Controlled Human Infection Model Challenge/Rechallenge: Shigella Flexneri 2a and S. Sonnei Cross-protective Antigens Discovery in Healthy Adults in the United States
Phase: PHASE1
Conditions: Shigellosis
Sponsor: PATH

Protocol v4.2
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1
Clinical Study Protocol
Protocol Title: 
Controlled Human Infection Model Challenge/Rechallenge: 
Shigella flexneri 2a and S. sonnei cross-protective antigens 
discovery in healthy adults in the United States
Version 4.2 16 January 2023
PATH CVIA 088 
17015 
00017454 
Protocol Version/Date:
Sponsor Number/Name: 
FDA IND Number: 
JHU IRB Number:
CIR Number:
CIR337 
Funder:
The Bill and Melinda Gates Foundation
Sponsor: 
PATH 
Principal Investigator:
Kawsar Talaat, MD
Center for Immunization Research
Department of International Health
Johns Hopkins Bloomberg School of Public Health
624 N. Broadway, HH, Room 205
Baltimore, MD 21205
Associate Investigators:
David Sack, MD
Jane Halpern, MD, MPH, Dr PH
Subhra Chakraborty PhD
Brittany Feijoo, MSN, FNP-BC
Kamal Dhanjani MPAS, PA-C, FCCM
Arthi Rameshkumar, MSPH
Barbara DeNearing, RN, BSN, CCRC
Bridgett Finley, BSN, RN
Center for Immunization Research
Johns Hopkins Bloomberg School of Public Health
Daniel Selig, MD
Walter Reed Army Institute of Research (WRAIR)
Janine Danko, MD
Naval Medical Research Center
Epidemiology
Chad Porter, PhD, MPH
Head, Translational and Clinical Research Department (TraCR)
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Naval Medical Research Center 
Research Monitor: 
 
Anna P. Durbin, MD 
Professor, International Health 
Johns Hopkins Bloomberg School of Public Health 
 
 
Data Management: 
 
The Emmes Company, LLC  
 
 
 
 
401 N. Washington Street, Suite 700 
 
 
 
 
Rockville, MD 20850 
 
 
 
 
 
 
Study Sites: 
 
 
Center for Immunization Research 
 
 
 
 
Department of International Health 
 
 
 
 
Johns Hopkins Bloomberg School of Public Health 
 
 
 
 
624 N Broadway, HH Room 117 
 
 
 
 
Baltimore, MD 20215 
 
 
 
 
 
 
 
 
 
Center for Immunization Research Inpatient Unit 
 
 
 
 
301 Building, 4th Floor 
 
 
 
 
Johns Hopkins Bayview Medical Center 
 
 
 
 
301 Mason Lord Drive 
 
 
 
 
Baltimore, MD 21224 
 
 
 
 
 
Center for Immunization Research 
 
 
 
 
1101 North Point Blvd 
Suites 101,103, 112 
Baltimore, MD 21224 
 
 
 
 
Laboratories: 
 
 
Quest Diagnostics, Inc.,  
1901 Sulphur Spring Rd.,  
Baltimore, MD 21227 
 
 
Johns Hopkins Bloomberg School of Public Health,  
615 N. Wolfe Street 
Baltimore, MD 21205 
 
Johns Hopkins Hospital 
600 N. Wolfe Street / Meyer B-130 
Baltimore, MD 21287–7070 
 
Johns Hopkins Bayview Medical Center 
4940 Eastern Ave. 
Baltimore, MD 21224 
 
Immunology Laboratories 
Robert J. Cybulski Jr, PhD, D(ABMM) LTC(P), MS, US Army 
 
 
 
 
Director, Bacterial Diseases Branch, WRAIR 
 
 
 
 
Assistant Professor in Medicine, Uniformed Services University 
 
 
 
 
Deputy Microbiology Consultant to the US Army Surgeon General 
 
 
 
 
 
 

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Subhra Chakraborty, PhD, MS, MPH  
CIR Enteric Research Laboratory (JHSPH)  
615 N Wolfe Street / W5616  
Baltimore, MD 21205  
 
Research Laboratories  
Johns Hopkins Biological Repository 
Johns Hopkins Bloomberg School of Public Health 
Room W6620 / 615 North Wolfe St. 
Baltimore, MD 21205 
 
 
 
 
 
