2: Maximum Severity of Solicited Adverse Events in Subjects Receiving WRSs2 
WRSs2 Dose 
(n) 
Diarrhea 
Fever 
Headache Cramps Vomiting Myalgia Arthralgia 
Mild 
Mod-
Severe 
Any 
Any 
Any 
Any 
Any 
Any 
103 (8) 
1 
0 
0 
1 
1 
0 
1 
0 
104 (8) 
1 
0 
0 
3 
2 
1 
0 
1 
105 (8) 
0 
0 
0 
4 
2 
0 
0 
0 
106 (8) 
1 
0 
0 
1 
1 
0 
0 
0 
107 (8) 
2 
1 
1 
(38.4°C) 
4 
2 
0 
1 
0 
Placebo (9) 
0 
0 
0 
2 
1 
0 
1 
1 
 
S. sonnei 53G 
S. sonnei strain 53G is a virulent strain that causes illness ranging from mild (watery diarrhea 
being the main symptom) to severe (fever and abdominal cramping with frequent small volume 
blood streaked stools containing mucus). Complaints may include nausea, vomiting, headache, 
anorexia, muscle and/or joint aches and malaise. Symptoms typically are self-resolving in 5-7 
days. However, administration of an effective antibiotic (as will occur in the current study) will 
significantly reduce, or eliminate, symptoms within 24 hours of initiation. Subjects will be 
closely monitored, and anyone determined by a study physician to meet criteria for severe illness 
will be offered antibiotic treatment at that time rather than the scheduled day 5.  
Shigella is not commonly associated with significant dehydration as stool volume typically is not 
large. The S. sonnei 53G challenge strain is known to be susceptible to multiple antimicrobial 
agents including TMP-SMX and quinolones. There is a risk of bacteremia, although Shigella 
bacteremia rarely occurs in adult populations with wild-type Shigella3. In a study we recently 
conducted to determine the dose of 53G that would produce illness in approximately 60% of 
recipients, 56 subjects were given an oral dose of 53G ranging from 0.5x103 cfu to 1.76x103 
cfu29. Symptoms experienced by subjects were typical of Shigella and are listed above in Table 
1.  
 

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Other Shigella-related AEs 
Post-Infectious Irritable Bowel Syndrome (PI-IBS) 
PI-IBS, a functional bowel disorder characterized by unexplained abdominal discomfort or pain 
associated with changes in normal bowel patterns, has been described to occur 6-7 times more 
frequently after an acute enteric infection compared to similar matched controls without such a 
history30, 31. The risk of PI-IBS associated with an attenuated Shigella vaccine is unknown. To 
minimize study risks, subjects with a history of abnormal bowel patterns who might be at higher 
risk for this post-infectious sequelae will be excluded. 
Post-Infectious Reactive Arthritis/Arthropathy 
A post-dysenteric reactive arthritis, with ocular and/or urethral inflammation, which has an onset 
typically 1 to 3 weeks after the onset of diarrhea, has been reported to occur in patients with 
shigellosis. Data suggest that the risk of reactive arthritis following shigellosis may be higher in 
persons carrying the human leukocyte antigen (HLA)-B27, thus the exclusion of subjects with a 
positive HLA-B27 test at screening.  
Secondary Spread of Vaccine 
Our experience, and that of others, have shown that secondary spread of the vaccine strain is 
highly unlikely. As part of the WRSs2 study discussed above; 40 subjects received WRSs2, 40 
received WRSs3 and 9 received placebo. Subjects were housed in an inpatient unit with 
antibiotics prescribed to everyone on Day 8 post vaccination. Of the WRSs2 vaccinees, 28 (70%) 
shed the vaccine while 25 (63%) of the subjects who received WRSs3 shed the strain. Subjects 
typically shed the vaccine strain in their stool for 2-7 days although some subjects shed until the 
initiation of antibiotic treatment. In contrast, despite the daily close contact, no placebo subject 
shed Shigella in their stool, strongly suggesting that the risk of secondary transmission is low. 
Similarly, a previous live oral S. sonnei vaccine candidate, WRSS1, that was derived from the 
same parental strain Moseley as WRSs2 was evaluated in an inpatient and multiple outpatient 
Phase 1 trials to further support the contention that secondary spread of the vaccine strain is 
extremely rare. WRSS1 initially was tested in clinic-based trials at the University of Maryland 
Center for Vaccine Development16. Subsequently, a community-based, outpatient trial of 
WRSS1, under FDA oversight, was conducted in Tel Aviv, Israel17. Three groups of 15 subjects 
received a dose of WRSS1 ranging from 103 to 105 cfu in a dose-escalating manner. For each 
vaccinee, a person who did not receive the vaccine but shared the same living space and toilet 
facilities was enrolled. Both vaccinees and housemates were asked to collect a daily rectal swab 
for up to 28 days that was immediately placed in Cary-Blair transport media and stored in a cool 

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place until delivered to the clinic for culture. While most vaccine recipients shed WRSS1 for 
approximately 5-6 days, WRSS1 was not isolated from any household contact. 
The combination of data from these various S. sonnei live-attenuated vaccine trials strongly 
indicate that secondary spread of the vaccine strain is extremely unlikely and thus the vaccine is 
safe to administer in the outpatient setting. 
Acquisition/Spread of COVID-19 
To minimize the risk of subjects acquiring and/or spreading COVID-19, all subjects will be 
encouraged to be vaccinated against SARS-CoV-2. Additionally, all subjects must have a 
negative test for SARS-CoV-2 at the time of admission to the inpatient unit. SARS-CoV-2 
testing will be repeated at any time a subject exhibits symptoms of COVID-19 illness during the 
inpatient stay. Subjects with symptoms of COVID-19 will be quarantined to their room until 
results of testing are available. Anyone testing positive for COVID-19 will remain quarantined in 
their room until it is medically safe for the subject to be escorted off the inpatient unit. Current 
COVID-19 guidelines for the study site will be followed.  
Other risks 
The inclusion and exclusion criteria have been constructed as to select for a population of 
healthy adults which should minimize the risks to study subjects. However, unrecognized risks 
still may be associated with administration of the vaccine. Because the risks of the vaccine to a 
pregnant or nursing woman or fetus are unknown, pregnancy is an exclusion and women of 
childbearing potential will be counseled against becoming pregnant during their participation in 
the study. 
Rectal swabs 
In the event a subject is unable to produce a bulk stool, rectal swabs will be collected. Rectal 
swabs may cause discomfort for patients and in case of special conditions (e.g., hemorrhoids, and 
rectal polyps) bleeding might happen. Rectal swabs are only collected when it is not possible to 
obtain a stool sample that will be needed to document presence/absence of the vaccine and/or 
challenge strain in the stool. Study staff will instruct the subjects on how to self-collect the rectal 
swab sample. 
Bicarbonate administration  
Mild gastrointestinal symptoms may occur due to ingestion of the bicarbonate solution.  
 
 

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Risk related to antibacterial therapy 
Ciprofloxacin is the desired antibiotic given to all subjects on the 5th day after challenge and is 
generally well-tolerated. The most frequently reported drug related events for ciprofloxacin 
therapy have been nausea, diarrhea, abnormal liver function tests, vomiting, and rash which are 
all mild to moderate in most cases. Fluoroquinolones, including ciprofloxacin, are associated 
with an increased risk of tendonitis and tendon rupture in all ages, and in high-risk patients can 
increase risk of aortic tear or rupture, and QT prolongation. Other symptoms rarely seen include 
hypersensitivity, dizziness, pseudomembranous colitis, and peripheral neuropathy.  
In the case of a subject being allergic to ciprofloxacin, trimethoprim-sulfamethoxazole (Bactrim) 
will be the alternate antibiotic administered. The most common adverse effects of Bactrim are 
gastrointestinal disturbances (nausea, vomiting, and anorexia) and allergic skin reactions (such as 
rash and urticaria). 
Spread of the S. sonnei 53G Challenge Strain 
To minimize the likelihood of transmission of bacteria from a subject to a study staff, “Contact 
Precautions” (good hand hygiene along with wearing gowns and gloves for activities involving 
contact with subjects) will be observed by all study personnel. If the staff are working with a 
subject who is actively vomiting, face masks will be worn.  
Subjects will be advised to wash their hands frequently, especially after toilet use and before 
meals. Subjects will receive counseling and education about gastroenteritis and the necessary 
precautions to minimize the potential for spread of disease to others prior to discharge.  
2.3.2 Potential Benefits 
There are no direct medical benefits to the subject as a result of study participation.  
The subject, by participating in this study will help provide data about the tolerability, 
immunogenicity, and efficacy of a candidate vaccine against S. sonnei infection. These data will 
be instrumental in further development of a Shigella vaccine for licensure.  
3 STUDY DESIGN, OBJECTIVES AND ENDPOINTS OR 
OUTCOME MEASURES 
3.1 Study Design Description 
This is a double-blind, placebo controlled, randomized trial to evaluate the safety, reactogenicity, 
immunogenicity and efficacy of up to a 106 cfu dose of an oral live-attenuated oral S. sonnei 

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vaccine candidate, WRSs2, in males and non-pregnant females aged 18-49, inclusive at the time 
of the first vaccination. This is a two-phase study, an outpatient WRSs2 vaccination phase and an 
inpatient S. sonnei 53G challenge phase (Table 3).  
Table 3A: Initial Vaccine Administration, Challenge and Follow-up 
 
Initial Vaccine Administration 
Arm 
Outpatient 
(Vaccination Phase) 
Inpatient (CCHMC) 
(Challenge Phase) 
Outpatient 
Study Day 
Day 4-57 
7 visits  
Day 57 
(+2) 
Day 58-65 
(planned 
discharge 
on study 
day 65) 
Day 71-180 
3 visits, 
final 
contact  
1  
29 +2d 
1 
WRSs2 
WRSs2 
Safety, vaccine 
fecal shedding, 
blood, and stool 
collection for 
immunogenicity  
challenge 
with  
S. sonnei 
53G 
Safety, 
challenge, 
fecal 
shedding, 
blood, and 
stool 
collection 
for immune 
responses 
post 
challenge  
Blood and 
stool 
collection 
for immune 
responses 
post 
challenge  
Final 
contact for 
safety 
2 
Placebo 
WRSs2 
3 
Placebo 
Placebo 
 
Up to 120 subjects will be enrolled and randomized 1:1:1 into 3 vaccination arms. Arm 1 
received 2 doses of WRSs2, Arm 2 received placebo followed by WRSs2, and Arm 3 received 
two doses of placebo. Twenty-eight (+2) days after the second dose of study agent, subjects will 
be admitted to the inpatient unit and given an oral challenge of approximately 1500 cfu of 53G. 
The goal will be to have 90 subjects receive a challenge dose of 53G.    
For the amendment, the remainder of subjects will be randomized into 2 arms at 2:1 (vaccine: 
placebo) to receive two doses of 5x105 vaccine or placebo. Neither the vaccination regimen nor 
the 53G challenge dose are being amended. The table below outlines the updated study design.  
 

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Table 4B: Amended Vaccine Administration, Challenge and Follow-up 
 
Amended Vaccine Administration  
Arm 
Outpatient 
(Vaccination Phase) 
Inpatient (CCHMC) 
(Challenge Phase) 
Outpatient 
Study Day 
Day 4-57 
7 visits  
Day 57 
(+2) 
Day 58-65 
(planned 
discharge 
on study 
day 65) 
Day 71-
180 
3 visits, 
final 
contact  
1  
29 +2d 
1 
WRSs2 
WRSs2 
Safety, vaccine 
fecal shedding, 
blood, and stool 
collection for 
immunogenicity  
challenge 
with  
S. sonnei 
53G 
Safety, 
challenge, 
fecal 
shedding, 
blood, and 
stool 
collection 
for immune 
responses 
post 
challenge  
Blood and 
stool 
collection 
for immune 
responses 
post 
challenge  
Final 
contact for 
safety 
2 
Placebo 
Placebo 
 
Screening Phase (Outpatient) 
During the screening period, Day -60 to Day -1 subjects providing informed consent will have 
their medical history recorded and be screened for general health status, vital signs, and 
abbreviated physical exam. In addition, subjects will be asked to provide specimens for urine, 
blood, and stool testing. Subjects unable to produce a stool will be asked to use a swab to collect 
the sample to be used for detection of Shigella in the stool. In addition, serum samples will be 
collected to determine eligibility based on Shigella LPS-specific IgG levels. 
 
 
Enrollment/Vaccination Phase (Outpatient) 
Eligibility for enrollment will be confirmed by reviewing inclusion and exclusion criteria, review 
of medical history and concomitant medications, vital signs assessment, negative pregnancy test 

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for females of child-bearing potential within 24 hours prior to dosing, and targeted physical 
exam to ensure that there are no contraindications for vaccination. 
Vaccination Days 1 and 29  
Eligible subjects will be randomized to a vaccination arm per the Internet Data Entry System
(IDES) in a 1:1:1 ratio to receive either WRSs2/WRSs2; Placebo/ WRSs2; or Placebo/Placebo.  
All participants enrolled after update of protocol V7.0 will be randomized 2:1 in vaccine or 
placebo groups. 
Subjects will be required to be NPO for 90 minutes prior to dosing. Prior to administration of the 
vaccine or placebo, subjects will drink approximately 150mL of bicarbonate buffer solution to 
neutralize the stomach acidity. Within 5 minutes, subjects will drink approximately 31 mL of 
saline containing the vaccine, or placebo (see current MOP for detail instruction of vaccine 
dilution and administration). Subjects who vomit the bicarbonate buffer solution before drinking 
the study agent (vaccine or placebo) may be re-dosed with bicarbonate. If a subject vomits the 
vaccine (WRSs2 or placebo) within 5 minutes of ingestion, the subject may be re-dosed once and 
allowed to remain in the study.   
Subjects will be asked to remain on site and observed for AEs for at least 90 minutes following 
vaccination to ensure post dose NPO status, have vital signs reassessed at the end of the 90-
minute observation period, and to ensure they are stable before they go home. 
Subjects will be provided with a thermometer, memory aid and instructions and site contact 
information. The memory aid will be used to collect all solicited and unsolicited AEs, and 
medications for the first 7 days after each vaccination starting the evening of administration of 
the vaccine/placebo. From 8 days until 28 days following each vaccination, only unsolicited 

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AEs, solicited adverse reactions that continue beyond the 7th day, and related medications will be 
collected during follow up. 
Also, subjects will be instructed on stool collection and storage as well as instructed on the days 
to return to the clinic with their stool samples.   
Through 7 days following each vaccination, if by the evening a subject has not entered data for 
the day, the subject will be sent automatic email reminders to enter their data.  
Subjects will be asked to follow up at the outpatient center post vaccination doses for safety, 
vaccine fecal shedding, and blood and stool collection for immunogenicity per schedule in 
Appendix A1 .  
Challenge Phase (Inpatient)  
Approximately 28 days after the 2nd dose of study agent (28 + 2 days), subjects will be scheduled 
for admission to the inpatient unit for about 9 days for challenge with live S. sonnei 53G by oral 
administration. Upon arrival to the inpatient unit, subjects will be assessed as to whether they 
meet all criteria for the challenge phase. Those who meet criteria for challenge will have SARS-
CoV-2 testing performed the result of which must be negative to be eligible to receive the 
challenge dose of 53G. Any subject positive for SARS-CoV-2 on the test prior to challenge will 
remain quarantined in room until it is medically safe to be escorted off the inpatient unit and will 
not receive a challenge dose. However, the subject still would be asked to complete safety 
follow-ups per protocol.   
Biological sample collection will occur per the schedule of events on Day 56 or Day 57 prior to 
challenge.  
Female subjects of child-bearing potential must have a negative pregnancy test within 24 hours 
prior to the challenge.  
 
 
 
Subjects will be NPO for 90 minutes prior to receiving the S. sonnei 53G challenge inoculum. A 
targeted oral challenge dose of approximately 1.5x103 cfu of S. sonnei 53G will be administered 
per MOP instructions.  
Subjects who vomit the bicarbonate buffer solution before drinking the study agent (challenge) 
may be re-dosed with bicarbonate. A subject who vomits following ingestion of the challenge 
agent will not be re-dosed with the challenge agent. A note will be made in the subject source 

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documents regarding the apparent amount of the vomitus (small, moderate, or large) along with 
the time after challenge that the vomiting started. The subject will continue in the study and 
undergo all study related procedures.  
Subjects will be observed for at least 90 minutes following challenge and have vital signs taken 
at the end of the observation period and be assessed for any AEs. 
Post-Challenge (Day 57 to Discharge Day 65(±1)), blood and stool collections will occur, and 
subjects will be monitored according to the schedule of events. If a subject is unable to produce a 
stool each day between midnight and 2100, a rectal swab will be obtained to be used for 
qualitative culturing of Shigella. To be eligible for discharge, the subject must have two stool 
samples that are culture negative for Shigella to confirm that the subject is no longer shedding.  
On the 5th day after challenge (Day 62), subjects will begin to receive 500 mg of ciprofloxacin 
twice daily for 3 days. Subjects with contraindication to ciprofloxacin may receive as second-line 
treatment trimethoprim-sulfamethoxazole (160 mg/800 mg twice daily for 5 days), or any 
antibiotic that is deemed suitable according to local guidelines and the investigator’s assessment 
can be used. 
A subject may be treated with antibiotics before the 5th day after challenge if any of the 
following occur32: 
1. Subject meets the primary endpoint of shigellosis and continues to feel ill,  
OR 
2. In the judgement of the study physician, the subject is too ill to continue without 
medical intervention.  
A subject receiving early antibiotic treatment will remain on the unit until the planned day of 
discharge. On that day, if a subject has met discharge criteria (clinically well and has had at least 
2 consecutive negative stool cultures, collected at least 6 hours apart, for S. sonnei), she/he will 
be discharged.  
A subject that, on the planned day of discharge, still has S. sonnei isolated from their stools will 
be asked to remain in the unit until she/he is no longer shedding. A subject still shedding 48 
hours after initiation of ciprofloxacin will be switched to trimethoprim-sulfamethoxazole. If a 
subject is still positive 48 hours after the second line antibiotic is administered, a culture and 
antibiotic sensitivity test will be obtained, and therapy initiated based on sensitivity results. If a 
subject has two consecutive negative stool cultures and meets the criteria to be discharged from 
the inpatient stay but did not finish the treatment course, she/he can be given the remaining doses 
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Following discharge, subjects will return at Study Day 71, Study Day 85, and Study Day 113 
post challenge for outpatient assessment, occurrence of pregnancy, AE and SAE, and collection 
of study samples. On Day 180, subjects will complete a final safety contact to assess for 
occurrence of pregnancy and SAE.   
3.2 Study Objectives 
 
3.2.1 Primary 
Estimate combined vaccine efficacy of 2 doses of WRSs2 (106 cfu or 5x105 cfu) in 
preventing shigellosis, following challenge with S. sonnei strain 53G.  
3.2.2 Secondary  
Estimate vaccine efficacy of 1 dose of 106 cfu, 2 doses of 106 cfu, and 2 doses of 5x105 
cfu of WRSs2 in preventing shigellosis following challenge with S. sonnei 53G 
Safety evaluations of WRSs2 
Evaluate immune responses following vaccination (immunogenicity) with WRSs2 and 
after challenge with S. sonnei strain 53G by serum anti-LPS and anti-Invaplex IgG and 
IgA by ELISA 
Determine fecal shedding of S. sonnei after WRSs2 vaccination and 53G challenge by 
qualitative stool culture 
3.2.3 Exploratory  
Evaluate fecal shedding of S. sonnei post vaccination and post challenge by colony 
immunoblot and PCR 
Evaluate S. sonnei antigen (LPS and Invaplex) specific IgG and IgA-ASC following 
vaccination and challenge 
Evaluate fecal IgA following vaccination and challenge 
3.2.4 Secondary Research 
Blood and stool samples will be collected and stored to characterize in depth innate and 
acquired immune response to WRSs2 and Shigella infection.  

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3.3 Study Endpoints or Outcome Measures 
3.3.1 Primary 
The occurrence of shigellosis (as outlined in Table 4) following challenge with S. 
sonnei strain 53G through Day 63 in the pooled group of subjects receiving two 
doses of 106 cfu or 5x105 cfu of WRSs2 compared to subjects receiving two doses 
of placebo   
 
Table 5: Primary Endpoint  
 Shigellosis 
Definition 
1. Severe Diarrhea 
≥6 stools classified as 3-5 in consistency in 24 hours OR >800 gm stool 
classified as 3-5 in 24 hours 
2. Moderate Diarrhea with 
additional signs/symptoms 
4-5 stools classified as 3-5 in consistency in 24 hours OR 400-800 gm 
stools classified as 3-5 in 24 hours with one or more of the following: 
oral temperature ≥38.0˚C†; ≥1 moderate constitutional/enteric symptom‡; 
≥2 episodes of vomiting in 24 hours 
3. Dysentery with additional 
signs/symptoms 
≥2 stools classified as 3-5 in consistency with gross blood (hemoccult 
positive) in 24 hours with one or more of the following: oral 
temperature ≥38.0˚C; ≥1 moderate constitutional/enteric symptom; ≥2 
episodes of vomiting in 24 hours 
† Confirmed by two separate readings at least five minutes apart 
‡  Moderate constitutional/enteric symptoms include nausea, abdominal pain/cramping, myalgia/arthralgia, malaise 
(does not include anorexia, chills, headache) 
3.3.2 Secondary 
The occurrence of shigellosis following challenge with S. sonnei strain 53G through Day 
63 in subjects receiving 1 dose of 106 cfu, 2 doses of 106 cfu, or 2 doses of 5x105 cfu of 
WRSs2 compared to subjects receiving two doses of placebo 
Occurrence of solicited systemic AEs through 7 days after each study vaccination 
Occurrence of vaccine-related unsolicited AEs through 28 days post last vaccination 
Occurrence of SAEs through Study Day 180 or until resolution or stabilization even if 
this extends beyond the study-reporting period 
Number of subjects with ≥ 4-fold rise from pre-vaccination in Shigella sonnei LPS-
specific and Invaplex-specific serum IgG and IgA by ELISA at Days 15, 29, 43, and 56 

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Maximum S. sonnei LPS-specific and Invaplex-specific serum IgG and IgA titer by 
ELISA post-vaccination through Day 56 
Peak fold-rise in S. sonnei LPS-specific and Invaplex-specific serum IgG and IgA titer by 
ELISA from pre-vaccination through Day 56 
Number of subjects with ≥ 4-fold rise from pre-challenge (Day 56) in S. sonnei LPS-
specific and Invaplex-specific serum IgG and IgA by ELISA at Days 64, 71, 85, and 113 
Maximum S. sonnei LPS-specific and Invaplex-specific serum IgG and IgA titer by 
ELISA post-challenge through Day 113 
Peak fold-rise in S. sonnei LPS-specific and Invaplex-specific serum IgG and IgA titer by 
ELISA from pre-challenge through Day 113 
Number of subjects shedding vaccine strain in their stool by culture pre-vaccination as 
well as at Days 4, 8, 15, 29, 32, 34, 36 and 43 
Number of subjects shedding 53G in their stool by culture post-challenge through Day 65 
Duration of shedding S. sonnei post-vaccination through Day 56 by culture 
Duration of shedding 53G post-challenge through Day 65 by culture 
3.3.3 Exploratory 
Duration of S. sonnei shedding post-vaccination through Day 56 by immunoblot and PCR 
Maximum S. sonnei CFU per gram of stool post-vaccination through Day 56 by 
immunoblot  
Duration of shedding 53G post-challenge through Day 65 by immunoblot and PCR 
Maximum S. sonnei CFU per gram of stool post-challenge through Day 65 by 
immunoblot  
Number of S. sonnei LPS-specific and Invaplex-specific IgG and IgA ASCs per 106 
PBMCs by ELISpot pre-vaccination and at Days 8, 29, 36, 56, 60, and 64 
Number of subjects with ≥10 IgG or IgA ASCs per 106 PBMCs (S. sonnei LPS-specific 
and Invaplex-specific) by ELISpot at any time post-vaccination through Day 56 
Number of subjects with ≥10 IgG or IgA ASCs per 106 PBMCs (S. sonnei LPS-specific 
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Number of subjects with ≥4-fold rise from pre-vaccination in S. sonnei LPS-specific and 
Invaplex-specific fecal IgA and total fecal IgA by ELISA at Day 8, 15, 29, 36, 43, 56, 61, 
64, 71, and 85 
Maximum fold rise post-vaccination of S. Sonnei LPS-specific and Invaplex-specific 
fecal IgA and total fecal IgA by ELISA through Day 56 
3.3.4 Secondary Research  
To be determined, endpoints vary based on innate and acquired immunological assays to be used. 
4 INTERVENTION/INVESTIGATIONAL PRODUCT 
4.1   Study Product Description 
WRSs2, S. sonnei vaccine candidate, is a live, attenuated vaccine that has been manufactured 
under cGMP conditions at the WRAIR PBF and it must only be administered to subjects 
participating in an FDA-approved clinical study according to the inclusion and exclusion criteria 
specified in the study protocol. 
The wild type S. sonnei strain was isolated from a laboratory worker “Moseley,” accidentally 
infected in 1975 with a laboratory S. sonnei strain obtained from an infected monkey.  The 
infected worker showed classic signs and symptoms of shigellosis such as fever, severe intestinal 
cramps, diarrhea, and dysentery.  The isolated strain termed Moseley is stored as lyophilized 
vials at WRAIR, and this material was used to manufacture (8 June 2000) a Production Cell 
Bank (PCB) of Moseley 
, which is stored at -80°C at the WRAIR, PBF.  
A vial (#56) of Moseley strain 
, received on July 2004 was the starting material 
for the construction of WRSs2.  Specific gene deletion was carried out to obtain the research 
seed of WRSs233. The method of lambda red recombineering was used to sequentially 
delete the entire open reading frames (ORFs) of senA, senB, and virG(icsA). Finally, the 
strain was made tetracycline sensitive (tet-S) by growth on fusaric acid33. The loss of these 3 
genes is expected to make this strain when given orally, to be safe, immunogenic, and 
protective against challenge in volunteers. It is also sensitive to tetracycline and other 
commonly used antibiotics. The strain constructed was named WRSs215, 25, 33.  
Research seed of WRSs2 was used to manufacture the lyophilized WRSs2 vaccine products for 
clinical use. Master Cell Bank (MCB) seeds and PCB seeds were manufactured and stored at -
. A single vial of PCB was used to 
 

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 The product WRSs2 
, was lyophilized in 2 mL aliquots (4.8 x 108 cfu/mL) and is stored at -80ºC ± 10ºC 
under continuous temperature monitoring at WRAIR, PBF. All vials were inspected under extra 
light and magnification
 
 
 
 
Lyophilized, frozen vials of WRSs2 (
), are labeled as: 
Shigella sonnei WRSs2 
BPR No.: BPR-887-00   
Lot No.: 
 
Contents: 2.0 ml + 5% (Lyoph.)    
Storage: -80 + 10°C 
Caution: New drug limited by Federal Law to investigational use only. 
Date of Mfg.: 04 Aug 2008 
Manufactured By: WRAIR, Silver Spring, MD 20910 
Placebo (0.9% Sterile Normal Saline, USP) 
0.9% sterile normal saline for human use will be used as the placebo for this trial. The USP grade 
0.9% sodium chloride (normal saline) is a sterile, nonpyrogenic, isotonic solution; each mL of 
fluid contains 9 mg of sodium chloride. It contains no bacteriostatic agent, antimicrobial agent, 
preservatives, or added buffer and is supplied only in single-dose containers. The solution may 
contain hydrochloric acid and/or sodium hydroxide for pH adjustment (pH 5.3, range 4.5-7.0). 
0.9% sterile normal saline will be stored at 20ºC to 25ºC (68°F to 77ºF) [See USP Controlled 
Room Temperature]. Study product will remain on site stored as indicated until final 
accountability is completed.  Used and unused product vials will remain on site stored as 
indicated until final accountability is completed. At study completion the site is given direction 
from DMID regarding disposition of the product. 
The study product will be labeled according to manufacturer or regulatory specifications and 
include the statement “Caution: New Drug – Limited by Federal Law to Investigational Use.” 
 Challenge (S. sonnei 53G) 
53G is a virulent S. sonnei strain that was initially isolated from a child with diarrhea in Tokyo in 
1954. The seed was maintained at the Center for Vaccine Development (CVD), University of 
Maryland. In Sep 1998, a research seed vial was streaked out on a 
 
 plate at the CVD and transferred to WRAIR PBF for the purpose of manufacturing a 
--
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MCB -
) and PCB -
) under cGMP. The MCB and PCB are 1 mL glycerol 
cultures that are stored at -80°C ±10°C and in liquid nitrngen at the WRAIR PBF. fu 2013, one 
vial of the PCB 
) was plated 
This standardized, lyophilized lot of S. sonnei 53G 
was manufactured 25 Feb. 2013 under cGMP conditions at the WRAIR PBF and is 
stored at -80°C ± 10°C at the PBF. 
The S. sonnei 53G 
the vials appears as 
was lyophilized in 2 mL aliquot (2 x 109 cfu/mL) and content of 
The study product is labeled according to manufacturer or regulato1y specifications and include 
the statement "Caution: New Dmg - Limited by Federal Law to fuvestigational Use." (See label 
below). 
Shigella sonnei 63G 
BPR No.: BPR-1094-40 
Lot No.: -
Cootents: 2.0 ml (Lyoph.} 
Storage: -80 :1: 10 •c 
Caution: New Drug - Limited by Federal (or Untted 
States) law to investigational use. 
Date of Mfg.: 25 Fell 13 
Manufactured By: WRAIR, Silver Spring, MD 20910 
4.1.1 
Product Storage and Stability 
The temperature of the storage units at study sites must be manually recorded daily ( excluding 
non-business days and holidays, as applicable) and continuously monitored and recorded during 
the course of this trial per the paiiicipating VTEU site standard operating procedures (SOPs), and 
documentation will be maintained. If the temperature fluctuates outside of the required range, the 
affected study product(s) must be quai·antined at the coITect storage temperature and labeled as 
'Do Not Use' (until fuii her notice). The pa1iicipating VTEU site's research pha1m acist must ale1i 
the site principal investigator and study coordinator, if the temperature fluctuates outside of the 
required range. fu the event the temperature fluctuates outside of the required range, including 
accidental deep-freezing or dismption of the cold chain, the affected study product(s) must not 
be administered. The site principal investigator or responsible person should immediately contact 
the DMID Product Support Team at DMIDProductSuppo1iTeam@niaid.nih.gov for fuither 
instructions before any additional study products ai·e administered. Based on the infonnation 
collected, DMID and/or the manufacturer will detennine whether the affected study product(s) 
can be used. If it cannot be used, the site will receive specific insti11ctions on how to return the 
affected study product(s) to the DMID CMS or destroy it on site. Additional instmctions for 
quarantine and DMID contact info1mation are provided in the protocol-specific MOP. 
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WRSs2 Vaccine Stability  
The study vaccine, Shigella sonnei WRSs2, 
, is stored at -80°C ±10°C. Once 
reconstituted, vaccine must be kept on ice and used within 2 hours. 
Details regarding results of Viability and Stability Testing are provided in the WRSs2 
Vaccine Investigator’s Brochure.  
 
Challenge strain (S. sonnei 53G)  
The challenge material, Shigella sonnei strain 53G, is stored at -80 °C ±10°C. Once 
reconstituted, the maximum hold time is 2 hours on ice between the reconstitution and 
administration of the product to the subjects. 
Details regarding results of Viability and Stability Testing are provided in the Shigella 
sonnei strain 53G Investigator’s Brochure.  
 
Placebo (0.9% Sterile Normal Saline, USP) 
 
0.9% sterile normal saline will be stored at 20ºC to 25ºC (68°F to 77ºF) [See USP Controlled 
Room Temperature]. For excursions between 15-30°C (59°F to 86°F), the product can continue 
to be used (no quarantine) but the site needs to complete the DMID Study Product Support Team 
Temperature Excursion Reporting Form and submit to PST. Based on USP guidelines, 
excursions between 15°C and 30°C are allowed and will not be considered deviations. Study 
product will remain on site stored as indicated until final accountability is completed. Used and 
unused product vials will remain on site stored as indicated until final accountability is 
completed. At study completion the site is given direction from DMID regarding disposition of 
the product. See protocol-specific MOP for further instructions.  
4.2   Acquisition/Distribution 
Vaccine (WRSs2, lot# 
)  
The product will be shipped on dry ice with a temperature monitor from WRAIR to the DMID 
Clinical Agents Repository Contract, Fisher BioServices, which will then distribute them to the 
study site(s). Upon delivery to sites, the vaccine vials will be stored in the Investigational 
Pharmacy in a -80°C ±10°C freezer that is consistently monitored for temperature.  
Placebo (0.9% Sterile Normal Saline, USP)  
0.9% sterile normal saline will be stored at 20ºC to 25ºC (68°F to 77ºF) [See USP Controlled 
Room Temperature]. Study product will remain on site stored as indicated until final 
accountability is completed. Used and unused product vials will remain on site stored as 

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indicated until final accountability is completed. At study completion the site is given direction 
from DMID regarding disposition of the product. See protocol-specific MOP for further 
instructions.  
Challenge strain (S. sonnei 53G, 
)                
The product will be shipped on dry ice with a temperature monitor from WRAIR to the DMID 
Clinical Agents Repository Contract, Fisher BioServices, which will then distribute them to the 
study site(s). Upon delivery to sites, the 53G challenge strain vials will be stored at the 
Investigational Pharmacy in a -80°C ±10°C freezer that is consistently monitored for 
temperature. 
 
Upon request by DMID, WRSs2 and 53G, will be transferred to the following address: 
DMID Clinical Materials Services (CMS) Contract  
Fisher BioServices 
20439 Seneca Meadows Parkway 
Germantown, MD 20876 
Phone: 
 
Fax: 
 
Email: DMID.CMS@thermofisher.com 
0.9% sterile normal saline will be obtained by the DMID Clinical Materials Services (CMS) 
Contract, Fisher BioServices. 
WRSs2, S. sonnei 53G and 0.9% sterile normal saline will be provided through the DMID CMS 
to the participating VTEU sites prior to the start of this trial upon request and with prior approval 
from DMID. Should the site principal investigator require additional WRSs2, S. sonnei 53G and 
0.9% sterile normal saline during this trial, further instructions are provided in the protocol-
specific MOP.  
Once received, the vaccine, placebo (0.9% sterile normal saline), and challenge material will be 
stored in and dispensed by the Investigational Pharmacy.  
Sterile water for injection (SWI), sodium bicarbonate, ciprofloxacin 500 mg tablets, and 
trimethoprim-sulfamethoxazole will be provided by the study site, stored per manufacturer 
instructions, and dispensed by the Investigational Pharmacy. 

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4.3   Protocol-Specified Medications/Treatments other than Study Products 
Ciprofloxacin (500 mg tabs) for human use will be purchased from a commercial vendor with 
packaging, labeling and storage as per manufacturer’s instructions. Subjects will receive 500 mg 
of ciprofloxacin twice daily for 3 days.                                                                                                   
Bactrim (trimethoprim (160)-sulfamethoxazole (800)) for human use will be purchased from a 
commercial vendor with packaging, labeling and storage as per manufacturer’s instructions. 
Trimethoprim-sulfamethoxazole will be dosed as 1 pill twice daily for 5 days, or any antibiotic 
that is deemed suitable according to local guidelines and the investigator’s assessment can be 
used. 
Sterile water for injection (SWI) for human use will be purchased by the study site and will be 
stored and prepared per manufacturer’s instructions.  
Sodium bicarbonate for human use will be purchased by the study site and will be stored and 
prepared per manufacturer’s instructions.  
4.4   Dosage/Regimen, Preparation, Dispensing and Administration of Study 
Intervention/Investigational Product 
Vaccine WRSs2 
Lyophilized, frozen vials of S. sonnei WRSs2 (
) will be maintained in Investigational 
Pharmacy and stored at -80°C ±10°C until the day of vaccination. On the day of vaccination, the 
requisite number of vials will be removed from the freezer and placed on ice and allowed to thaw 
for 30 minutes. Then 2 mL of sterile water for injection (SWI) will be added to each vial, this 
reconstitution will result in 4.8x108 cfu/mL. To ensure complete rehydration of the vaccine strain 
along with homogeneous mixing of each vial’s contents, the vials will remain on ice for another 
15 minutes with intermittent swirling of the suspension. All vials then will be combined and 
diluted in sterile normal saline 0.9% to arrive at a concentration of 5x105 cfu/mL. The diluted 
vaccine will be kept on ice until administration. At the time of reconstitution, an aliquot will be 
removed for testing by colony count to be able to document the actual dose administered to the 
subjects. Time of reconstitution will be noted. A second aliquot will be collected and frozen at    
-80°C ±10°C until testing is performed to confirm that the bacteria administered to the subjects 
still possessed the original mutations. Detailed vaccine dilution procedure is described in the 
current version of the MOP. 
One mL of saline containing 106 cfu as per version 7.0, and for the final cohort, 5x105 cfu of the 
WRSs2 vaccine, will be added to 30 mL of sterile normal saline (0.9% NaCl) and placed in a 

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container. 150 mL of a sodium bicarbonate solution (2 grams of sodium bicarbonate, NaHCO3, 
in 150 mL of SWI) will be placed in a second container. Placebo will consist of 31 mL sterile 
normal saline. 
Subjects will be NPO for 90 minutes prior to and after receiving the vaccine. At the indicated 
time, the subjects will be gathered together, and doses distributed. Subjects will drink the sodium 
bicarbonate solution to neutralize gastric acidity and then drink the vaccine suspension or 
placebo within 5 minutes of drinking the sodium bicarbonate. Care must be taken to ensure that a 
minimum amount of time, not to exceed 2 hours, is spent between the reconstitution of the 
vaccine and the oral administration of the vaccine to the subjects.  
 