 
Daniel Leung, MD MsC 
30 N 1900 E 
University of Utah, Infectious Diseases, 4B319 
Salt Lake City, UTԜ 84132 
 
Antigen Discovery Inc (ADi) 
1 Technology Drive 
Irvine, CA 92618 
 
Dennis Burton, PhD 
IAVI Neutralizing Antibody Center 
The Scripps Research Institute 
La Joya, CA 
 
Institutional Review Board 
JHSPH IRB Office 
615 N. Wolfe Street 
Suite E1100 
Baltimore, Maryland 21205 
Phone: 410-955-3193 
Toll-Free: 1-888-262-3242 
Fax: 410-502-0584 
Email: JHSPH.irboffice@jhu.edu 
 
 
 
 
 

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Investigator’s Agreement 
 
 
Controlled Human Infection Model Challenge/Rechallenge: Shigella flexneri 2a and S. sonnei 
cross-protective antigens discovery in healthy adults in the United States 
 
 
 
“I have read this protocol and agree to conduct the study as outlined herein in accordance with 
International Conference on Harmonization Good Clinical Practice Guideline and FDA and DoD 
Regulations.” 
 
 
 
 
Kawsar R. Talaat, MD 
 
 
 
 
 
 
   Date 
Principal Investigator 
 
 
 

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I. Table of Contents 
I. 
Table of Contents ......................................................................................................... 5 
II. 
ABBREVIATIONS ........................................................................................................ 10 
III. CLINICAL PROTOCOL SYNOPSIS ........................................................................... 13 
IV. BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE ........................... 21 
1. 
Background Information-Clinical Significance and Pathogenicity of Shigella .......... 21 
2. 
Rationale for Current Study .............................................................................................. 21 
3. 
Previous Experience .......................................................................................................... 21 
S. flexneri 2a Strain 2457T ............................................................................................................................... 21 
S. sonnei Strain 53G ......................................................................................................................................... 22 
4. 
Dose and Administration Rationale ................................................................................. 22 
V. STUDY HYPOTHESIS ................................................................................................. 22 
VI. OBJECTIVES ............................................................................................................... 22 
1. 
Primary Objective: ............................................................................................................. 22 
2. 
Secondary Objective: ........................................................................................................ 22 
3. 
Exploratory Objectives:..................................................................................................... 22 
VII. ENDPOINTS ................................................................................................................. 23 
1. 
Primary Endpoint ............................................................................................................... 23 
2. 
Secondary Endpoints ........................................................................................................ 23 
VIII. STUDY DESIGN ........................................................................................................... 24 
1. 
Overview ............................................................................................................................. 24 
2. 
Description of Study Design ............................................................................................. 25 
IX. STUDY POPULATION ................................................................................................. 26 
1. 
Selection and Enrollment of Participants ....................................................................... 26 
2. 
Inclusion Criteria: ............................................................................................................... 26 
3. 
Exclusion Criteria: ............................................................................................................. 26 
4. 
Continuing Eligibility Criteria to Proceed to Cohort 1B or 2B ...................................... 27 
5. 
Access to Medical Records .............................................................................................. 27 
X. STUDY PROCEDURES ............................................................................................... 28 
1. 
Screening ............................................................................................................................ 28 
2. 
Clinical Evaluations ........................................................................................................... 28 
i. 
Monitoring During Inpatient Phase ........................................................................................................ 28 
ii. 
Wearable Devices and PVT Monitoring ................................................................................................ 29 
iii. 
Rehydration Procedures.......................................................................................................................... 29 

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iv. 
Treatment for Vomiting ............................................................................................................................ 30 
v. 
Treatment for Fever or Pain .................................................................................................................... 30 
vi. 
Other Treatments ..................................................................................................................................... 30 
3. 
Routine Discharge ............................................................................................................. 30 
4. 
Early Discharge .................................................................................................................. 31 
5. 
Monitoring During Outpatient Phase ............................................................................... 31 
6. 
Concomitant Medications/Treatments ............................................................................ 31 
XI. STUDY SCHEDULE .................................................................................................... 31 
1. 
Recruitment, Screening, and Enrollment of Study Volunteers: ................................... 31 
2. 
JH200 Screening: ............................................................................................................... 31 
3. 
Study-Specific Screening: ................................................................................................ 32 
4. 
Enrollment ........................................................................................................................... 33 
5. 
Study Days .......................................................................................................................... 33 
i. 
Day -2 or -1, Admission to the Inpatient Isolation Unit (Day 89 challenge 2) .................................. 33 
ii. 
Day 1, Challenge Day (Day 90 challenge 2) ........................................................................................ 34 
iii. 
Challenge .................................................................................................................................................. 34 
iv. 
Days 1 – 8, Planned Inpatient Days (Days 90-97 challenge 2) ......................................................... 34 
v. 
Day 9, Planned Discharge Day (Day 98 challenge 2) ........................................................................ 35 
vi. 
Day 15 Outpatient Follow-Up Visit (+/- 2 Days) (Day 104 challenge 2) ........................................... 35 
vii. 
Day 29 Outpatient Follow-Up Visit (+/- 4 Days) (Day 118 challenge 2) ........................................... 35 
viii. Day 57 Outpatient Follow-Up Visit (+/- 4 Days) (Day 146 challenge 2) ........................................... 35 
ix. 
Day 90 (-7/+28 days) Outpatient Visit OR readmission to the inpatient unit for second challenge: 
(Day 180 challenge 2) ............................................................................................................................. 36 
x. 
Re-Admission for Second Challenge .................................................................................................... 36 
xi. 
Day 180/270 Follow-Up Visit .................................................................................................................. 37 
6. 
Antibiotic Treatment .......................................................................................................... 37 
7. 
Early Termination ............................................................................................................... 37 
8. 
Laboratory procedures ...................................................................................................... 38 
9. 
Microbiology: ...................................................................................................................... 39 
10. Research Labs: .................................................................................................................. 39 
11. Retention of Study Samples ............................................................................................. 39 
XII. STUDY INTERVENTION/INVESTIGATIONAL PRODUCT ....................................... 40 
1. 
Shigella Challenge Inoculum/Product Characterization ............................................... 40 
S. flexneri 2a strain 2457T ................................................................................................................................ 40 
S. sonnei strain 53G .......................................................................................................................................... 40 
2. 
Packaging and Labeling .................................................................................................... 41 
3. 
Product Acquisition, Storage and Transfer .................................................................... 41 
S. flexneri 2a strain 2457T ................................................................................................................................ 41 
S. sonnei strain 53G .......................................................................................................................................... 42 
4. 
Product Preparation .......................................................................................................... 42 

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S. flexneri 2a strain 2457T ................................................................................................................................ 42 
S. sonnei strain 53G .......................................................................................................................................... 42 
5. 
Product Administration ..................................................................................................... 43 
6. 
Accountability Procedures for the Investigational Products ....................................... 43 
XIII. ASSESSMENT OF SAFETY ....................................................................................... 43 
1. 
Safety Monitoring-Vital Signs ........................................................................................... 44 
2. 
Physical Examination ........................................................................................................ 45 
3. 
Assessment of Stools ....................................................................................................... 45 
4. 
Laboratory Assessments .................................................................................................. 45 
5. 
IND Safety Reporting - Definitions ................................................................................... 47 
Adverse Event .................................................................................................................................................... 47 
Suspected Adverse Reaction ........................................................................................................................... 47 
Solicited and Anticipated Adverse Events (Reactogenicity Events) ........................................................... 47 
Adverse Events of Special Interest ................................................................................................................. 48 
Serious Adverse Event ...................................................................................................................................... 48 
6. 
Relationship to Investigational Product (Assessment of Causality) ........................... 49 
7. 
Recording Adverse Events – Method/Timing ................................................................. 50 
8. 
Duration of Follow-Up of Participants after Adverse Events ....................................... 50 
9. 
Safety Assessments .......................................................................................................... 51 
10. Reporting Adverse Events ................................................................................................ 52 
11. Outcome Adjudication Committee ................................................................................... 52 
12. Reporting Serious and Unexpected Adverse Events .................................................... 53 
13. Reporting to the Sponsor.................................................................................................. 53 
14. Reporting to the IRB .......................................................................................................... 54 
15. Immediately Reportable Events - Pregnancy ................................................................. 55 
16. AE-Related Withdrawal of Consent ................................................................................. 55 
17. Pending Inspections/Issuance of Reports ...................................................................... 56 
18. IND reporting ...................................................................................................................... 56 
Annual .................................................................................................................................................................. 56 
Final ………………………………………………………………………………………………………………………………………………………….……56 
19. Safety Criteria for Stopping Further Challenges ............................................................ 56 
20. Study Termination.............................................................................................................. 57 
XIV. STUDY MONITORING ................................................................................................ 57 
1. 
Monitoring ........................................................................................................................... 57 
2. 
Data Entry ........................................................................................................................... 57 
3. 
Data Management/Collection/Source Documents ......................................................... 57 