Challenge S. sonnei 53G 
Lyophilized, frozen vials of S. sonnei 53G will be maintained in Investigational Pharmacy and 
stored at -80°C ±10°C until the day of challenge.  
On the day of challenge, one vial will be removed from the freezer and placed on ice and 
allowed to thaw for 30 minutes. Then 2 mL of cold sterile water (2-8°C) for injection (SWI) will 
be added to the vial. To ensure complete rehydration of the vaccine strain along with 
homogeneous mixing of the vial’s contents, the vial will remain on ice for another 15 minutes 
with intermittent swirling of the suspension. The content of the vial will then be diluted in cold, 
sterile normal saline 0.9% to arrive at the desired concentration of approximately 1.5x103 
cfu/mL. The diluted challenge strain will be kept on ice until administration. At the time of 
reconstitution, an aliquot will be removed for testing by colony count to be able to document the 
actual dose administered to the subjects. Time of reconstitution will be noted. The maximum 
hold time is 2 hours on ice between the reconstitution and administration of the challenge 
product to the subjects. See MOP for details. 
One mL of prepared challenge will be added to 30 mL of sterile USP 0.9% saline in a plastic 
drinking cup. The maximum hold time for placebo will be 2 hours. 
Subjects will be NPO for 90 minutes prior to receiving approximately 120 mL solution of 
bicarbonate buffer (2 gm bicarbonate in 120 mL water). Within 5 minutes of the buffer, subjects 
will drink approximately 31 mL of saline containing approximately 1.5x103 cfu of the S. sonnei 
53G suspension.  
Pre-determined Modification of Study Intervention/Investigational Product for an 
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If a subject vomits the vaccine (WRSs2 or placebo) within 5 minutes of ingestion, the subject 
may be re-dosed once and allowed to remain in the study. Subjects who vomit the dose of study 
agent the second time will not be administered additional doses of study agent, nor will they be 
replaced, but will be asked to remain in the study to follow for safety. Subjects who vomit after 
receipt of the challenge dose of 53G will not be re-dosed due to safety concerns for potentially 
administering higher than intended dose of virulent Shigella.  
4.5   Accountability Procedures for the Study Intervention/Investigational 
Product(s) 
The Food and Drug Administration (FDA) requires accounting for the disposition of all 
investigational products. The Investigator is responsible for ensuring that a current record of 
product disposition is maintained, and product is dispensed only at an official study site 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 drug was administered.  
The Investigational Pharmacist will be responsible for maintaining accurate records of the 
shipment and dispensing of the investigational product. The pharmacy records must be available 
for inspection by the DMID monitoring contractors and is subject to inspection by a regulatory 
agency (e.g., FDA) at any time. An assigned Study Monitor will review the pharmacy records. 
Used and unused of study products will be retained until monitored and released for disposition, 
as applicable.  Final disposition of the unused study products will be determined by DMID and 
communicated to the participating sites by the DMID Clinical Project Manager. 
The WRSs2 (
) vaccine vials will be stored in the Investigational Pharmacy in a                   
-80± 10 °C freezer that is under a temperature-monitoring program. All vials of used and unused 
product will remain on site stored as indicated until final accountability is completed. At study 
completion the site is given direction from DMID regarding disposition of the product.  
Lyophilized, frozen vials of S. sonnei 53G (
) will be maintained in Investigational 
Pharmacy and stored at -80 °C ±10 °C. Used and unused product vials will be stored in the 
Investigational Pharmacy until final accountability is completed. At study completion the site is 
given direction from DMID regarding disposition of the product.  
0.9% sterile normal saline will be stored at 20ºC to 25ºC (68°F to 77ºF) [See USP Controlled 
Room Temperature].  Study product will remain on site stored as indicated until final 
accountability is completed.  Used and unused product vials will remain on site stored as 
indicated until final accountability is completed. At study completion the site is given direction 
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5 SELECTION OF SUBJECTS AND STUDY ENROLLMENT 
AND WITHDRAWAL 
5.1   Eligibility Criteria 
Up to 120 individuals, male or non-pregnant female 18 to 49 years, inclusive at the time of the 
first vaccination, who are in good health by history and screening values and meet all of the 
inclusion criteria and none of the exclusion criteria, will be enrolled and randomized to a study 
group using IDES. 
Inclusion and exclusion criteria are applied for enrollment of subjects into the study. The target 
population should reflect the community at large at each of the participating VTEU sites.  
Subject inclusion and exclusion criteria must be assessed by a study clinician licensed to make 
medical diagnoses and listed on the Form FDA 1572 as the site principal investigator or sub-
investigator. 
No exemptions are granted on subject inclusion or exclusion criteria in DMID-sponsored studies. 
Questions about eligibility should be directed toward the DMID Medical Officer. 
5.1.1 Subject Inclusion Criteria 
1. Provide informed consent prior to initiation of any study procedures. 
2. Are able to understand and comply with planned study procedures and be available for all 
study visits. 
3. Is 18-49 years of age inclusive and in sufficiently good health* to be safely enrolled in 
this study as determined by medical history, medication use, and abbreviated physical 
exam. 
 
*Good health is defined by the absence of any exclusionary medical conditions. If the subject has another 
current, ongoing medical condition, the condition cannot meet any of the following criteria: 1) first 
diagnosed within 3 months of enrolment; 2) is worsening in terms of clinical outcome in last 6 months; or 
3) involves need for medication that may pose a risk to subject’s safety or impede assessment of AEs or 
immunogenicity if they participate in the study. Topical, nasal, and inhaled medications (with the exception 
of inhaled corticosteroids as outlined in the Subject Exclusion Criteria [see Section 5.1.2]), herbals, 
vitamins, and supplements are permitted. 
4. Oral temperature is less than 100.4°F. 
5. Pulse is 50 to 100 beats per minute (bpm), inclusive.    
6. Systolic blood pressure is 90 to 140 mmHg, inclusive.  
7. Diastolic blood pressure is 55 to 90 mmHg, inclusive. 

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8. Females of childbearing potential** may enroll if subject has practiced adequate 
contraception*** > 30 days prior to enrollment and agrees to continue adequate 
contraception for the entire study. 
  **Child-bearing potential is defined as not 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 of the last menses if menopausal. 
  ***Adequate contraception includes non-male sexual relationships, abstinence from sexual intercourse 
with a male partner, monogamous relationship with vasectomized partner who has been vasectomized for 
180 days or more prior to the subject enrollment, barrier methods such as condoms or diaphragms with 
spermicide, effective intrauterine devices, NuvaRing®, and licensed hormonal methods such as implants, 
injectables, or oral contraceptives (“the pill”). 
9. Females of childbearing potential must have a negative urine or serum pregnancy test 
within 24 hours prior to enrollment. 
10. Drug screen for opiates is negative. 
11.  BMI between 18 and 40 kg/m^2.  
 
5.1.2 Subject Exclusion Criteria 
1. Have any disease or medical condition that, in the opinion of the site principal 
investigator or appropriate sub-investigator, is a contraindication to study participation+. 
 
+Including acute or chronic disease or medical condition that would place the subject at an unacceptable risk 
of injury, render the subject unable to meet the requirements of the protocol, or may interfere with the 
evaluation of responses or the subject’s successful completion of this trial. These include: 
 
• History of inflammatory bowel disease (IBD) (including ulcerative colitis, Crohn’s disease, 
indeterminate colitis, or celiac disease). 
• Irritable bowel syndrome (IBS) within the past 12 months or any active uncontrolled gastrointestinal 
disorders or diseases as assessed by the investigator. Including: symptoms or evidence of active gastritis 
or gastroesophageal reflux disease, gastric surgery, or gastric acid hyper-secretory disorders (e.g., 
Zollinger-Ellison syndrome), gastrointestinal obstruction, ileus, gastric retention, bowel perforation, 
toxic colitis, persistent infectious gastroenteritis, persistent or chronic diarrhea of unknown etiology, 
Clostridium difficile infection. 
• 
Known active neoplastic disease^, a history of any hematologic malignancy, or have used anticancer 
chemotherapy/radiation therapy (cytotoxic) within 3 years prior to study enrollment.  
^Non-melanoma, treated, skin cancers are permitted. 
• 
Personal or family history of reactive arthritis. 
• 
Reported history of HIV, Hepatitis B, or Hepatitis C  

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• 
History of immunodeficiency due to congenital or hereditary causes, underlying illness, or treatment. 
2. Positive serology results for HLA-B27, HIV, HBsAg, or HCV antibodies. 
3. Have clinically significant abnormalities as determined by study investigator in other 
screening laboratory tests, as outlined in the protocol.   
4. Have participated in a previous Shigella challenge study or reports having received 
vaccination for Shigella previously.   
5. Have a previously laboratory confirmed case of disease caused by S. sonnei or serology 
positive (>1:2500) for anti S. sonnei LPS IgG ELISA titer at screening. 
6. Has a history of diarrhea in the 14 days prior to enrollment.  
7. Have fewer than 3 stools per week or more than 3 stools per day as the usual frequency.  
8. Recent history/current use of immunosuppressive/immunomodulating disease therapy.  
9. Known hypersensitivity to ciprofloxacin or trimethoprim-sulfamethoxazole; sodium 
bicarbonate; or any components of vaccine, placebo, or challenge material.   
10. Received or plan to receive a licensed live vaccine within 30 days prior to enrollment. 
11. Received or plan to receive a licensed, inactivated vaccine, COVID-19 vaccine, or an 
influenza vaccine within + 7 days from receipt of study product. 
12. Have a history of severe reactions following previous immunization with any licensed or 
unlicensed vaccine. 
13. Received Ig or other blood products (with exception of Rho D Ig) within 90 days prior to 
enrollment. 
14. Have taken oral or parenteral (including intra-articular) corticosteroids of any dose, or 
high-dose inhaled corticosteroids= within 30 days prior to enrollment. 
=High-dose defined per age as using inhaled high dose per reference chart Estimated Comparative Daily 
Dosages 
https://www.nhlbi.nih.gov/files/docs/guidelines/asthma_qrg.pdf   
15. Have taken systemic antibiotics within 7 days prior to enrollment. 
16. Have taken prescription and/or OTC medication containing loperamide, acetaminophen, 
aspirin, ibuprofen, or other non-steroidal anti-inflammatory < 48 hours prior to 
enrollment. 
17. Have a history of alcohol or drug abuse within 1 year prior to enrollment. 
18. Have been hospitalized for psychiatric illness, history of suicide attempt, or confinement 
for danger to self or others within 5 years prior to enrollment. 
19. Work or plan to work in either a health care setting, day care center, or as a food handler 
or have known daily contact with individuals with possible increased susceptibility$ to 
Shigella within 14 days after discharge from inpatient challenge.  
 
$Immunocompromised, elderly persons aged 70 years or more, diapered individuals, persons with 
disabilities, children <2 years old, a woman known to be pregnant or nursing, or anyone with diminished 

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immunity. Known daily contact includes contact at home, school, day-care, nursing home, or similar 
places. 
 
20. Are pregnant, breastfeeding, or plan to become pregnant or breastfeed at any given time 
during the study. 
21. Have fever or an acute illness! as determined by the site principal investigator or 
appropriate sub-investigator, within 72 hours prior to enrollment 
 
! An acute illness which is nearly resolved with only minor residual symptoms remaining is allowable if, in the opinion 
of the site principal investigator or appropriate sub-investigator, the residual symptoms will not interfere with the 
ability to assess safety parameters as required by the protocol. 
 
22. Received an investigational agent& within 30 days prior to the first study vaccination or 
expect to receive an investigational product during the study period which might affect 
safety or assessment of study endpoints. 
 
&Including vaccine, drug, biologic, device, blood product, or medication, other than from participation in 
this trial. 
 
23. Taking prescription or over-the-counter medication for weight reduction.  
5.2   Withdrawal from the Study, Discontinuation of Study Product, or Study 
Termination  
5.2.1 Withdrawal from the Study or Discontinuation of the Study Product 
Subjects may voluntarily withdraw their consent for study participation at any time without 
penalty or loss of benefits to which they are otherwise entitled.   
An investigator may also withdraw a subject from receiving the study product for any reason. 
Follow-up safety evaluations will be conducted if the subject agrees. If a subject withdraws or is 
withdrawn prior to completion of the study, the reason for this decision must be recorded in the 
case report forms (CRFs).  
The reasons for withdrawal or discontinuation of study product, might include, but are not 
limited to the following: 
Subject meets halting criteria  
Subject becomes noncompliant 

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Disease or condition, or new clinical finding(s) for which continued participation, in the 
opinion of the investigator might compromise the safety of the subject, interfere with the 
subject’s successful completion of this study, or interfere with the evaluation of responses 
Subject lost to follow-up 
Subject becomes pregnant 
Determined by a physician’s discretion to require additional therapy not indicated in the 
protocol to ensure subject’s health and well-being (or treatment failure, if applicable) 
The investigator should be explicit regarding study follow-up (e.g., safety follow-up) that might 
be carried out despite the fact the subject will not receive further study product. If the subject 
consents, every attempt will be made to follow all AEs through to resolution or stabilization.  
The investigator will inform the subject that already collected samples and data will be retained 
and analyzed even if the subject withdraws from this study. 
5.2.2 Handling of Withdrawals 
In the case of subjects who are lost to follow-up, extensive effort (i.e., three documented contact 
attempts via phone calls, emails, text messages, etc., made on separate occasions and followed 
by a certified letter) will be made to locate or recall them, or at least to determine their health 
status. These efforts will be documented. Every attempt will be made to follow all AEs, 
including solicited systemic AEs, unsolicited non-serious AEs, and SAEs, ongoing at the time of 
early withdrawal through resolution as per applicable collection times defined for the specific 
type of AE. 
Subjects who withdraw, or are withdrawn from this study, or are lost to follow-up after signing 
the informed consent form (ICF) and after administration of the study product will not be 
replaced. Subjects who withdraw, or are withdrawn from this study, or are lost to follow-up after 
signing the ICF but before administration of the study product may be replaced.   
5.2.3 Study Termination 
If the study is prematurely terminated by the sponsor, any regulatory authority, or the 
investigator for any reason, the investigator will promptly inform the study subjects and assure 
appropriate therapy or follow-up for the subjects, as necessary. The investigator will provide a 
detailed written explanation of the termination to the IRB/IEC. If any subject’s private 
information will continue to be collected for this study, the IRB/IEC must approve a consent 
form with the study procedures, any risks and discomforts, and applicable elements, and the 
investigator or designee will re-consent the subjects as approved by the IRB/IEC. 

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5.2.4 Study Discontinuation  
Each subject has the right to withdraw from the trial at any time for any reason without affecting 
their right to treatment by the investigator. The investigator also has the right to withdraw the 
subjects in the event of intercurrent illnesses, AEs, a subject’s failure to comply with study 
procedures, or if it is determined to be in the subject’s best interest. If a subject withdraws for 
safety reasons, an AE or SAE will be reported as described in the protocol. 
If a subject withdraws for other reasons, the withdrawal will be reported as part of the annual 
review process. If a subject has been vaccinated but does not participate in the challenge phase, 
they will be discontinued and followed for safety including an approximately 6-month post 
vaccination study contact. If a subject chooses to withdraw from the study after challenge but 
before the time of planned administration of antibiotics, they will be asked to inform either the PI 
for the trial or an Associate investigator of the study and antibiotic therapy will be initiated. They 
will be encouraged to remain in the Inpatient Unit until a three-day course of treatment can be 
completed. If they choose not to, the study staff will observe the first dose of antibiotics and 
instruct the subjects how to complete the antibiotic therapy. They will also be encouraged to 
complete all remaining outpatient visits and be followed for safety including a 6-month study 
contact. No further blood samples for immunology will be collected if a subject is terminated at 
any time during the study. 
6 STUDY PROCEDURES 
6.1   Screening 
After signing consent, demographic information will be obtained, and subjects will be carefully 
screened to ensure they are in good physical and mental health. Screening may be completed 
between Day -60 and Day -3 (-2), at one or more visits, depending on the subject and the timing 
of specimen collection.    
Screening evaluations will include: 
Obtaining informed consent followed by review of the inclusion/exclusion criteria, collection of 
demographic data, measurement of vital signs (including temperature, blood pressure, pulse, 
height, and weight) and collection of medical history, including concomitant medications.  
An abbreviated physical examination will be performed.  

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Urine will be collected for health screening values. Blood will be collected to be used for health 
screening labs as well as baseline values for immunological response to vaccine. Stool will be 
collected to be used for immunological response and shedding of vaccine.  
Additionally, subjects will be provided educational materials.  
No subject may be screened more than twice due to a screening failure result as defined 
above. The Study Sponsor and Primary Investigator will be contacted for clarification or 
questions regarding screening failures. All eligibility criteria must be satisfied before a subject is 
enrolled.  
Labs collected pre-vaccination include:  
o Urine 
• Urine dip for protein and urine toxicology screen for opiates. 
• Urine pregnancy testing (human chorionic gonadotropin/ hCG) for women of 
child-bearing potential. 
o Blood: 
• Hematology (white cell count, hemoglobin, platelets, absolute neutrophil 
count/ANC). 
• Chemistry (total bilirubin, ALT, creatinine, sodium, potassium).  
• Serology including HIV antibodies, HBsAg, and HCV antibodies.  
• HLA-B27. 
• Antibody (IgA and IgG) serology assays 
• ASC  
• Secondary research samples (per Appendix A1) 
o Stool: 
• Culture for shedding of Shigella 
• Fecal IgA 
• Secondary research (per Appendix A1) 
Acceptable screening values, including laboratory values are those that do not fall within Grade 
2 or 3 toxicity ranges as defined in Appendix B. A low creatinine value, low bilirubin, or a low 
ALT value are acceptable for study inclusion as they are not considered to be clinically 
significant. Subjects with a laboratory abnormality may be re-screened once if there is a 
suspected inter-current, short-term medical illness or a suspected laboratory error. If the repeat 
value remains out of range but determined by the study investigator not to be clinically 
significant, the subject may continue the screening process.  