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XV. STATISTICAL CONSIDERATIONS ............................................................................ 58 
1. 
Introduction ........................................................................................................................ 58 
2. 
Volunteer Selection ........................................................................................................... 58 
3. 
Definitions of populations to be analyzed ...................................................................... 58 
4. 
Analysis ............................................................................................................................... 59 
Baseline Data ..................................................................................................................................................... 59 
Rates of Shigellosis ........................................................................................................................................... 59 
Safety Analyses ................................................................................................................................................. 59 
Immunology Analyses ....................................................................................................................................... 59 
5. 
Sample Size Considerations ............................................................................................. 60 
XVI. OBLIGATIONS AND ROLES OF THE SPONSOR, INVESTIGATOR, AND 
STUDY PERSONNEL .................................................................................................. 61 
XVII. QUALITY CONTROL AND ASSURANCE ............................................................... 61 
XVIII. PROTOCOL DEVIATIONS ....................................................................................... 61 
XIX. PROTECTION OF HUMAN SUBJECTS .................................................................... 62 
1. 
Insurance and Indemnity................................................................................................... 62 
2. 
Risks/Benefits .................................................................................................................... 62 
Risks Associated with Shigella Infection ............................................................................................................. 62 
Risks Associated with Transmission of Challenge ............................................................................................... 62 
Risks Associated with Antibiotics ........................................................................................................................ 62 
Risks Associated with Saliva Collection .............................................................................................................. 63 
Risks Associated with Nasal Swab: Risks occasionally associated with nasal swabs may include discomfort with 
procedure, nasal irritation ........................................................................................................................... 63 
Risks Associated with Venipuncture or IVs ......................................................................................................... 63 
Risks Associated with Wearable Data Collection: Risks may include feeling uncomfortable about wearing a 
wrist device in public. The wrist band may also cause some minor wrist irritation. .................................. 63 
Risks with Isolation .............................................................................................................................................. 63 
Risks Associated with Laboratory Results ........................................................................................................... 63 
Risks Associated with Collection of Stool ........................................................................................................... 63 
Risks Associated with Post-Infectious Irritable Bowel Syndrome (PI-IBS) ......................................................... 63 
Risk of Breach of Confidentiality ......................................................................................................................... 64 
Risk of SARS-CoV-2 Exposure and Risk Mitigation ........................................................................................... 64 
3. 
Risk Mitigation Strategies ................................................................................................. 65 
4. 
Benefits ............................................................................................................................... 66 
XX. INFORMED CONSENT ................................................................................................ 66 
1. 
Safeguards for Vulnerable Participants: ......................................................................... 67 
2. 
Recruitment ........................................................................................................................ 67 
XXI. COMPENSATION ........................................................................................................ 67 
1. 
Compensation for Participation ....................................................................................... 67 
2. 
Compensation for Investigators....................................................................................... 68 

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3. 
Fair and Equitable Selection of Participants .................................................................. 68 
XXII. PRIVACY AND CONFIDENTIALITY ......................................................................... 68 
1. 
Storage of Data and Samples ........................................................................................... 68 
2. 
Provisions Protecting Privacy and Confidentiality ........................................................ 68 
XXIII. PROTOCOL REVIEW PROCESS ............................................................................ 68 
XXIV. PUBLICATION POLICY ........................................................................................... 68 
XXV. CONFLICT OF INTEREST POLICY ......................................................................... 69 
XXVI. REFERENCES .......................................................................................................... 70 
 