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Subjects will be provided educational materials and administered a written test of understanding. 
Subjects must pass with a score of at least 70%. Anyone not passing the test initially may retake 
the test. Subjects will have 2 chances to pass successfully. If subjects are unable to score 70% or 
greater on the test after the second attempt, they will be excluded from the study. 
A subject may be re-screened if screened eligible previously but was not enrolled (e.g., 
scheduling conflict with inpatient admission days, vacation).  
If screening labs are collected more than 60 days before vaccination, the screening labs may be 
re-collected to determine if the subject remains eligible for study participation. HLA-B27 will 
not be repeated on rescreen labs if drawn with initial screen.        
Subjects will be informed of their screening test results by a member of the study team. Those 
with results that exclude them from eligibility and are sensitive in nature will be asked to return 
to the clinic to receive test results and counseling regarding follow-up care in the clinic.   
6.2   Enrollment 
Subjects who pass screening and meet all of the inclusion criteria and none of the exclusion 
criteria may be considered for study enrollment.  
6.3   Planned Study Visits 
VACCINATION VISITS  
Study Day 1 (Enrollment/Vaccination Dose 1) 
A two-day time period is provided to accommodate the vaccination of all eligible subjects of a 
cohort. However, there is no window for this visit.   
Review of inclusion and exclusion criteria will be completed to confirm eligibility. 
An interim medical history and review of concomitant medications will be completed, and if 
indicated, a targeted physical exam will be performed. 
After eligibility has been confirmed, subjects will be randomized in Advantage eClinical® prior 
to receiving study product.   
Pre-vaccination and post-vaccination reactogenicity assessment and vital signs will be obtained. 
Urine will be collected within 24 hours of vaccination for βHCG pregnancy testing on females of 
child-bearing potential.  
If not collected during the screening visit, blood and stool will be collected (prior to 
vaccination) for: 

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o Blood: 
• Antibody (IgA and IgG) serology assays 
•  ASC 
• Secondary research (per Appendix A1) 
o Stool:  
• Culture for shedding of Shigella 
• Fecal IgA 
• Secondary research (per Appendix A1) 
Subjects will remain in clinic for at least 90 minutes prior to dosing and after dosing to ensure 
they remain NPO for at least 90 minutes before and after dose is given. Vaccination will be given 
orally; procedures for vaccine preparation and administration are detailed in the MOP. Subjects 
will remain in clinic at least 90 minutes post vaccination to also observe for immediate reactions, 
complete a post vaccination assessment and record any AE/SAEs on the appropriate data 
collection form prior to discharge from clinic. 
Staff will review with subjects use of memory aid, how to complete a web based “e-Memory 
aid,” and are expected to enter information in the e-Memory aid each day. 
Subjects using the e-Memory aid will also be provided a paper memory aid for their use in the 
event they are unable to access the web-based system. The subjects will be asked to enter the 
information from the paper memory aid into the e-Memory aid once they are able to access the 
web-based system. The paper memory aids will not be retained as a source document.  
Subjects also will be given a thermometer and instructed how to obtain a temperature and record 
the result.  
Subjects will be asked to complete the memory aid from Day 1 through 7 days post vaccination 
(Day 8).  
Subjects will be instructed to contact the site for any Grade 3 event, changes to health, 
emergency room visit or hospitalization, pregnancy, or SAE.      
 
Study Days 1 through 7 following vaccination (Contact/Memory Aid) 
Subjects will be prompted via the memory aid to complete daily reactogenicity assessment.  
Additionally, the memory aid will include a reminder on when the subject should collect a stool 
and bring the stool to clinic visits 03 (study day 4±1) and 05 (study day 8±1). (See appendix A1).    
 

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Study Day 4 ± 1 (3 days following Vaccination Dose 1) 
An interim medical history and review of concomitant medications will be completed, if 
indicated. 
Vital signs and a targeted physical exam will be performed, if either are indicated. 
Staff will review and confirm memory aid information.  
Subjects will be reminded to complete the memory aid.  
A stool collection kit and instructions will be given to the subjects. 
Review changes to health, pregnancy, AE, or SAE.  
Subjects will be instructed to contact the site for any Grade 3 event, changes to health, 
emergency room visit or hospitalization, pregnancy, or SAE.         
Samples will be collected for: 
o Blood  
• Secondary research (per Appendix A1) 
o Stool 
• Culture for shedding of Shigella 
• immunoblot and PCR 
• Secondary research (per Appendix A1) 
 
Study Day 8 ±1 (7 days following Vaccination Dose 1) 
An interim medical history and review of concomitant medications will be completed. 
Vital signs and a targeted physical exam will be performed, if either are indicated. 
Staff will review and confirm memory aid information.  
Review changes to subject’s health, pregnancy, AE, or SAE.  
Subjects will be instructed to contact the site for any Grade 3 event, changes to health, 
emergency room visit or hospitalization, pregnancy, or SAE.         
A stool collection kit and instructions will be given to the subjects, and they will be asked to 
bring a stool sample with them for the next study visit. 
Samples will be collected for: 

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o Blood 
• Safety labs  
 
Hematology, chemistry 
• ASC  
• Secondary research (per Appendix A1) 
o Stool 
• Culture for shedding of Shigella 
• immunoblot and PCR 
• Fecal IgA 
• Secondary research (per Appendix A1) 
 
Study Day 15 ±2 (14 days following Vaccination Dose 1) 
An interim medical history and review of concomitant medications will be completed. 
Vital signs and a targeted physical exam will be performed, if either are indicated. 
Review changes to subject’s health, pregnancy, AE, or SAE.    
Subjects will be instructed to contact the site for any Grade 3 event, changes to health, 
emergency room visit or hospitalization, pregnancy, or SAE.         
A stool collection kit and instructions will be given to the subjects, and they will be asked to 
bring a stool sample with them for the next study visit. 
Samples will be collected for: 
o Blood 
• Antibody (IgA and IgG) serology assays  
• Secondary research (per Appendix A1) 
o Stool 
• 
Culture for shedding of Shigella 
• 
immunoblot and PCRFecal IgA 
• 
Secondary research (per Appendix A1) 
 
 Study Day 29 (+2) (Vaccination Dose 2/28 days following Vaccination Dose 1) 
An interim medical history and review of concomitant medications will be completed, and if 
indicated, a targeted physical exam will be performed. If the subject is noted to have any disease 

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or medical condition that, in the opinion of the site principal investigator or appropriate sub-
investigator, is a contraindication to further study participation, Dose 2 will not be given. In 
addition, a subject will not be given Dose 2 if any of the following are noted: 
- 
Have diarrhea within 14 days before Dose 2 
- 
Use of immunosuppressive/immunomodulating disease therapy since enrollment 
- 
Received or plan to receive a licensed live vaccine within 30 days prior to Dose 2 
- 
Received or plan to receive a licensed, inactivated vaccine, COVID-19 vaccine, or an 
influenza vaccine + 7 days of Dose 2. 
- 
Received Ig or other blood products (with exception of Rho D Ig) since enrollment 
- 
Have taken oral or parenteral (including intra-articular) corticosteroids of any dose, or 
high-dose inhaled corticosteroids within 30 days before Dose 2 
- 
Have taken systemic antibiotics within 7 days before Dose 2 
- 
Have taken prescription and/or OTC medication containing loperamide, acetaminophen, 
aspirin, ibuprofen, or other non-steroidal anti-inflammatory <48 hours prior to Dose 2 
- 
Have fever or an acute illness within 72 hours before Dose 2 
- 
Received an investigational agent (including vaccine, drug, biologic, device, blood 
product, or medication outside of the current study) since enrollment which might affect 
safety or assessment of study endpoints. 
If a previous AE is ongoing at dose 2, the subject may be vaccinated if the AE does not exceed 
the definition of AE Grade 1 and the AE is considered to be stable by a study investigator.  If a 
Grade 2 or Grade 3 AE occurs between vaccine dose 1 and 2 and is determined not to be related 
to investigational product and returns to Grade 1 or lower before the next dose, the subject may 
receive the second dose. 
Review changes to subject’s health, pregnancy, AE, or SAE.   
Subjects will be instructed to contact the site for any Grade 3 event, changes to health, 
emergency room visit or hospitalization, pregnancy, or SAE.      
Pre-vaccination and post-vaccination reactogenicity assessment and vital signs will be obtained.  
Samples (prior to vaccination) will be collected for: 
o Blood 
• Antibody (IgA and IgG) serology assays 
• ASC  
• Secondary research (per Appendix A1) 
o Urine βHCG pregnancy test (for females of child-bearing potential)  
o Stool 

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• Culture for shedding of Shigella 
• immunoblot and PCR 
• Fecal IgA 
• Secondary research (per Appendix A1) 
Subjects will remain in clinic for at least 90 minutes prior to dosing and after dosing to ensure 
they remain NPO for at least 90 minutes before and after dose is given. Vaccination will be given 
orally; procedures for vaccine preparation and administration are detailed in the MOP. Subjects 
will remain in clinic at least 90 minutes post vaccination to also observe for immediate reactions 
and to complete a post vaccination assessment prior to discharge from clinic. 
Staff will review with subjects use of memory aid, how to complete a web based “e-Memory 
aid” and are expected to enter information in the e-Memory aid each day. 
Subjects using the e-Memory aid will also be provided a paper memory aid for their use in the 
event they are unable to access the web-based system. The subjects will be asked to enter the 
information from the paper memory aid into the e-Memory aid once they are able to access the 
web-based system. The paper memory aids will not be retained. 
Subjects also will be given a thermometer and instructed how to obtain a temperature and record 
the result.  
Subjects will be asked to complete the memory aid from Day 29 (±1) through 7 days post 
vaccination.  
A stool collection kit and instructions will be given to the subjects, and they will be asked to 
attempt to collect stool samples per study schedule and bring them for the next study visit. 
 
Study Day 30 through 35 following vaccination (Contact/Memory Aid) 
Subjects will be prompted via the e-Memory aid to complete daily reactogenicity assessment. 
Additionally, the e-Memory aid will include home stool collection reminders. The memory aid 
will be brought to the clinic at visits 09 (study day 32±1) and 11 (study day 36±1) for review by 
staff. (See appendix A1).  
 
Study Day 32±1 (3 days following Vaccination Dose 2) 
An interim medical history and review of concomitant medications will be completed, if 
indicated. 

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Vital signs and a targeted physical exam will be performed, if indicated. 
Staff will review and confirm memory aid information.  
Subjects will be reminded to complete the memory aid.  
Stool collection kits and instructions will be given to the subjects, and they will be asked to 
collect stool samples per study schedule and bring with them to the next study visit. 
Review changes to subject’s health, pregnancy, or SAE.   
Subjects will be instructed to contact the site for any Grade 3 event, changes to health, 
emergency room visit or hospitalization, pregnancy, or SAE.      
Samples will be collected for: 
o Blood 
• 
Secondary research (per Appendix A1) 
o Stool 
• 
Culture for shedding of Shigella 
• 
immunoblot and PCR 
• Secondary research (per Appendix A1) 
 
Study Day 36 ±1 (7 Days Following Vaccination Dose 2) 
An interim medical history and review of concomitant medications will be completed. 
Vital signs and a targeted physical exam will be performed, if either are indicated. 
Review changes to subject’s health, pregnancy, AE, or SAE.   
Subjects will be instructed to contact the site for any Grade 3 event, changes to health, 
emergency room visit or hospitalization, pregnancy, or SAE.      
Staff will review and confirm memory aid information.  
A stool collection kit and instructions will be given to the subjects, and they will be asked to 
bring a stool sample with them for the next study visit. 
Samples will be collected for: 
o Blood 
• 
Safety labs  
 Hematology, chemistry 

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• 
ASC   
• 
Secondary research (per Appendix A1) 
o Stool  
• 
Culture for shedding of Shigella 
• 
immunoblot and PCR 
• 
Fecal IgA 
• 
Secondary research (per Appendix A1) 
 
Study Day 43 ±2 (14 days following Vaccination Dose 2) 
An interim medical history and review of concomitant medications will be completed. 
Vital signs and a targeted physical exam will be performed, if either are indicated. 
Review changes to subject’s health, pregnancy, AE, or SAE.   
Subjects will be instructed to contact the site for any Grade 3 event, changes to health, 
emergency room visit or hospitalization, pregnancy, or SAE.      
A stool collection kit and instructions will be given to the subjects, and they will be asked to 
bring a stool sample with them for the next study visit. 
Samples will be collected for: 
o Blood 
• Antibody (IgA and IgG) serology assays  
• Secondary research (per Appendix A1) 
o Stool 
• Culture for shedding of Shigella 
• immunoblot and PCR 
• Fecal IgA 
• Secondary research (per Appendix A1) 
 
CHALLENGE ADMISSION (Study Day 56) 
Challenge will be administered 28 days (+/-2) after 2nd vaccination. 
During the stay in the inpatient facility, visitors will not be permitted, and subjects will not be 
allowed to leave the inpatient facility until they have been cleared by a member of the study 
team.  

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Meals and any medications needed to treat clinical symptoms associated with Shigella illness 
will be provided for the study subjects during their stay in the inpatient facility. Prescribed 
medicines routinely taken by subjects may be taken per their usual routine and are reviewed by 
the investigator. 
Subjects will be challenged as a single group.  
Upon arrival to the inpatient unit, the subject will have SARS-CoV-2 testing performed.  
Subjects with a negative result will be eligible for study continuation.   
Upon admission to the inpatient unit the following will be performed prior to challenge 
administration:  
An interim medical history and review of concomitant medications will be completed. If  the 
subject is noted to have any disease or medical condition that, in the opinion of the site principal 
investigator or appropriate sub-investigator, is a contraindication to further study participation, 
challenge will not be given. In addition, a subject will not be given challenge if any of the 
following are noted: 
- 
Have diarrhea within 14 days before challenge 
- 
Use of immunosuppressive/immunomodulating disease therapy since Dose 2 
- 
Received or plan to receive a licensed live vaccine within 30 days prior to challenge 
- 
Received or plan to receive a licensed, inactivated vaccine, COVID-19 vaccine, or an 
influenza vaccine within 7 days of challenge. 
- 
Received Ig or other blood products (with exception of Rho D Ig) since Dose 2 
- 
Have taken oral or parenteral (including intra-articular) corticosteroids of any dose, or 
high-dose inhaled corticosteroids within 30 days before challenge 
- 
Have taken systemic antibiotics within the past 7 days before challenge 
- 
Have taken prescription and/or OTC medication containing loperamide, acetaminophen, 
aspirin, ibuprofen, or other non-steroidal anti-inflammatory <48 hours prior to challenge 
- 
Have fever or an acute illness within the past 72 hours of challenge 
- 
Have positive SARS-CoV-2 test at the time of admission to the inpatient unit. 
- 
Received an investigational agent (including vaccine, drug, biologic, device, blood 
product, or medication outside of the current study) since Dose 2 which might affect 
safety or assessment of study endpoints.  
- 
Work or plan to work in either a health care setting, day care center, or as a food handler, 
or have known daily contact with individuals with possible increased susceptibility$ to 
Shigella within 14 days after discharge from inpatient challenge  
$Immunocompromised, elderly persons aged 70 years or more, diapered individuals, persons with 
disabilities, children <2 years old, a woman known to be pregnant or nursing, or anyone with diminished 

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immunity. Known daily contact includes contact at home, school, day-care, nursing home, or similar 
places.  
If a previous AE is ongoing at the time of challenge, the subject may be given the challenge dose 
if the AE does not exceed the definition of AE Grade 1 and the AE is considered to be stable by 
a study investigator. If a Grade 2 or Grade 3 AE occurs between vaccine dose 2 and challenge 
and is determined not to be related to investigational product and returns to Grade 1 or lower 
before the challenge, the subject may receive the challenge dose. 
Perform a urine pregnancy test for women of childbearing potential (test may be done up to 24 
hours prior to challenge). The pregnancy result must be negative and known prior to challenge. 
Perform an abbreviated physical exam and record pre-challenge weight. 
Collect vital signs including temperature, blood pressure, and pulse. 
Pre-challenge samples will be collected for: 
o Blood 
• Safety labs  
 Hematology, chemistry 
• Antibody (IgA and IgG) serology assays  
• ASC  
• Secondary research (per Appendix A2) 
o Urine βHCG pregnancy test (for females of child-bearing potential)  
o Stool 
• Culture for shedding of Shigella 
• Fecal IgA 
• Secondary research (per Appendix A2) 
o  SARS-CoV-2 testing  
 
Challenge (Day 57) 
Pre-challenge and post-challenge reactogenicity assessment and vital signs will be obtained.   
Dietary Requirements Prior to Challenge 
A light, clear liquid breakfast may be consumed the morning of challenge, then nothing by 
mouth at least 90 minutes before and after the Challenge Dose.   

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Administer 120 mL of a sodium bicarbonate solution (2 g in 120 mL of SWI) by mouth 
approximately five minutes before the Challenge Dose. Challenge dose will be given orally; 
procedures for challenge inoculum preparation and administration are detailed in the MOP. 
Challenge Dose   
Nothing by mouth for 90 minutes after dosing. 
Procedures following the Challenge Dose 
Subjects will be observed closely during the 90 minutes after receipt of the challenge by a study 
team member to ensure NPO status is maintained and to detect and treat any immediate adverse 
reactions. Vital signs will be taken at approximately 90 minutes after challenge.  
Assessments and symptom directed physical examinations will be performed daily, or more often 
as clinically indicated, by qualified medical staff.  
Assessment of reactogenicity symptoms (e.g., fever, headache, arthralgia, nausea, 
pain/abdominal cramps, myalgia, malaise/fatigue, anorexia/loss of appetite, chills) will be 
completed once in the evening of challenge administration and then at least daily until discharge. 
Vomiting (number of episodes per day and weights), and diarrhea (as defined per protocol) with 
stool consistency, number of stools per day, and stool weights during the inpatient stay will be 
recorded. Stool will be collected for Shigella detection. 
Review of AEs and SAEs 
Stool collection:  
All stools will be classified for consistency (see Appendix D, Diarrhea Grading Classification for 
details), and all stools consistency classified as 3-5 (i.e., those that conform to the shape of the 
container) will be weighed and visually assessed for gross blood. Samples with gross blood will 
be confirmed by a hemoccult test. 
For each subject, at least one stool sample per day will be saved for testing (the first stool 
produced each 24-hour period) and up to two stools with blood present by visual inspection in a 
24- hour period will be tested by Hemoccult. 
For each stool saved, aliquots will be made, labeled, and stored at -70 °C or colder. Any 
remaining stool will be disinfected with bleach in the plastic stool collector and discarded in the 
hazardous waste.  
Inpatient Post-Challenge (Days 58-65)  
Subjects will remain in the inpatient facility for a minimum of eight days following challenge.  

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Inpatient monitoring. 
Nurses and other clinical staff will provide day and night supervision of the subjects.   
Assessments and symptom directed physical examinations will be performed daily, or more 
often as clinically indicated, by qualified medical staff. 
Vital signs (pulse, blood pressure and temperature) will be obtained at least daily, more often as 
clinically indicated.  
If gastrointestinal illness occurs, intake and output will be measured and recorded.  
If illness occurs, serum chemistry (sodium, potassium, blood urea nitrogen/BUN and creatinine) 
and urine specific gravity testing may be performed at the direction of the investigator and daily 
as indicated. Testing will be conducted until any clinically significant abnormal tests return to 
normal, baseline, or are acceptable.  
Review of AEs and SAEs. 
At least daily assessment of reactogenicity symptoms (e.g., fever, headache, arthralgia, nausea, 
pain/abdominal cramps, myalgia, malaise/fatigue, anorexia/loss of appetite, chills).  
Antibiotic treatment will be given to subjects on the 5th day after challenge.  
Review concomitant medications taken. 
Collect study samples as described in table of events and as described below: 
Inpatient Study Days  
o Stool will be collected per schedule of events. 
• Every day, an aliquot of stool will be collected for testing. If no stools are passed 
on a given day, 1 rectal swab will be collected for culture. All stools will be 
classified for consistency (see Appendix D, Diarrhea Classification for details), and 
all stools classified as 3-5 (i.e., those that conform to the shape of the container) 
will be weighed and visually assessed for gross blood. Samples with gross blood 
will be confirmed by hemoccult test. 
o Blood will be collected per schedule of events.  
o Nasal swab will be collected for SARS-CoV-2 at any time a subject exhibits 
symptoms of COVID-19 illness during the inpatient stay.  
o Emesis All emesis will be measured and recorded then disinfected with bleach and 
discarded in the hazardous waste.  
Treatment of vomiting and/or diarrhea: 

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Subjects who develop diarrhea will be asked to drink 1.5 cc of oral rehydration solution (ORS) 
for each gram of diarrheal stool that they produce. Subjects developing vomiting will be asked to 
drink a volume of ORS equivalent to the amount of the emesis. Subjects unable to maintain their 
hydration status may be provided with intravenous fluids to prevent and treat dehydration. 
Therapies including intravenous fluids and other medications (excluding ORS) will be recorded 
as concomitant medications. Subjects will be allowed to continue eating solid foods as tolerable. 
At the investigator’s discretion and at the subject’s request, subjects may be prescribed 
acetaminophen and/or NSAIDS (or their equivalent) for fever, headache, muscle aches, 
abdominal pain, or other symptoms. Loperamide (Imodium A-D®) may be prescribed if the study 
physician feels the subject is at high risk for volume depletion.  
Anti-emetic medication may be administered by PI discretion only. Ondansetron – (Orange Book 
#020781, Zofran® Oral Disintegrating Tablet) or its licensed equivalent may be administered as 
prescribed by the investigator.  
If at any time during the inpatient facility stay, the subject develops illness requiring medical 
care that is beyond the care available in this facility, the subject will be transferred to a hospital 
capable of providing this level of care. The hospital and physicians accepting the subject for care 
will be informed of the subject’s participation in this research study so that the subject can be 
managed in appropriate isolation precautions. 
 
Criteria for Discharge from Inpatient Facility 
Subjects will be discharged from the inpatient unit if they have taken their antibiotic treatment, 
are symptom free (or improving and able to care for themselves) and passed 2 consecutive stools 
culture-negative for Shigella at least six hours apart. In the event stool cultures are positive for 
Shigella, subjects will remain in the inpatient unit until they have at least two stools that are 
culture-negative for the Shigella and will continue to have daily assessments per protocol.  
Planned Discharge (Study Day 65) 
Subjects will be discharged from the inpatient unit on Day 65 after they have met all of the 
following criteria: 
Antibiotic treatment has been initiated on Day 62 (or earlier if protocol criteria are 
met) 
Two stool samples (collected at least six hours apart) are negative for S. sonnei 
Illness symptoms are resolved or resolving 

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The subject is able to tolerate eating and drinking   
Subjects who receive IV hydration during the inpatient admission will be required 
to have clinically acceptable electrolyte values for sodium and potassium prior to 
discharge. 
Subjects will be provided with verbal and written instructions, a memory aid, and thermometer to 
take home and record symptoms and body temperature daily starting at discharge and for the 
next 5 days. Staff will review with subjects’ use of memory aid, how to complete a web based 
“e-Memory aid” and are expected to enter information in the e-Memory aid each day. 
Subjects using the e-Memory aid will also be provided with a paper memory aid for their use in 
the event they are unable to access the web-based system. The subjects will be asked to enter the 
information from the paper memory aid into the e-Memory aid once they are able to access the 
web-based system. The paper memory aid, if used, will not be retained as a source document. The 
e-Memory Aid will include prompts for the subject to indicate if they have other symptoms to 
report or changes to their medications. Study staff will review the responses to these prompts with 
the subject and enter AEs or medications on the appropriate eCRFs as needed. Subjects will be 
instructed to take any remaining doses of study antibiotic per the written schedule provided.  
Subjects will be asked to notify the study staff promptly if they develop fever, vomiting, or diarrhea 
during the 5 days after discharge and may be asked to return to the study site for evaluation.    
Subjects will be instructed to contact the site for any Grade 3 event, changes to health, 
emergency room visit or hospitalization, pregnancy, or SAE.  
A stool collection kit and instructions will be given to the subjects, and they will be asked to bring 
a stool sample with them for the next study visit. 
6.3.1 Follow-up 
Follow-Up Visit Post Challenge, Day 71 (14 Days post-challenge ±2) 
Following discharge from the inpatient facility, subjects will return to the outpatient clinic on 
Day 71. (If a subject, due to persistent symptoms, remains in the inpatient unit longer than the 
prescribed time, study procedures may be performed while inpatient).  
At this visit, the following procedures will be performed: 
Review interim medical history and record concomitant medications. 
Perform a targeted physical exam if indicated. 
Collect vital signs, if indicated, including temperature, blood pressure, and pulse. 

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Review of pregnancies, AEs, and SAEs.  
Subjects will be instructed to contact the site for any Grade 3 event, changes to health, 
emergency room visit or hospitalization, pregnancy, or SAE.      
Review and confirm reactogenicity information from subject’s 5-day memory aid.  
Remind subject to record any unsolicited AEs through Day 85 (28 days after study challenge 
administration).  
A stool collection kit and instructions will be given to the subjects, and they will be asked to bring 
a stool sample with them for the next study visit. 
The following samples will be collected: 
o Blood  
• Safety labs  
− Hematology, Chemistry 
• Antibody (IgA and IgG) serology assays 
• Secondary research  (per Appendix A2) 
o Stool  
• Fecal IgA 
• Secondary research (per Appendix A2) 
 
 
Follow-up Visit Day 85 (28 Days post challenge ±2) 
The following procedures will be performed at this visit: 
Review interim medical history and record concomitant medications 
Perform a targeted physical exam if indicated 
Collect vital signs, if indicated, including temperature, blood pressure, and pulse  
Review of pregnancies, AEs, and SAEs. 
Subjects will be instructed to contact the site for any Grade 3 event, changes to 
health, emergency room visit or hospitalization, pregnancy, or SAE. 
A stool collection kit and instructions will be given to the subjects, and they will 
be asked to bring a stool sample with them for the next study visit. 
The following samples will be collected: 

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o Blood  
• Safety labs, if indicated  
 Hematology, Chemistry 
• Antibody (IgA and IgG) serology assays 
• Secondary research (per Appendix A2) 
o Stool 
• Fecal IgA (per Appendix A2) 
 
Follow-up Visit Day 113 (56 Days post challenge ±4) 
Review interim medical history and concomitant medications regarding occurrence of SAEs, any 
other illnesses/changes to health or pregnancies. 
Perform a targeted physical exam if indicated. 
Collect vital signs, if indicated, including temperature, blood pressure, and pulse. 
Subjects will be instructed to contact the site for any Grade 3 event, changes to health, 
emergency room visit or hospitalization, pregnancy, or SAE. 
The following samples will be collected: 
o Blood  
• Antibody (IgA and IgG) serology assays 
• Safety labs, if indicated  
 Hematology, Chemistry 
• Secondary research (per Appendix A2) 
o Stool 
• Secondary research (per Appendix A2) 
 
6.3.2 Final Study Visit 
Final Study Contact Day 180 (123 days post challenge ±14)     
On Day 180, subjects will complete a final safety contact by phone or email to find out about the 
occurrence of any pregnancies or SAE.  
6.3.3 Early Termination Visit  
If, after admission to the inpatient unit and receipt of the Shigella challenge, a subject decides to 
withdraw consent and leave the unit early, he/she will be counseled about the risk of 
transmission of Shigella to close contacts. Prior to leaving the unit, the subject will be required to 

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be directly observed by the study staff to swallow the 500 mg dose of ciprofloxacin.  The subject 
will be sent home with five additional 500 mg tablets of ciprofloxacin to complete the 3-day 
course of therapy at home. Subjects with contraindication to ciprofloxacin may receive treatment 
trimethoprim-sulfamethoxazole (160 mg/800 mg twice daily for 5 days). Prior to leaving the 
unit, the subject will be required to be directly observed by the study staff to swallow the first 
dose of trimethoprim-sulfamethoxazole. The subject will be sent home with 9 additional tablets 
of trimethoprim-sulfamethoxazole to complete the 5-day course of therapy at home. This will be 
documented in the subject source document. 
If the subject needs to be transferred to a hospital for additional care during his/her inpatient 
facility stay, the study staff will ask permission of the subject to obtain additional medical 
records to follow the subject until the symptoms of this SAE resolves or the subject’s condition 
becomes stable. 
Regardless of when a subject decides to withdraw consent, it will be requested that the subject 
return for safety evaluations prior to withdrawal. 
6.4   Unscheduled Study Visits 
Subjects will be asked to notify the study staff promptly if they develop any illness suggestive of 
recurrence of Shigella infection (vomiting and/or diarrhea) or possible dehydration. If the study 
staff determines the symptoms are potentially significant, the subject will be asked to come to the 
clinic for an evaluation. Subjects will be asked to complete an unscheduled visit for any event 
that warrants follow-up. Per PI discretion, safety labs may be collected to further evaluate subject 
well-being. All events will be followed to resolution or until determined to be stable. 
6.5   Protocol Deviations 
A protocol deviation is any noncompliance with the clinical trial protocol, cGCP, or protocol-
specific MOP requirements. The noncompliance may be either on the part of the subject, the site 
principal investigator, or the site personnel. As a result of deviations, corrective actions are to be 
developed by the site and implemented promptly. 
These practices are consistent with ICH E6: 
4.5 
Compliance with Protocol, Sections 4.5.1, 4.5.2, and 4.5.3 
5.1 
Quality Assurance and Quality Control, Section 5.1.1 
5.20 
Noncompliance, Sections 5.20.1, and 5.20.2. 
It is the responsibility of the site principal investigator and personnel to use continuous vigilance 
to identify and report deviations within five working days of identification of the protocol 

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deviation, or within five working days of the scheduled protocol-required activity. All deviations 
must be promptly reported to DMID per the DCC protocol deviation reporting procedures. 
All protocol deviations, as defined above, must be addressed in study subject case report forms. 
A completed copy of the DMID Protocol Deviation Form must be maintained in the Regulatory 
File, as well as in the subject’s chart. Protocol deviations must be sent to the local IRB/IEC per 
their guidelines. The site principal investigator and personnel are responsible for knowing and 
adhering to their IRB requirements. 
7 DESCRIPTION OF CLINICAL AND LABORATORY 
EVALUATIONS 
7.1   Clinical Evaluations 
Following consent, a screening evaluation will be performed.  
Medical History 
Medical history will be obtained by direct interview and will include a review of concomitant 
medications, supplements, and over-the-counter medications. Subjects will be queried regarding 
a history of significant medical disorders of the head, eyes, ears, nose, throat (HEENT), mouth, 
cardiovascular system, lungs, gastrointestinal tract, liver, pancreas, kidney, urologic, nervous 
system, blood, lymph glands, endocrine system, musculoskeletal system, skin, and genital/ 
reproductive tract. A history of any allergies, cancer, gastrointestinal disorders, 
immunodeficiency, psychiatric illness, substance abuse, and autoimmune disease will be 
solicited.  
Physical Examination 
An abbreviated physical examination will be conducted at the screening visit. An abbreviated 
physical examination is distinguished from a complete physical exam in that all assessments are 
not required (e.g., genito-urinary and rectal exams). A rectal examination may be performed at 
screening if, in the opinion of the investigator, this assessment is warranted based on the 
subject’s medical history. A targeted physical examination may be performed at all other study 
visits if indicated based on assessment, reactogenicity, or interim medical history review. 
Inpatient Evaluations 
During the Inpatient Stay, the following clinical evaluations will occur: 

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Assessments and targeted physical examinations will be performed daily, or more often as 
clinically indicated, by qualified medical staff.  
Assessment of reactogenicity symptoms will be completed every evening beginning post 
challenge through discharge. 
Vomiting (number of episodes per day and weights) and diarrhea (as defined per protocol) with 
stool consistency, number of stools per day and stool weights during the inpatient stay will be 
recorded. Stool will be collected for Shigella detection and stools with gross blood will be tested 
for occult blood. 
Subjects will be asked to collect every stool passed during the inpatient portion of the study.   
Pre-challenge body weight will be measured on admission to the inpatient unit and repeated 
during the inpatient stay as clinically indicated to evaluate for dehydration.  
Vital signs including oral temperature, blood pressure and pulse will be obtained at least daily.  
Daily evaluations will be performed by an investigator, or more frequently as clinically 
indicated. 
Evaluation for dehydration. A clinician designated on the 1572 will evaluate subjects who are 
symptomatic and have any of the following:  
o Tachycardia (resting heart rate >100 beats/min). 
o Hypotension (systolic pressure <90 mm Hg or diastolic pressure <60 mm 
Hg accompanied by symptoms of orthostasis). 
o Greater than 1000 mL deficit in ORS replacement in 24 hours. 
Illness labs may be collected at the discretion of the investigator (serum chemistry, including 
sodium and potassium, blood urea nitrogen/BUN, and creatinine) and as indicated thereafter until 
symptoms of gastroenteritis resolve and any clinically significant abnormal tests return to 
normal, baseline, or are acceptable. A urine dipstick may be performed to capture urine specific 
gravity results in real time to help determine hydration status of a subject. 
7.1.1 Assessment of Concomitant Medications/Treatments other than Study Product 
Administration of any medications or vaccines will be documented in the appropriate eCRF. All 
concomitant medications, taken in the 30 days prior to study enrollment through Day 28 
following challenge or early termination, whichever occurs first, will be recorded. All 
prescription and over-the-counter medications as well as vitamins and supplements will be 
recorded. 

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Medications which may interfere with the evaluation of the investigational product should not be 
used unless absolutely necessary. Assessment of whether the subject qualifies to receive the dose 
will include a review of permitted and prohibited medications as described in flow of study visit. 
Taking prescription or over-the-counter weight-loss medication is prohibited for the duration of 
the study and is exclusionary for enrollment. 
Use of new medications should prompt evaluation for the presence of an AE or new chronic 
medical condition.  
Any medications considered for treatment of fever, AE, or reactogenicity will be given only at 
the discretion of a study investigator.  
7.1.2 Non-Research Standard of Care  
If at any time during the inpatient facility stay, the subject develops illness requiring medical 
care that is beyond the care available in this facility, the subject will be transferred to a hospital 
capable of providing this level of care. The hospital and physicians accepting the subject for care 
will be informed of the subject’s participation in this research study so that the subject can be 
managed in appropriate isolation precautions.  
7.2   Laboratory Evaluations 
7.2.1 Clinical Laboratory Evaluations 
Safety laboratory evaluations will be performed by the local lab of the performance site and sites 
will follow the FDA toxicity table (see Appendix B) for guidance.  
Clinical safety lab testing will include hematology, serology, and chemistry.   
Hematology: white blood cell count (WBC), absolute neutrophil count (ANC), hemoglobin 
(Hgb), and platelet count 
Chemistry: sodium, potassium, creatinine, ALT, total bilirubin, and blood urea nitrogen/BUN if 
indicated during the inpatient admission.  
Subjects who receive IV hydration during the inpatient admission will be required to have 
clinically acceptable electrolyte values for sodium and potassium prior to discharge. 
Urine pregnancy testing will be performed in the clinic by a qualified study team member using a 
commercially available test.  
For females of childbearing potential, a urine pregnancy test will be performed at screening. 
Urine pregnancy testing will be performed within 24 hours before vaccination and challenge 

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administration. Pregnancy test results must be negative for subjects to be eligible for enrollment 
and qualify to receive vaccination and challenge doses.   
Subjects will be tested for HLA-B27, HIV, Hepatitis B surface antigen, and antibody to          
Hepatitis C at screening only. Any subject having HIV, Hepatitis B, or Hepatitis C, or positive 
HLA-B27 will not be enrolled into the study. 
During screening urinalysis will be done to test for urine protein via dipstick in the clinic. Clean-
catch, mid-stream, urine specimen will be collected in a sterile urine cup. Urine specific gravity 
may be performed during the inpatient admission, if indicated. 
Urine test for opiates: A clean-catch, mid-stream urine specimen will be collected in a sterile 
urine cup and transported to the clinical laboratory for processing and examination 
A SARS-CoV-2 test will be performed on all subjects upon entry into the challenge unit. Anyone 
with a positive test will remain quarantined in their room until it is medically safe for the subject 
to be escorted off the inpatient unit. In addition, a SARS-CoV-2 test will be performed at any 
time during the inpatient stay if the participant is demonstrating symptoms consistent with 
SARS-CoV-2. 
Stool Culture, immunoblot and PCR:  
The local laboratory at each site, using fresh stool samples, will conduct an assay for the 
presence of S. sonnei. The first procedure is qualitative and involves streaking swabs containing 
fecal material to Hektoen Enteric Agar (HEA) plates. All HEA plates will be incubated at 37±1o 
C overnight. For each subject from the qualitative cultures, up to two blue-green (non-lactose 
fermenting) colonies will be picked from swab-streaked HEA plate and tested by agglutination 
by S. sonnei polyvalent Group D antiserum. Colonies that agglutinate will be recorded as 
potential positive for presence of S. sonnei. The second procedure (to be performed at WRAIR) 
is quantitative (determined by immunoblot) and involves plating serial dilutions of frozen-
thawed stool suspensions (in BGS) to HEA plates. HEA plates used for the serial dilutions that 
contain blue-green colonies will be processed using a colony blot procedure specific for the 
detection of S. sonnei. The number of positive colonies for each dilution will be recorded and 
used to calculate the cfu per gram of stool. These procedures will follow the MOP for culture 
isolation, immunoblot and identification for S. sonnei from stool specimens. Stool will be 
analyzed at WRAIR by PCR to detect WRSs2 vaccine and challenge strain S. sonnei 53G.  
Microbiology and Immunology:  
Biosamples (serum, PBMCs, stool) for immunological assays will be collected, processed, 
cryopreserved and stored as detailed in the protocol MOP. All immunological assays will be 

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performed in batches after all samples have been collected for a given assay within a cohort. To 
minimize variability, all pre- and post-vaccination/challenge samples of a subject will be assayed 
on the same day as detailed in the MOP. 
Antibody secreting cells (ASC):  
At times specified in the protocol and the MOP, the peripheral blood mononuclear cells 
(PBMCs) will be isolated from whole blood and cryopreserved to be used in ASC. An ELISPOT 
assay will be used to enumerate S. sonnei LPS and S. sonnei Invaplex specific IgA and IgG 
ASCs per 106 PBMC. The IgA and IgG ASC assays will be performed at CCHMC. 
 