 
List of Tables 
 
TABLE 1: DESCRIPTION OF STUDY GROUPS ....................................................................................................... 24 
TABLE 2: REFERENCE RANGES AND ADVERSE EVENT CODING FOR VITAL SIGNS PARAMETERS ..... 44 
TABLE 3: REFERENCE RANGES AND ADVERSE EVENT CODING FOR CLINICAL HEMATOLOGY 
PARAMETERS ........................................................................................................................................... 46 
TABLE 4: REFERENCE RANGES AND ADVERSE EVENT CODING FOR BLOOD CHEMISTRY 
PARAMETERS ........................................................................................................................................... 46 
TABLE 5: CHALLENGE INPATIENT PHASE SHIGELLA INFECTION SOLICITED ADVERSE 
EVENT/ENDPOINT .................................................................................................................................... 52 
TABLE 6: CLASSIFICATION OF INTENSITY OF DIARRHEA DURING THE OUTPATIENT PHASE .............. 52 
TABLE 7: STUDY CONTACTS FOR REPORTING SERIOUS ADVERSE EVENTS ............................................. 54 
TABLE 8: SAE INFORMATION TO BE REPORTED TO THE SPONSOR .............................................................. 54 
TABLE 9: SCHEDULE OF PROCEDURES FOR SUBJECTS RECEIVING ONE CHALLENGE ........................... 73 
TABLE 10: SCHEDULE OF PROCEDURES FOR SUBJECTS RECEIVING TWO CHALLENGES ...................... 75 
 
List of Figures 
 
FIGURE 1: STUDY SCHEMA ................................................................................................................................ 25 
FIGURE 2: PRODUCTION CELL BANK FOR 2457T S. FLEXNERI 2A ........................................................................... 41 
FIGURE 3: SHIGELLA SONNEI STRAIN 53G ........................................................................................................... 41 
 
 
 
 
 

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II. ABBREVIATIONS 
Abbreviation 
Definition 
ADi 
Antigen Discovery, Inc 
AE 
Adverse event 
AESI 
Adverse event of special interest 
ALT 
Alanine aminotransferase 
ALS 
Antibody from lymphocytes supernatant 
ASC 
Antigen stimulated cells  
BMI 
Body mass index 
BP 
Blood pressure 
BPR 
Batch Production Record 
BUN 
Blood urea nitrogen 
CBC 
Complete blood count 
CFU 
Colony forming units 
cGMP 
Current Good Manufacturing Practice 
CHIM 
Controlled Human Infection Model  
CIR 
Center for Immunization Research, Johns Hopkins School of Public 
Health 
CLIA 
Clinical Laboratory Improvement Amendments of 1988 
CR 
Congo red 
eCRF 
Electronic Case Report Form  
CRF 
Case Report Form 
CVIA 
Center for Vaccine Innovation and Access 
DMP 
Data management plan 
DoD 
Department of Defense 
DSMB 
Data safety monitoring board 
EDC 
Electronic data capture 
ELISA 
Enzyme-linked immunosorbent assay 
GCP 
Good Clinical Practice 
h 
Hours  
H2 blockers 
H2 receptor agonists 
HBsAg 
Hepatitis B surface antigen 
HCV 
Hepatitis C virus 
HIV-1 
Human Immunodeficiency Virus 1 
HLA 
Human leukocyte antigen 
IATA 
International Air Transport Association 
ICF 
Informed Consent Form 

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ICH 
International Conference on Harmonisation 
IgG; IgA 
Immunoglobulin G; Immunoglobulin A 
IP 
Investigational Product 
IRB 
Institutional Review Board 
IV 
Intravenous 
JHSPH 
Johns Hopkins Bloomberg School of Public Health 
JHU 
Johns Hopkins University 
JHH 
Johns Hopkins Hospital 
LPS 
Lipopolysaccharide 
MCB 
Master cell bank 
mL 
Milliliter 
mm 
Millimeter 
MOP 
Manual of procedures 
NMRC 
Naval Medical Research Center 
ORS 
Oral rehydration solution 
PCB 
Production cell bank 
PBF 
Pilot Bioproduction Facility (WRAIR) 
PBMC 
Peripheral blood mononuclear cells 
PBS 
Phosphate buffered saline 
PE 
Physical Exam 
PI 
Principal Investigator 
PI-IBS 
Post-infectious irritable bowel syndrome 
PSRT 
Protocol Safety Review Team 
PVT 
Psychomotor vigilance test 
QA 
Quality assurance 
QC 
Quality control 
RNA 
Ribonucleic acid 
SAE 
Serious adverse event 
SAP 
Statistical analysis plan 
SBA 
Serum bactericidal antibody 
SMP 
Safety Monitoring Plan 
SOP 
Standard Operating Procedure  
SSP 
Study Specific Procedure 
SWI 
Sterile water for injection 
TD 
Travelers’ diarrhea 
TMF 
Trial Master File 
TSA 
Trypticase soy agar 

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TSB 
Tryptic soy broth 
VS 
Vital signs 
WBC 
White blood cell 
WRAIR 
Walter Reed Army Institute of Research 
 