Serology:  
Blood will be collected for IgA and IgG assay by ELISA25. The serum will be separated and 
stored frozen for determination of specific antibody responses against S. sonnei LPS and 
Invaplex antigens. The serum IgA and IgG ELISA assays will be performed at CCHMC. 
Fecal IgA:  
The stool sample (4-5gm) will be placed in a 30 mL Oakridge tube and frozen at -70ºC or colder 
until ready for extraction using soybean trypsin inhibitor-EDTA procedure. Total and Shigella-
antigen specific IgA will be measured in stool extracts using ELISA procedures. Fecal total and 
antigen-specific IgA ELISA assays will be performed at CCHMC. 
7.2.2 Secondary Research Assays 
Stool and blood will be preserved for future studies. Details of assays, methodology and location 
of testing will be determined at a later date. 
7.2.3 Laboratory Specimen Preparation, Handling, and Storage 
Whole blood will be collected by the venous route using aseptic techniques. Depending on the 
testing performed, aliquots of blood will be sent to the local Clinical Laboratory and/or the site’s 
local research for processing. 
Stool samples will be collected using specially designed stool collection kits. Study team 
members will use appropriate contact isolation procedures to minimize their risk of handling the 
stool specimens. If visible spillage is present on the outside of the stool container, the container 
will be wiped with an appropriate disinfectant and dried prior to the study labels being placed on 
the tubes. Any spillage will be handled in the same manner. The samples will then be placed in 
sealed specimen bags and sent to the site’s local research laboratory for processing and storage 
until testing is performed. Instructions for Specimen Preparation, Handling, and Storage 

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Instructions for specimen preparation, handling, and storage are included in the protocol-specific 
MOP. Instructions for specimen shipment are included in the protocol-specific MOP. 
7.2.4 Laboratory Specimen Shipping 
Instructions for Specimen Preparation, Handling, and Storage Instructions for specimen 
preparation, handling, and storage are included in the protocol-specific MOP. Instructions for 
specimen shipment are included in the protocol-specific MOP. 
8 ASSESSMENT OF SAFETY 
8.1   Assessing and Recording Safety Parameters 
Safety will be assessed by the frequency and severity of:   
1. SAEs occurring from the time of the first vaccination through Day 180. 
2. Solicited AEs – reactogenicity events occurring from the time of each study vaccination 
through 7 days following vaccination doses: 
a. Reactions following vaccination to include headache, arthralgia, nausea, 
vomiting, diarrhea, pain/abdominal cramps, myalgia (body aches, muscular pain), 
malaise/fatigue, anorexia/loss of appetite, chills, and fever 
3. Solicited AEs- from time of challenge through 5 days after discharge following challenge 
administration. 
a. Reactions following challenge to include headache, arthralgia, nausea, 
pain/abdominal cramps, myalgia (body aches, muscular pain), malaise/fatigue, 
anorexia/loss of appetite, chills, vomiting, diarrhea, and fever. 
4. Unsolicited AEs – non-serious events occurring from the time of each vaccination and 
challenge through approximately 28 days post vaccination doses and challenge 
administration. 
5. Clinical safety laboratory AEs occurring from the time of each study vaccination through 
7 days following vaccination doses and challenge administration, respectively:  
Parameters to be evaluated include WBC, ANC, hemoglobin, platelet count, potassium, 
sodium, creatinine, ALT, and total bilirubin. 
 
The memory aid will be used to collect all solicited and unsolicited AEs, and medications for the 
first 7 days after each vaccination starting the evening of administration of the vaccine/placebo. 
From 8 days until 28 days following each vaccination, only unsolicited AEs, solicited adverse 
reactions that continue beyond the 7th day, and related medications will be collected during 
follow up. 

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Subjects will be asked to contact the study site for fever, any Grade 3 event, emergency room 
visits, hospitalizations, pregnancy, or changes in their baseline health since vaccination. 
8.1.1 Adverse Events (AEs)   
Adverse Event (AE): (ICH) E6 defines an AE as any untoward medical occurrence in a patient or 
clinical investigation subject administered a pharmaceutical product regardless of its causal 
relationship to the study treatment. FDA defines an AE as any untoward medical occurrence 
associated with the use of a drug in humans, whether or not considered drug related. 
An AE can therefore be any unfavorable and unintended sign (including an abnormal laboratory 
finding), symptom, or disease temporally associated with the use of medicinal (investigational) 
product. The occurrence of an AE may come to the attention of study personnel during study 
visits and interviews of a study recipient presenting for medical care, or upon review by a study 
monitor.  
Beginning on the day of the first dose of study agent, AEs, including solicited systemic 
(subjective and quantitative) reactions, will be captured on the appropriate eCRF. Information to 
be collected for unsolicited non-serious AEs includes event description, date of onset, assessment 
of severity, relationship to study product and alternate etiology (assessed only by those with the 
training and authority to make a diagnosis and listed on the Form FDA 1572 as an investigator), 
date of resolution or stabilization, seriousness, and outcome. AEs occurring during the trial 
collection and reporting period will be documented appropriately regardless of relationship. AEs 
will be followed through resolution or stabilization. 
Any medical condition that is present at the time that the subject is screened and does not exceed 
the protocol definition of a Grade 1 AE, will be considered as baseline, documented as medical 
history for the subject, and will not be reported as an AE. However, if at any visit after 
administration of the study product the severity of any pre-existing medical condition increases 
or is graded greater than a Grade 1, it will be recorded as an AE.  
8.1.2 AE Grading 
All AEs (laboratory and clinical symptoms) will be graded for severity and assessed for 
relationship to study product (see definitions). AEs characterized as intermittent require 
documentation of onset and duration of each episode. The start and stop date of each reported 
AE will be recorded on the appropriate eCRF.  
Severity of Event   

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AEs will be assessed by the investigator using a protocol-defined grading system (toxicity table 
included as an appendix).  For events not included in the protocol-defined grading system, the 
following guidelines will be used to quantify severity: 
• Mild (Grade 1):  Events that are usually transient and may require only minimal or no 
treatment or therapeutic intervention and generally do not interfere with the subject’s usual 
activities of daily living. 
• Moderate (Grade 2):  Events that are usually alleviated with additional specific therapeutic 
intervention.  The event interferes with usual activities of daily living, causing discomfort but 
poses no significant or permanent risk of harm to the research subject. 
• Severe (Grade 3):  Events interrupt usual activities of daily living, or significantly affects 
clinical status, or may require intensive therapeutic intervention.  Severe events are usually 
incapacitating. 
AEs will also be graded according to the following Grading Scales: 
• Appendix B: Toxicity Table/Laboratory AE Grading Scale 
• Appendix C: Vital Signs AE Grading Scale  
• Appendix D: Diarrhea Classification 
• Appendix E: Reactogenicity Grading Scale 
AEs characterized as intermittent require documentation of onset and duration of each episode. 
Changes in the severity of an AE will be documented to allow an assessment of the duration of 
the event at each level of intensity. 
Relationship to Study Products 
The assessment of the relationship of an AE to the administration of study product is made only 
by those with the training and authority to make a diagnosis and listed on the Form FDA 1572 as 
an investigator based on all available information at the time of the completion of the eCRF. 
Whether the AE is related or not, is not a factor in determining what is or is not reported in this 
trial. If there is any doubt as to whether a clinical observation is an AE, the event should be 
reported.   
In a clinical trial, the study product must always be suspect. To help assess, the following 
guidelines are used. 
• Related – There is a reasonable possibility that the study product caused the AE. Reasonable 
possibility means that there is evidence to suggest a causal relationship between the study 
product and the AE. 

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• Not Related – There is not a reasonable possibility that the administration of the study 
product caused the event. 
8.1.3 Reactogenicity 
Reactogenicity is AEs that are common and known to or expected to occur following the 
administration of the study vaccination and challenge products. Subjects will be assessed for 
reactogenicity at baseline and following vaccination/placebo administration and following 
challenge administration.  
The Toxicity Grading Scales to be used to guide the grading of reactogenicity are in Appendix D 
and Appendix E. 
8.1.4 Serious Adverse Events (SAEs) 
An AE or suspected adverse reaction is considered “serious” if, in the view of either the 
investigator or sponsor, it results in any of the following outcomes:  
Death 
A life-threatening AE1  
Inpatient hospitalization or prolongation of existing hospitalization  
A persistent or significant incapacity or substantial disruption of the ability to 
conduct normal life functions  
A congenital anomaly/birth defect   
Important medical events that may not result in death, be life-threatening, or 
require hospitalizations may be considered serious when, based upon appropriate 
medical judgment they may jeopardize the patient or subject and may require 
medical or surgical intervention to prevent one of the outcomes listed in this 
definition. Examples of such medical events include allergic bronchospasm 
requiring intensive treatment in an emergency room or at home, blood dyscrasias 
or convulsions that do not result in inpatient hospitalization, or the development 
of drug dependency or drug abuse. 
1  
Life-threatening AE. An AE is considered “life-threatening” if, in the view of either the site principal 
investigator or sponsor, its occurrence places the patient or subject at immediate risk of death. It does not include an 
AE, had it occurred in a more severe form, might have caused death. 
SAEs will be: 
Assessed for severity and relationship to study product and alternate etiology 

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Recorded on the appropriate SAE data collection form and eCRF 
Followed through resolution or stabilization 
Reviewed and evaluated by the DSMB (periodic review unless related), DMID, 
and the IRB 
8.2   Specification of Safety Parameters 
Safety will be based on the frequency and severity of: 
8.2.1 Solicited Events  
Solicited Events are AEs that are common and known to or expected to occur following the 
administration of the study product. Subjects will be assessed for AEs following vaccination and 
challenge administration (see Appendix E) and will complete an e-memory aid to record 
symptoms for seven days following each vaccination and five days after discharge from the 
isolation facility. During the in-patient phase, solicited events will be reviewed and assessed by 
the clinical research team. The subjects will be provided with a thermometer. The subject e-
memory aids will be reviewed with the subject at subsequent visits. Any symptoms still present 
after the solicited event collection period (Day 8 post vaccination doses and 5 days post 
discharge from the inpatient isolation unit) will continue to be followed until resolution or 
determined to be stable per investigator.   
For baseline laboratory results that are abnormal according to the local laboratory reference 
range and fall within Grade 1 toxicity table range, these will not be considered laboratory AE 
and will thus not be graded. However, if baseline clinical labs fall within Grade 1 range, then a 
laboratory AE is reported only if the value changes such that it falls into Grade 2 or higher when 
subsequent safety laboratory testing is done. 
8.2.2 Unsolicited Events 
Unsolicited, or unexpected events, are any other AEs that occur following administration of 
vaccination doses or challenge. Subjects will be asked to record any unsolicited AEs through 
Day 29 after receipt of vaccine and challenge study product administration. AEs will be followed 
until resolution or determined to be stable per investigator discretion.   
8.3   Reporting Procedures 
8.3.1 Reporting SAEs 
SAEs will be followed until resolution or stabilization even if this extends beyond the study-
reporting period. Resolution of an AE is defined as the return to pretreatment status or 
stabilization of the condition with the expectation that it will remain chronic.  

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Any AE that meets a protocol-defined serious criterion must be submitted immediately 
(within 24 hours of site awareness) on an SAE form to the DMID Pharmacovigilance 
Group, at the following address:  
 
DMID Pharmacovigilance Group 
Clinical Research Operations and Management Support (CROMS) 
6500 Rock Spring Dr. Suite 650 
Bethesda, MD 20817, USA 
SAE Hot Line: 
 (US) or 
 (outside US) 
SAE FAX Number: 
 (US) or 
 (outside US) 
SAE Email Address: 
 
In addition to the SAE form, select SAE data fields must also be entered into the DCC system 
(for example: Advantage eClinical®). Please see the protocol-specific MOP for details regarding 
this procedure.  
Other supporting documentation of the event may be requested by the DMID Pharmacovigilance 
Group and should be provided as soon as possible.  
The DMID Medical Monitor and DMID Clinical Project Manager will be notified of the SAE by 
the DMID Pharmacovigilance Group. The DMID Medical Monitor will review and assess the 
SAE for regulatory reporting and potential impact on study subject safety and protocol conduct. 
At any time after completion of the study, if the site principal investigator or appropriate sub-
investigator becomes aware of an SAE that is suspected to be related to study product, the site 
principal investigator or appropriate sub-investigator will report the event to the DMID 
Pharmacovigilance Group. 
8.3.2 Regulatory Reporting for Studies Conducted Under DMID-Sponsored IND 
Following notification from the investigator, DMID, the Investigational New Drug (IND) 
sponsor, will report any suspected adverse reaction that is both serious and unexpected. DMID 
will report an AE as a suspected adverse reaction only if there is evidence to suggest a causal 
relationship between the drug and the AE. DMID will notify FDA and all participating 
investigators (i.e., all investigators to whom the sponsor is providing drug under its IND(s) or 
under any principal investigator’s IND(s)) in an IND safety report of potential serious risks from 
clinical trials or any other source, as soon as possible, but in no case later than 15 calendar days 
after the sponsor determines that the information qualifies for reporting as specified in 21 CFR 

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Part 312.32. DMID will also notify FDA of any unexpected fatal or life-threatening suspected 
adverse reaction as soon as possible, but in no case later than 7 calendar days after the sponsor’s 
initial receipt of the information. Relevant follow-up information to an IND safety report will be 
submitted as soon as the information is available. Upon request from FDA, DMID will submit to 
FDA any additional data or information that the agency deems necessary, as soon as possible, 
but in no case later than 15 calendar days after receiving the request. 
All serious events designated as “not related” to study product(s), will be reported to the FDA at 
least annually in a summary format. 
8.3.3 Reporting of Pregnancy 
Pregnancy is not an AE but is a collectible event.  Pregnancies occurring in study subjects will be 
reported in IDES. Pregnancy will be confirmed with serum pregnancy testing. No further 
vaccinations or challenge will be administered to pregnant subjects. With the subject’s 
permission all planned study samples will be collected and the subject will continue in follow-up 
for safety events. Efforts will be made to follow all pregnancies reported during the study to 
pregnancy outcome with the subject’s permission. 
8.4   Type and Duration of Follow-up of Subjects after AEs 
All AEs and SAEs will be followed to resolution or until determined to be stable. 
AEs will be assessed and followed from initial recognition of the AE through end of the protocol 
defined follow-up period.   
SAEs will be followed up through resolution even if duration of follow-up goes beyond the 
protocol-defined follow-up period. 
Resolution of an AE is defined as the return to pre-treatment status or stabilization of the 
condition with the expectation that it will remain chronic. 
8.5   Procedures to be Followed in the Event of Abnormal Laboratory Test 
Values or Abnormal Clinical Findings 
The site principal investigator or appropriate sub-investigator is responsible for recording all 
AE/SAEs that are observed or reported during this trial, regardless of the relationship to study 
product. AE/SAEs, abnormal laboratory test values, or abnormal clinical findings will be 
collected, assessed, documented, reported, and followed appropriately. Grading of the laboratory 
AEs will be based on the Appendix B toxicity table. 

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8.6   Study Halting Rules 
Further study enrollment and dosing at 5x105 will be halted for DSMB review/recommendation 
if any of the following Halting Rules are met at this dose: 
Death of an enrolled subject or any SAE if the SAE is considered by the Medical Monitor 
to be related to the study products (WRSs2 or S. sonnei 53G), at any time during the 
study 
Any Grade 3 AE (of the same parameter or MedDRA Preferred Term) in two subjects 
prior to challenge 
Any Grade 3 AE (of the same parameter or MedDRA Preferred Term) in two subjects 
post-challenge, unless it is expected due to shigellosis (diarrhea, vomiting, fever, nausea, 
abdominal pain/cramping, myalgia/arthralgia, headache, anorexia, chills, and malaise). 
The study may also be suspended because of safety findings such as an overall pattern of 
symptomatic, clinical, or laboratory events that the Medical Monitor considers related to 
the study products (WRSs2 or S. sonnei 53G) and that may appear minor in terms of 
individual events, but that may collectively represent a serious potential concern for 
safety  
8.6.1 Individual Halting Criteria 
A subject may be removed from the study if an investigator deems it in the best interest of the 
subject. A subject also may halt (discontinue participation) at any time. If a subject meets an 
individual halting criterion, it will not halt the study but halt the subject’s involvement. 
8.7   Safety Oversight (DSMB, as applicable) 
Safety oversight will be conducted by a Data and Safety Monitoring Board (DSMB) which is an 
independent group of experts that monitors subject safety and advises the IND sponsor and study 
investigators.  
8.7.1 Data and Safety Monitoring Board (DSMB) 
Safety oversight will be conducted by a DSMB that is an independent group with expertise to 
interpret data from this study and will monitor subject safety and advise DMID. The DSMB 
members will be separate and independent of study personnel participating in this study and 
should not have scientific, financial, or other conflict of interest related to this study. DSMBs 
must consist of at least three voting members, including a biostatistician experienced in statistical 
methods for clinical trials and a clinician with relevant expertise. 

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The DSMB will review safety data at the following time points: 
Before study initiation and after all challenge cohorts have been completed   
Ad hoc when a halting rule is met or as needed 
At least annually 
Final review meeting: 6 to 8 months after clinical database lock to review the cumulative 
unblinded safety data for the study. The data will be provided in a standard summary format. The 
DSMB may be asked to provide recommendations in response to questions posed by the IND 
sponsor. 
Procedures for DSMB reviews/meetings will be defined in a charter. The DSMB will review 
applicable data to include but not limited to enrollment, demographic information, dosing, 
laboratory, and safety data which may include solicited and unsolicited AE/SAEs, concomitant 
medications, medical history, clinical laboratory values and any physical examinations. Safety 
data will be reviewed by the DSMB per the DSMB charter for this study. The DSMB may 
receive data in aggregate and presented by group. The DSMB may be unblinded to study group 
assignment, as needed, to assess safety issues. As an outcome of each review/meeting, the 
DSMB will make a recommendation as to the advisability of proceeding with vaccinations (as 
appropriate), and to continue, modify or terminate the study. 
The DSMB will review data at specified times during the course of the study for subject and 
overall study progress and will conduct ad hoc reviews as appropriate when a halting rule is met 
or for immediate concerns regarding observations during this study. 
The IND sponsor or the DSMB chair may convene the DSMB on an ad hoc basis according to 
protocol criteria or if there are immediate concerns regarding observations during the course of 
the study. The Medical Monitor is empowered to stop the study at any time if AEs that meet the 
halting criteria are reported. The Medical Monitor will be responsible for reviewing SAEs in real 
time. 
 