 
 
 

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III. CLINICAL PROTOCOL SYNOPSIS 
Protocol Title 
Controlled Human Infection Model Challenge/Rechallenge: Shigella flexneri 2a 
and S. sonnei cross-protective antigens discovery in healthy adults in the United 
States 
 
Name of the 
investigational 
products 
x 
Shigella flexneri 2a 2457T (lot 1617) 
x 
Shigella sonnei 53G (lot 1794) 
Study Site 
Johns Hopkins Center for Immunization Research 
Principal 
Investigator 
Kawsar Talaat, M.D. 
Study Hypothesis  Challenge with one species of Shigella (Shigella flexneri 2a or Shigella sonnei) will 
induce some cross-protective immunity such that a subset of volunteers will be protected 
upon challenge with the second strain. 
Study Rationale  
The goal of this study is to better understand Shigella immunity. The study will compare 
the shigellosis rate in subjects previously challenged with a heterologous Shigella 
serotype to the attack rate in naïve subjects. Subsequent evaluations of the immune 
response will seek to identify cross-protective Shigella antigens which could improve 
upon the current requirement for serotype-specific O-antigens from the most common 
Shigella species. 
 
Study Population 
Up to 54 healthy participants (male or non-pregnant, non-nursing female), aged 18-50 
years (all inclusive) from the Baltimore, MD-Washington, DC region and adjacent 
regions. 
 
Study Design 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
This is a single-center controlled human infection model (CHIM) study designed to 
assess the cross-protective efficacy and markers of protection after challenge and 
heterologous rechallenge with Shigella strains of different serotypes in naïve healthy 
adult volunteers. 
 
As shown in Table S1, a group of up to 13 naïve participants will be challenged with 
1500 colony forming units (cfu) of S. sonnei 53G (Group 1A1). At least 3 months later, 
11 of these volunteers, who are willing and eligible for rechallenge, along with up to 8 
newly recruited naïve participants, will be challenged with 1500 cfu of S. flexneri 2a 
(Group 1B1). Additionally, nine newly recruited naïve participants will be challenged 
with 1500 cfu of S. sonnei 53G (Group 1A2) and later 8 of these volunteers willing and 
eligible for rechallenge will be rechallenged with 1500 cfu of S. flexneri 2a (Group 
1B2). An additional group of 13 naive participants will be challenged with 1500 cfu of 
S. flexneri 2a (Group 2A1). At least 3 months later, up to 11 of those volunteers from 
Group 2A1 willing and eligible to proceed to another challenge, along with 5 or more 
naïve participants, will be challenged with 1500 cfu of S. sonnei 53G (Group 2B1), and 
another group of up to 6 naive participants will be challenged with 1500 cfu of S. 
flexneri 2a (Group 2A2). Each volunteer will be followed for 6 months from the time of 
their last challenge. 
 
 
Table S1. Clinical trial study design. 
 

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Group/cohort N 
Sub-
group 
N naïve  
N veterans 
Challenge strain 
(1500 cfu/dose) 
1A 
13 
1 
13 
0 
S. sonnei 53G 
 
9 
2 
9 
0 
S. sonnei 53G 
1B 
19 
1 
Up to 8 
11 from 1A1 
S. flexneri 2a 2457T 
 
8 
2 
0 
8 from 1A2 
S. flexneri 2a 2457T 
2A 
13 
1 
13 
0 
S. flexneri 2a 2457T 
 
Up to 6 
2 
Up to 6 
0 
S. flexneri 2a 2457T 
2B 
Up to 16 
1 
5 
Up to 11 from 
2A1 
S. sonnei 53G 
 
Breakdown of study cohorts/groups: Each cohort is defined by which challenge agent is 
administered first (e.g., Cohort 1, S. sonnei, Cohort 2, S. flexneri 2a).  The groups refer 
to the admission; Group A is the first admission/challenge per cohort, B, the second 
admission/rechallenge. Within the A groups, all volunteers are naïve; within each of the 
B groups, up to 20 volunteers will be veterans from the A groups and up to10 volunteers 
will be naïve. The numbers after the letter e.g. 1A1, denote subgroups that occurred at 
different admissions. 
 