 

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9 HUMAN SUBJECTS PROTECTION  
9.1   Institutional Review Board/Independent Ethics Committee 
Each site principal investigator will obtain IRB approval for this protocol to be conducted at 
his/her research site(s) and send supporting documentation to the DMID before initiating 
recruitment of subjects. The investigator will submit applicable information to the IRB/IEC on 
which it relies for the review, to conduct the review in accordance with 45 CFR 46, ICH E6 
GCP, and as applicable, 21 CFR 56 (Institutional Review Boards) and 21 CFR 50 (Protection of 
Human Subjects), other federal, state, and local regulations. The IRB/IEC must be registered 
with OHRP as applicable to the research. DMID must receive the documentation that verifies 
IRB/IEC-approval for this protocol, associated informed consent documents, and upon request 
any recruitment material and handouts or surveys intended for the subjects, prior to the 
recruitment and enrollment of subjects.     
Any amendments to the protocol or consent materials will be approved by the IRB/IEC before 
they are implemented. IRB/IEC review and approval will occur at least annually throughout the 
enrollment and follow-up of subjects and may cease if annual review is no longer required by 
applicable regulations and the IRB/IEC. The investigator will notify the IRB/IEC of deviations 
from the protocol and reportable SAEs, as applicable to the IRB/IEC policy. 
Each institution engaged in this research will hold a current Federal-wide Assurance (FWA) 
issued by the Office of Human Research Protection (OHRP) for federally funded research. 
9.2   Informed Consent Process 
Informed consent is a process that is initiated prior to an individual agreeing to participate in a 
trial and continuing throughout the individual’s trial participation. Before any study procedures 
are performed, informed consent will be obtained and documented. Subjects will receive a 
concise and focused presentation of key information about the clinical trial, verbally and with a 
consent form. The explanation will be organized and presented in lay terminology and language 
that facilitates understanding why one might or might not want to participate. 
An investigator or designee will describe the protocol to potential subjects face-to-face. The key 
information about the purpose of the study, the procedures and experimental aspects of the study, 
risks and discomforts, any expected benefits to the subject, and alternative treatment will be 
presented first to the subject.   
Subjects will also receive an explanation that the trial involves research, and a detailed summary 
of the proposed study procedures and study interventions/products. This will include aspects of 

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the trial that are experimental, the probability for random assignment to treatment groups, any 
expected benefits, all possible risks (including a statement that the particular treatment or 
procedure may involve risks to the subject or to the embryo or fetus, if the subject is or may 
become pregnant, that are currently unforeseeable), the expected duration of the subject’s 
participation in the trial, alternative procedures that may be available and the important potential 
benefits and risks of these available alternative procedures.  
Subjects will be informed that they will be notified in a timely manner if information becomes 
available that may be relevant to their willingness to continue participation in the trial. Subjects 
will receive an explanation as to whether any compensation and any medical treatments are 
available if injury occurs, and, if so, what they consist of, or where further information may be 
obtained. Subjects will be informed of the anticipated financial expenses, if any, to the subject 
for participating in the trial, as well as any anticipated prorated payments, if any, to the subject 
for participating in the trial. They will be informed of whom to contact (e.g., the investigator) for 
answers to any questions relating to the research project.  
Information will also include the foreseeable circumstances and/or reasons under which the 
subject’s participation in the trial may be terminated. The subjects will be informed that 
participation is voluntary and that they are free to withdraw from the study for any reason at any 
time without penalty or loss of benefits to which the subject is otherwise entitled.   
The extent of the confidentiality of the subjects’ records will be defined, and subjects will be 
informed that applicable data protection legislation will be followed. Subjects will be informed 
that the monitor(s), auditors(s), IRB, NIAID, and regulatory authority(ies) will be granted direct 
access to the subject’s original medical records for verification of clinical trial procedures and/or 
data without violating the confidentiality of the subject, to the extent permitted by the applicable 
laws and regulations, and that, by signing an informed consent form, the subject is authorizing 
such access.  
Subjects will be informed that records identifying the subject will be kept confidential, and, to 
the extent permitted by the applicable laws and/or regulations, will not be made publicly 
available and, if the results of the trial are published, the subject’s identity will remain 
confidential. Subjects will be informed whether private information collected from this research 
and/or specimens will be used for additional research, even if identifiers are removed.   
Subjects will be allowed sufficient time to consider participation in this research trial and have 
the opportunity to discuss this trial with their family, friends, or legally authorized representative, 
or think about it prior to agreeing to participate. 

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Informed consent forms will be IRB approved and subjects will be asked to read and review the 
consent form. Subjects must sign the informed consent form prior to starting any study 
procedures being done specifically for this trial. 
Once signed, a copy of the informed consent form will be offered to the subject(s) for their 
records. The subject(s) may withdraw their consent at any time during the trial. The rights and 
welfare of the subject(s) will be protected by emphasizing to them that the quality of their 
medical care will not be adversely affected if they decline to participate in this study. 
Study personnel may employ recruitment efforts prior to obtaining study consent if a patient-
specific screening consent is on record or if the IRB has agreed that chart review is allowed 
without a fully executed screening consent. In cases where there is not a patient-specific 
screening consent on record, site Clinical staff may pre-screen via chart review and refer 
potential subjects to the Research staff. Research staff would obtain consent per the standard 
informed consent process before conducting protocol-specific screening activities. 
New information will be communicated by the site principal investigator to subjects who consent 
to participate in this trial in accordance with IRB requirements. The informed consent document 
will be updated, and subjects will be re-consented per IRB requirements, if necessary. Subjects 
will be offered a copy of all informed consent forms that they sign. 
9.3   Consent for Secondary Research of Residual Specimens and Data 
Residual samples are samples that are left over after the study has been completed and are stored 
for possible use in future research studies. Retention of residual samples for secondary research 
and the collection and storage of samples for use in future research studies is a condition of study 
participation. Subjects who sign the informed consent form for the study are consenting to allow 
the collection, storage, and use of both residual and secondary research samples.  
Residual samples will be maintained for possible use in future research studies, such as 
examining additional immunological assessments or testing for antibodies against other viruses 
or bacteria. These samples may be shared for purposes other than per protocol analysis with 
investigators at the participating VTEU site and with other investigators at other institutions once 
the clinical study report has been finalized.   
Secondary research samples are also being collected and will not become part of a subject’s 
medical record and will not be shared with their doctor. Secondary research studies may include 
but are not limited to non-traditional immune assay development, assessing innate immune 
factors, cytokines and other virologic evaluations. 

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Residual and secondary research samples will be stored indefinitely at a DMID designated 
central clinical storage facility. These samples will not be sold or used directly for production of 
any commercial product. Each sample will be encoded (labeled) only with a barcode and a 
unique tracking number to protect subject confidentiality. There are no benefits to subjects in the 
collection, storage, and subsequent use of their specimens for future research. Reports about 
future research done with subjects’ samples will NOT be kept in their health records.  
Residual samples will be available upon the completion of the study; however, secondary 
research samples may be requested from DMID and shipped from the DMID CMS at any time. 
Subjects may change their decision to participate in the study at any time by notifying the study 
doctors or nurses in writing. However, any data from a previously collected sample prior to the 
withdrawn consent will not be removed. Any secondary research of residual samples or 
collection of samples specifically for future research not yet collected/stored will not be stored 
for future/residual use. Any data from a previously collected sample prior to the withdrawn 
consent will not be removed.  
9.4   Exclusion of Women, Minorities, and Children (Special Populations) 
Special populations, e.g., non-English speakers, children, illiterate or non-writing individuals and 
vulnerable populations will not be enrolled in this study.  
9.5   Subject Confidentiality 
Subject confidentiality is strictly held in trust by the participating investigators, their staff, and 
the sponsor(s) and their agents. This confidentiality includes documentation, investigation data, 
subject’s clinical information, and all other information generated during participation in the 
study. No information concerning the study, or the data generated from the study will be released 
to any unauthorized third party without prior written approval of the DMID and the subject. 
Subject confidentiality will be maintained when study results are published or discussed in 
conferences. The study monitor, or other authorized representatives of the sponsor or 
governmental regulatory agencies 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 subjects in this study. The clinical study site will permit 
access to such records. 
All records will be kept locked, and all computer entry and networking programs will be carried 
out with coded numbers only and with password protected systems. All non-clinical specimens, 
evaluation forms, reports, and other records that leave the site will be identified only by a coded 
number. 

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Each subject will be assigned a unique study identifier. All data collection sheets will identify the 
subject by a unique identifier, and the date. Names will not be used on any samples or in any 
publication of this study. All efforts will be made to protect the privacy of subjects. 
This information and data will not be used by the site principal investigator or other study 
personnel for any purpose other than conducting the study. These restrictions do not apply to: (1) 
information which becomes publicly available through no fault of the site principal investigator or 
other study personnel; (2) information which is necessary to disclose in confidence to an IRB 
solely for the evaluation of the study; (3) information which it is necessary to disclose in order to 
provide appropriate medical care to a study subject; or (4) study results which may be published. 
9.6   Certificate of Confidentiality  
To protect privacy, we have received a Certificate of Confidentiality. With this Certificate, the 
researchers cannot be forced to release information that may identify the research subject, even 
by a court subpoena, in any federal, state, or local civil, criminal, administrative, legislative, or 
other proceedings. The researchers will use the Certificate to resist any demands for information 
that would identify the subject, except as explained below. 
The Certificate cannot be used to resist a demand for information from personnel of the United 
States Government that is used for auditing or evaluation of federally funded projects, like this 
study, or for information that must be released in order to meet the requirements of the Federal 
Food and Drug Administration (FDA). 
A Certificate of Confidentiality does not prevent the subject from voluntarily releasing 
information about themselves or their involvement in this research.  If any person or agency 
obtains a written consent to receive research information, then the researchers may not use the 
Certificate to withhold that information.   
The Certificate of Confidentiality does not prevent the researchers from reporting without the 
subject’s consent, information that would identify the subject as a participant in the research 
project regarding matters that must be legally reported including child and elder abuse, sexual 
abuse, or wanting to harm themselves or others.   
The release of individual private information or specimens for other research will only occur if 
consent was obtained from the individual to whom the information, document, or biospecimen 
pertains, or for the purposes of other research that is in compliance with applicable Federal 
regulations governing the protection of human subjects in research. 

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9.7   Costs, Subject Compensation, and Research Related Injuries 
There is no cost to subjects for the research tests, procedures, and study product while taking part 
in this trial. Procedures and treatment for clinical care may be billed to the subject, subject’s 
insurance or third party. Subjects may be compensated for their participation in this trial.   
Compensation will be in accordance with the local IRB’s policies and procedures, and subject to 
IRB approval. 
If it is determined by the site principal investigator that an injury occurred to a subject as a direct 
result of the tests or treatments that are done for this trial, then referrals to appropriate health care 
facilities will be provided to the subject. Study personnel will try to reduce, control, and treat any 
complications from this trial. Immediate medical treatment may be provided by the participating 
site. No financial compensation will be provided to the subject by the NIAID, NIH to the subject, 
or by the participating site for any injury suffered due to participation in this trial. 
10 STATISTICAL CONSIDERATIONS 
10.1 Study Hypotheses 
The primary objective of this study is to estimate the combined vaccine efficacy of 2 doses of 
WRSs2 (106 cfu or 5x105 cfu) in preventing shigellosis following challenge with S. sonnei strain 
53G. Vaccine efficacy (VE) will be estimated by the prevention of shigellosis, as defined in 
Table 4, among vaccine recipients compared to placebo recipients through Day 63 after receipt 
of challenge. 
𝑉𝑉𝑉𝑉
෢= 𝑃𝑃0 −𝑃𝑃1
𝑃𝑃0
 
P1 is the observed proportion of shigellosis among subjects receiving WRSs2 and P0 is the 
observed proportion among subjects receiving placebo.  
The null hypothesis is that the absolute vaccine efficacy is zero. The one-sided alternative 
hypothesis is that the absolute vaccine efficacy is greater than zero. 
10.2 Sample Size Considerations 
Assuming a rate of 70% in the placebo arm with shigellosis after challenge, and that the expected 
vaccine efficacy is 57%, this corresponds to an expected rate of 30% in a WRSs2 group.  
The study was originally designed with three arms (one dose of WRSs2, two doses of WRSs2, 
and placebo) with two primary hypotheses comparing each vaccine arm to placebo. It was 

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determined that 120 randomized subjects would be required to ensure at least 90 subjects were 
challenged, assuming approximately 25% of subjects who are randomized would drop-out or no 
longer qualify to receive challenge. Thirty (30) subjects per arm provided a power of ~82% for 
each hypothesis test, using a two-sided Chi-square test at alpha=0.025 (equivalent to a one-sided 
test at alpha=0.0125) utilizing a Bonferroni correction to adjust for the increase in Type I error 
due to having two primary hypotheses. 
After the study accrued with 69 subjects, the DSMB recommended lowering the dose due to a 
halting event. To meet the recommendation of the DSMB and evaluate the safety and efficacy 
signal, the decision was made to amend the protocol to discontinue further enrollment in the one 
dose vaccine arm and proceed with two arms (two lower doses of WRSs2 or two doses of 
placebo). It is anticipated that approximately 43 subjects will be randomized to either two 
administrations of the lower dose or placebo to achieve approximately 30 subjects challenged. 
The primary objective was modified to estimate the combined vaccine efficacy of two doses of 
WRSs2 at either the higher or lower dose in order to have enough power to detect vaccine 
efficacy with a smaller sample size than planned. Given the following assumptions: 
-70% Shigellosis rate in placebo arm 
-57% true vaccine efficacy of WRSs2 
-One hypothesis test for the combined vaccine efficacy of 2 doses of WRSs2 (eliminating the 
one dose arm from the primary analysis), with the following null and alternative hypothesis: 
-Null hypothesis: vaccine efficacy = 0 
-Alternative hypothesis: vaccine efficacy > 0 
-Placebo arm sample size: 25 (~15 from prior to study halt + ~10 after protocol amendment) 
-Combined 2 dose WRSs2 arm sample size: 35 (~15 from prior to study halt + ~20 after protocol 
amendment) 
A two-sided Chi-squared test at alpha=0.05 provides approximately 89% power to detect a 
combined vaccine efficacy greater than zero.  
10.3 Treatment Assignment Procedures 
10.3.1 Randomization Procedures 
It was planned to randomize up to 120 subjects to three vaccination arms (Arm 1: 
WRSs2/WRSs2, Arm 2: Placebo/WRSs2, and Arm 3: Placebo/Placebo) in a 1:1:1 ratio using 106 
cfu.  

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Based on recommendations of the DSMB, the dose of study vaccine was decreased to 5x105 cfu 
WRSs2 and the design modified to 2 arms with a randomization scheme of 2:1 (vaccine: 
placebo). Up to 3 sites may be utilized for this study and as such treatment assignments will be 
stratified by site. A permuted block randomization scheme will be developed to avoid the 
potential for serious imbalance in the number of subjects assigned to each arm, an imbalance that 
can occur in the simple randomization procedures. Subjects who withdraw, or are withdrawn 
from the study, or are lost to follow-up after randomization will not be replaced. 
The list of randomized treatment assignments will be prepared by statisticians at the Emmes 
Corporation and included in the enrollment module IDES for the trial. IDES will assign each 
subject a treatment code after demographic and eligibility data have been entered into the 
system. A designated individual at the site will be provided with a treatment key, which links the 
treatment code to the actual treatment assigned, which will be kept in a secure place.  
Instructions for use of the enrollment module will be included in the IDES User’s Guide. Manual 
back-up randomization procedures and instructions are provided for use in the event that the site 
temporarily loses access to the Internet, or the online enrollment system is unavailable.   
Per GCP, screening records will be kept at the study 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 IDES.   
10.3.2 Masking Procedures 
The Shigella vaccine and placebo will be prepared by the unblinded site pharmacist and will be 
administered by unblinded study staff on Day 1 and Day 29. The subjects, the study personnel 
who perform any study-related assessments after administration, data entry personnel at the sites, 
and laboratory personnel performing immunologic assays will be blinded to treatment 
assignment.  
The DSMB may receive data in aggregate and presented by treatment group, but without the 
treatment group identified. The DSMB may be unblinded to individual study treatment 
assignments, as needed to adequately assess safety issues. Refer to the MOP for unblinding 
procedures. 
10.4 Planned Interim Analyses  
No formal interim analysis involving the testing of a hypothesis is planned.   
10.4.1 Interim Safety Review  
As described in Section 8.7, the DSMB will meet and review safety data at specified intervals. 
The study will be monitored to determine if any of the safety halting rules described in Section 

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8.6 is met. The halting rules do not utilize any statistical criteria and no formal hypothesis testing 
is planned to occur for the safety reviews. 
10.4.2 Interim Immunogenicity or Efficacy Review 
No interim immunogenicity or efficacy review is planned.  
10.5 Final Analysis Plan 
A formal SAP will be developed and finalized prior to any analysis, or database lock.  
The final analysis will be performed, and clinical study report completed and distributed when 
all primary, secondary, and exploratory endpoint data are available. For purposes of scientific 
meeting presentation of study progress in advance of the final analysis, the protocol PI may 
request analysis of the demographics and blinded aggregate analyses of available primary and 
secondary endpoint data.  
10.5.1 Endpoint Review Committee 
For all enrolled subjects, all available clinical trial data necessary for determination of primary 
endpoint status (shigellosis) per Table 4 will be reviewed by a blinded endpoint review 
committee (ERC).   This committee will be comprised primarily of independent Shigella experts, 
the study PI, and the SDCC. The ERC will be responsible for making a final determination of 
endpoint status for each subject prior to database lock and study unblinding34. 
10.5.2 Analysis Populations 
The Safety Population includes all subjects who received at least one dose of study vaccination. 
Subjects will be analyzed according to the study product they actually received, not necessarily 
the study product to which a subject was randomized.  
The Immunogenicity Population consists of all subjects who received any study product and for 
whom immunogenicity endpoint data are available. Subjects will be analyzed according to the 
study product actually received, not necessarily the study product to which a subject was 
randomized to. 
The Full Analysis Population includes all randomized subjects with complete data for the 
primary efficacy endpoint. Subjects will be analyzed according to the study arm to which they 
were randomized. 
The Per Protocol (PP) Population includes all subjects in the Full Analysis Population with the 
following exclusions: 
• Subjects found to be ineligible at baseline. 

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• Subjects who received an incorrect study vaccination  
• Subjects with second study vaccination or challenge received out of window 
• Subjects who received any of the following prior to the assessment of the primary 
endpoint: 
o Non-study vaccines 
o Antibiotics 
o Oral, parenteral, or high-dose inhaled steroids 
o Immunosuppressive or cytotoxic therapy 
o Blood products or immunoglobulins 
o Experimental products 
In the case of mis-randomization, subjects in the PP population will be analyzed according to the 
study product actually received. 
10.5.3 Primary Efficacy Analysis 
The primary efficacy analysis will be conducted in the Full Analysis Population and the Per 
Protocol Population among subjects who were randomized to receive two doses of 106 cfu or 
5x105 cfu of WRSs2 or placebo.  
The combined absolute vaccine efficacy of two doses of 106 or 5x105 of WRSs2 will be 
estimated and presented with a 95% confidence interval, and the null hypothesis that the absolute 
vaccine efficacy is zero will be tested using a two-sided Chi-squared test. If the p-value is <0.05 
and the lower limit of the 95% confidence interval for VE is above zero, then we can conclude 
that the combined VE of two doses of WRSs2 is greater than zero.   
10.5.4 Secondary Efficacy Analysis 
Secondary efficacy analyses to estimate the vaccine efficacy of the individual vaccine arms (1 
dose of 106 cfu, 2 doses of 106 cfu, and 2 doses of 5x105 cfu) will be presented along with 95% 
confidence intervals. As the study is not powered for these individual comparisons to placebo, no 
formal hypothesis tests are planned, and no p-values will be presented. As such, no adjustments 
for multiplicity will be made.  
10.5.5 Safety Data 
Safety data will be summarized for the Safety Analysis Population. Subjects receiving a 
vaccination according to a study arm other than the study arm to which they were randomized, 
will be analyzed according to the vaccination(s) they actually received. Solicited AEs will be 
summarized by maximum severity for each day after each study vaccination for Days 1-8. The 
number, percentage (observed rate), and exact two-sided 95% confidence interval for subjects 
reporting each solicited AE within 8 days following vaccination will be summarized. In addition, 

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maximum severity for each solicited AE will be determined for each subject and reported by 
study arm and the resulting number and percentage of subjects will be summarized by severity 
grade (none, mild, moderate, severe). Summaries of solicited AEs will be presented separately 
by study arm for each study vaccination as well as overall study vaccinations. 
Vaccine-related unsolicited AEs will be coded by Medical Dictionary for Regulatory Activities 
(MedDRA®) for preferred term and system organ class (SOC). The number of SAEs are likely 
to be small in this trial and will be reported by detailed listings showing the event description, 
MedDRA® preferred term and SOC, relevant dates (study vaccinations/challenge and AEs), 
severity, relatedness, and outcome for each event. Non-serious unsolicited vaccine-related AEs 
will be summarized as number and percentage of subjects reporting at least one event in each 
MedDRA® preferred term and SOC, cross tabulated by severity and relationship to study 
product. Additionally, the proportion of subjects and exact 95% confidence intervals of AEs by 
study arm for each study vaccination and challenge as well as overall study vaccinations and by 
MedDRA® categories will be calculated.  
10.5.6 Immunogenicity Data 
Immunogenicity data summaries and analysis will be presented for the Immunogenicity 
Population. 
Immune responses for Shigella LPS-specific and Invaplex-specific IgA and IgG will be 
summarized at each time point by study arm. Geometric mean titers (GMTs), geometric mean 
fold rise (GMFR), and corresponding 95% confidence intervals will be reported. The maximum 
titer and peak-fold rise for the post-vaccination and post-challenge time period will be reported. 
The number and percentage of subjects with at least a 4-fold rise from pre-vaccination (or pre-
challenge) will be reported for each post-vaccination (and post-challenge) time point.  
Graphical presentations of immune response may include reverse cumulative distribution (RCD) 
curves and longitudinal presentation of GMTs. 
10.5.7 Missing Values and Outliers 
All attempts will be made to collect all data per protocol. As missing data are expected to be 
minimal, no imputation will be performed for missing values. Any data point that appears to be 
erroneous or inexplicable based on clinical judgment will be investigated as a possible outlier. If 
data points are identified as outliers, sensitivity analyses will be performed to examine the impact 
of including or excluding the outliers. Any substantive differences in these analyses will be 
reported.   
 
 

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11  SOURCE DOCUMENTS AND ACCESS TO SOURCE 
DATA/DOCUMENTS 
Each participating site will maintain appropriate medical and research records in compliance 
with ICH E6, Section 4.9 and regulatory and institutional requirements for the protection of 
confidentiality of subjects. Each site will permit authorized representatives of the DMID, its 
designees, and appropriate regulatory agencies to examine (and when required by applicable law, 
to copy) clinical records for the purposes of quality assurance reviews, audits, and evaluation of 
the study safety and progress. These representatives will be permitted access to all source data 
and source documents, which include, but are not limited to, hospital records, clinical and office 
charts, laboratory notes, memoranda, 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.  
The study uses direct data entry for the participating clinic site. Subjects will utilize a web-based 
e-Memory aid. Staff will review with subjects use of memory aid, how to complete a web based 
“e-Memory aid” and are expected to enter information in the e-Memory aid each day. Data 
entered by subjects into the e-Memory Aid are stored in the data system for clinic staff review 
during scheduled visits.  The e-Memory Aid is not considered source data. After clinic staff 
review and save the data, the data will be entered into Advantage eClinical as source.  
The site staff will be the data originators for the clinically reviewed data in Advantage eClinical® 
that will be used for the study endpoints.  
 
12  QUALITY CONTROL AND QUALITY ASSURANCE 
Following a written DMID-accepted site quality management plan, each participating site and its 
subcontractors are responsible for conducting routine quality assurance (QA) and quality control 
(QC) activities to internally monitor study progress and protocol compliance. The site principal 
investigator will provide direct access to all study-related sites, source data/data collection forms, 
and reports for the purpose of monitoring and auditing by the sponsor, and inspection by local 

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and regulatory authorities. The site principal investigator will ensure all study personnel are 
appropriately trained and applicable documentations is maintained on site. 
The DCC will implement quality control procedures beginning with the data entry system and 
generate data quality control checks that will be run on the database. Any missing data or data 
anomalies will be communicated to the participating site(s) for clarification and resolution. 
DMID-designated clinical monitors will verify that the clinical trial is conducted, and data are 
generated, documented (recorded), and reported in compliance with the protocol, ICH/GCP 
guidelines, and the applicable regulatory requirements. Clinical monitoring reports will be 
submitted to DMID. 
13  DATA HANDLING AND RECORD KEEPING 
13.1 Data Management Responsibilities  
The investigator is responsible for ensuring the accuracy, completeness, legibility, and timeliness 
of the data reported. All source documents should be completed in a neat, legible manner to 
ensure accurate interpretation of data. Black or blue permanent ink is required to ensure clarity of 
reproduced copies. When making changes or corrections, 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. 
Electronic case report forms will be created by the SDCC to record and maintain data for each 
subject enrolled in this study.   
The sponsor and/or its designee will provide guidance to the site principal investigators and other 
study personnel on making corrections to the data collection forms and eCRF. 
13.2 Data Coordinating Center/Biostatistician Responsibilities 
All electronic case report forms and laboratory reports must be reviewed by the clinical team and 
data entry personnel, who will ensure that they are accurate and complete.  AEs must be recorded 
on the appropriate electronic case report form, assessed for severity and relationship, and reviewed 
by the site principal investigator or appropriate sub-investigator. 
Data collection is the responsibility of the study personnel at the participating VTEU site under 
the supervision of the respective site principal investigator.  During this study, the site principal 
investigator must maintain complete and accurate documentation for this study. 

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The SDCC for this study will be responsible for data management, quality review, analysis, and 
reporting of the study data. 
13.3 Data Capture Methods 
Clinical data (including, but not limited to, AE/SAEs, concomitant medications, medical history, 
physical assessments, and clinical laboratory values) immunogenicity, and reactogenicity will be 
collected by study personnel then entered into eCRFs via a 21 CFR Part 11-compliant internet 
data entry system provided by the study data coordinating center. The data system includes 
password protection and internal quality checks, such as automatic range checks, to identify data 
that appear inconsistent, incomplete, or inaccurate. Clinical data will be entered directly into 
electronic case report forms by the study personnel. 
13.4 Types of Data 
Data for this trial will include clinical, safety, efficacy, and outcome measures (e.g., assessments, 
clinical laboratory values, reactogenicity, and immunogenicity data). 
13.5 Study Records Retention 
Study records and reports including, but not limited to, eCRFs, source documents, ICFs, 
laboratory test results, and study drug disposition records will be retained for 2 years after a 
marketing application is approved for the study product for the indication for which it is being 
investigated; or, if no application is to be filed or if the application is not approved for the study 
product, until 2 years after the investigation is discontinued and the FDA has been notified. 
These documents will be retained for a longer period, however, if required by local regulations. 
ICFs for secondary research will be maintained if the sample/specimen exists. 
No records will be destroyed without the written consent of the sponsor. The sponsor is 
responsible for informing the site’s principal investigator when these documents no longer need 
to be retained. The participating VTEU site must contact DMID for authorization prior to the 
destruction of any study records. 
 
14  CLINICAL MONITORING 
Site monitoring is conducted to ensure that the human subjects’ protections, study and laboratory 
procedures, study intervention administration, and data collection processes are of high quality 
and meet sponsor, ICH/GCP guidelines and applicable regulations, and that this trial is 
conducted in accordance with the protocol, protocol-specific MOP, and applicable sponsor 

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standard operating procedures. DMID, the sponsoring agency, or its designee will conduct site-
monitoring visits as detailed in the clinical monitoring plan. 
Site visits will be made at standard intervals as defined by DMID and may be made more 
frequently as directed by DMID. Monitoring visits will include, but is not limited to, review of 
regulatory files, accountability records, eCRFs, informed consent forms, medical and laboratory 
reports, and protocol and GCP compliance. Site monitors will have access to each participating 
site, study personnel, and all study documentation according to the DMID-approved site 
monitoring plan. Study monitors will meet with site principal investigators to discuss any 
problems and actions to be taken and will document site visit findings and discussions. 
15  PUBLICATION POLICY 
Following completion of the study, the lead Principal Investigator will publish the results of this 
research in a scientific journal. The Principal Investigators will submit or have submitted for 
them to the National Library of Medicine’s PubMed Central (http://www.ncbi.nlm.nih.gov/pmc/) 
an electronic version of their final, peer-reviewed manuscripts upon acceptance for publication, 
to be made publicly available no later than 12 months after the official date of publication in 
accordance with:  
NIH Public Access Policy, http://publicaccess.nih.gov/ 
NIH Office of Extramural Research (OER) Grants and Funding, 
http://grants.nih.gov/grants/oer.htm 
As of January 2018, all clinical trials supported by the NIH must be registered on 
ClinicalTrials.gov, no later than 21 days after the enrollment of the first subject. Results of all 
clinical trials supported by the NIH, generally, need to be submitted no later than 12 months 
following the primary completion date. A delay of up to 2 years is available for trials that meet 
certain criteria and have applied for certification of delayed posting. 
As part of the result posting a copy of this protocol (and its amendments) and a copy of the 
Statistical Analysis Plan will be posted on ClincialTrials.gov. 
For this trial the responsible party is DMID which will register the trial and post results.   
The responsible party, DMID, does not plan to request certification of delayed posting. 
Refer to: 
Public Law 110-85, Section 801, Clinical Trial Databases 
42CFR11 

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NIH NOT-OD-16-149 
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17  APPENDICES 
Appendix A: Schedule of Events 
Appendix A 1: Screening and Vaccination 
 
Appendix A 2: Inpatient Challenge and Follow-up 
Appendix B: Toxicity Table/Laboratory AE Grading Scale 
Appendix C: Vital Signs AE Grading Scale 
Appendix D: Diarrhea Classification 
Appendix E: Reactogenicity Grading Scale

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Plasma
Whole Blood PBMC for Tfh (FACS T/B/IR Cell 
Panel) and Phenotyping, mL
16
16
Paxgene whole blood, mL
2.5
2.5
2.5

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!  = On the 5th day after challenge (Day 62), subjects will begin to receive 500 mg of ciprofloxacin twice daily for 3 days. Subjects with contraindication to ciprofloxacin 
may receive as second-line treatment trimethoprim-sulfamethoxazole (160 mg/800 mg twice daily for 5 days), or any antibiotic that is deemed suitable according to local 
guidelines and the investigator’s assessment can be used. 
? = These are assays that may be performed.  
( ) = collected on day of vaccination or second screening visit if not collected during initial screening visit 
1  = Pre-vaccination vital signs and post vaccination dose if indicated 
 
2  = review use of memory aid, provide thermometer and instruct to contact site for AEs, changes to health, emergency room visit or hospitalization, SAE, or pregnancy   
3 = review and confirm information from memory aid 
4  = contact by various methods, including email, text, or phone call to remind subject to complete memory aid and instruct how to fill out memory aid  
5  = vaccination assessment completed at least 90 minutes post dose  
6  = Pre-challenge vital signs, post challenge dose per protocol +90 min, daily and additional assessment as indicated 
7  = Illness Labs collected per PI discretion include: serum chemistry, sodium, potassium, blood urea nitrogen/BUN, and creatinine. Urine dip for specific gravity, as 
indicated 
8  = All emesis will be measured and recorded then disinfected with bleach and discarded in the hazardous waste 
9 = A SARS-CoV-2 test will be performed on all subjects upon entry into the challenge unit. Anyone with a positive test will be quarantined in their room until they can 
be safely escorted out of the challenge unit. In addition, a SARS-CoV-2 test will be performed at any time a subject were to exhibit symptoms of COVID-19 illness during 
the inpatient stay. 
10 = All concomitant medications, taken in the 30 days prior to study enrollment through Day 28 following challenge or early termination, whichever occurs first, will be  
recorded.   
11= Height and weight collected to determine BMI at screening  
 
 

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Appendix B: Toxicity Table/Laboratory AE Grading Scale 
AE Grading 
This protocol is CDISC compliant. The FDA Toxicity Grading Scale for Laboratory 
Abnormalities below serves as a guideline. - Institutional normal reference ranges should be 
provided to demonstrate that they are appropriate. During screening, subjects will have blood 
drawn to determine if any clinically significant laboratory abnormalities exist that would 
preclude study participation. Following enrollment, the toxicity table will be used as a reference 
for grading severity of laboratory AEs.  
 
Mild  
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Serum  
 
 
 
Sodium – Hyponatremia mEq/L 
132 – 134 
130 – 131 
<130 
Sodium – Hypernatremia mEq/L 
144 – 145 
146 – 147 
>147 
Potassium – Hyperkalemia mEq/L 
5.1 – 5.2 
5.3 – 5.4 
>5.4 
Potassium – Hypokalemia mEq/L 
3.5 – 3.6 
3.3 – 3.4 
< 3.3 
Blood Urea 
Nitrogen BUN 
23 – 26 
27 – 31 
> 31 
Creatinine – mg/dL 
1.5 – 1.7 
1.8 – 2.0 
> 2.0 
Liver Function Tests ALT 
increase by factor 
1.1 – 2.5 x ULN** 2.6 – 5.0 x ULN 
>5.0 x ULN 
Bilirubin – when Liver Function 
Test is normal; increase by factor 
1.1 – 1.5 x ULN 
1.6 – 2.0 x ULN 
>2.0 x ULN 
Hematology  
 
 
 
Hemoglobin (Female) - gm/dL 
11.0 – 12.0 
9.5 – 10.9 
 < 9.5 
Hemoglobin (Male) - gm/dL 
12.5 – 13.5 
10.5 – 12.4 
 < 10.5 
WBC Increase - cell/mm3 
10,800 – 15,000 
15,001 – 20,000 
> 20, 000 
WBC Decrease - cell/mm3 
2,500 – 3,500 
1,500 – 2,499 
< 1,500 
Neutrophils Decrease - cell/mm3 
1,500 – 2,000 
1,000 – 1,499 
 < 1,000 
Platelets Decreased - cell/mm3 
125,000 – 140,000 100,000 – 124,000 
< 100,000 
Urine  
 
 
 
Protein 
Trace 
1+ 
2+ 
** ULN” is the upper limit of the normal range. 
 

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Appendix C: Vital Signs ADVERSE EVENT Grading Scale 
Vital Signs Grading 
AE 
Severity Parameter 
Fever 
1 
38.0 – 38.40C 
100.4 – 101.10F 
2 
38.5 – 38.90C 
101.2 – 102.00F 
3 
≥39.00C 
>1020F or ER visit or hospitalization 
Hypertension 
(systolic) 
1 
141-150 mm Hg 
2 
151-155 mm Hg 
3 
>155mm Hg  
Hypertension 
(diastolic) 
1 
91 – 95 mm Hg 
2 
96-100 mm Hg 
3 
>100 mm Hg  
Hypotension (systolic) 
1 
85-89 mm Hg 
2 
80-84 mm Hg 
3 
<80 mm Hg   
Hypotension 
(diastolic) 
1 
50-54 mm Hg 
2 
45-49 mm Hg 
3 
<45 mm Hg 
Bradycardia 
1 
45-49 bpm  
2 
40-44 bpm 
3 
<40 bpm   
Tachycardia  
1 
101-115 bpm 
2 
116-130 bpm 

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Vital Signs Grading 
AE 
Severity Parameter 
3 
>130 bpm   
  
Appendix D: Diarrhea Classification  
Diarrheal stools are loose or watery stools classified as 3-5 consistency. To assess diarrhea, study 
personnel will use the following definitions for classification of stool samples: 
1 (Normal stool) – Firm, tootsie roll consistency 
2 (Soft stool) - Pudding consistency, not firm but holds some shape 
3 (Loose stool) – Takes the shape of the container, thick gravy consistency/brown 
watery, opaque watery, chocolate milk consistency 
4 (Watery stool) – Opaque colored, watery consistency 
5 (“Rice water”) - soapy water consistency 
Episode of diarrhea- begins with the first 3 or higher stool classification that 
ultimately is part of a diarrhea episode and ends when the subject has not passed 
any stool classified as 3-5 for 24 hours  
Only stools classified as 3, 4 and 5 consistency will be considered in the criteria for diarrhea. 
Stools classified as consistency of 3, 4 and 5 within any 24-hour period will be added to 
determine the highest severity for the episode. When there are no stools classified as 3-5 
consistency for 24 hours, the episode will be considered resolved.  The end date for the episode 
will be the date and time of the last stool consistency was classified as 3, 4, or 5. For subjects 
who experience diarrhea, all stools classified as 3-5 consistency will be weighed and evaluated 
by qualified medical personnel for grossly visible blood.  
Dysentery- ≥2 stools classified as consistency of 3-5 with gross blood (hemoccult positive) in 24 
hours with one or more of the following: oral temperature ≥38.0˚C; ≥1 moderate 
constitutional/enteric symptom; ≥2 episodes of vomiting in 24 hours. 
See Appendix E for diarrhea grading scale.