Clinical Monitoring 
After challenge, participants will be monitored for shigellosis by daily medical checks, 
vital sign determinations, and grading and weighing of all stools. Monitoring for fecal 
shedding of the challenge strain will occur daily after challenge by standard 
microbiological and molecular assays (detailed description given in separate study 
specific procedure [SSP]). If a participant is unable to provide a stool sample by 1300 
hours, s/he will be asked to obtain a rectal swab. Swabs will be used starting the day 
after challenge. 
 
Mucosal and systemic antibody responses to the challenge strains will be assessed 
throughout the study, as shown in the schedule of events. 
 
Antibiotic Treatment 
Routine antibiotic treatment with antibiotics to which the strains are susceptible will 
commence for all participants approximately 120 hours post-challenge. All participants 
will be treated with trimethoprim 160 mg / sulfamethoxazole 800 mg orally twice daily 
for five days, or ciprofloxacin (500 mg orally twice daily for three days), or, 
alternatively, ampicillin (500 mg orally four times daily for three days). 
Early antibiotic treatment after challenge may commence when any of the following 
criteria are identified and a study physician considers it to be warranted: 
 
- 
When volunteers meet the primary endpoint (clinical definition of 
shigellosis) 
  OR 
- 
Oral temperature ≥ 39°C 
  OR 

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- 
Any other reason warranting the early treatment in the physician’s 
opinion 
If, because of illness, a participant is unable to take oral antibiotics, intravenous 
antibiotics (e.g., ciprofloxacin) may be given at an appropriate dose based on weight and 
clinical status. 
 
Discharge Criteria 
All participants will be discharged from the inpatient unit when they are well, have taken 
at least 2 doses of antibiotics, and have had at least two consecutive stool cultures 
negative for the challenge strain. 
 
Post-Challenge Follow-Up 
Participants will return for post-challenge assessments at 15, 29, 57, and 90 days post-
challenge. A follow-up visit approximately 6 months after challenge will be performed 
to inquire about the occurrence of any new chronic health conditions (such as reactive 
arthritis, conjunctivitis, urethritis), serious health events, or hospitalizations, and to 
complete a functional bowel survey to assess for new-onset chronic bowel problems. 
Study Procedures 
Screening and Consenting 
Initial screening will occur under a separately approved general screening protocol over 
the course of 1 – 2 visits. Baseline laboratories will be drawn, including sera for the 
analysis of the S. flexneri- and S. sonnei-IgG levels within 90 days of enrollment. Only 
participants who have low titers to the challenge strains, as determined by O-antigen-
specific antibodies in the serum, will be eligible for enrollment. 
 
Eligible and willing participants will be invited to participate in an informed consent 
process where study information is presented in writing (ICF), view an audio/visual 
study brief, complete a comprehension assessment, and consent to study participation by 
signing the informed consent document.  
 
Inpatient Admission and Challenge 
Each group will be admitted to the inpatient unit on Day -2 or -1. The day after 
admission, Day 1, each participant will be challenged with approximately 1500 cfu of 
the fully virulent Shigella strain after a ≥90-minute fast and immediately following 
ingestion of a bicarbonate solution. Volunteers will fast for ≥90 additional minutes after 
challenge. 
 
Participants will be selected for the rechallenge randomly, if they are eligible and willing 
to continue with the study. The duration of the active study period for participants who 
receive one dose of challenge is approximately 6 months; for participants who receive 
both challenges, the active study period is about 9 months from enrollment, not 
including up to 60 days for screening. 
 
Follow-Up Phase 
Safety assessments and blood for immunology will be performed at all study visits. The 
timeframe for the collection of AEs begins at the time of Investigational Product 
administration through 28 days after receipt of the challenge strain. 
Additionally, Adverse Events of Special Interest (AESI) and Serious Adverse Events 
(SAEs) will be collected for approximately 180 days from last challenge. 
 
Outcome Adjudication Committee 

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To obtain an unbiased determination of the efficacy outcomes, a blinded independent 
outcome adjudication committee will evaluate challenge outcome data after completion 
of the inpatient phase of the study for all cohorts. 
 
Primary 
Objective 
To evaluate the cross-species protection conferred by a re-challenge with a Shigella 
species of a different serotype. 
 
Primary 
Endpoint 
The primary endpoint for outcomes following initial challenge and heterologous re-
challenge is shigellosis [1], defined as: 
 
- 
severe diarrhea 
OR 
- 
moderate diarrhea with fever OR with one or more moderate 
constitutional or enteric symptom OR ≥2 episodes of vomiting in a 24-
hour period 
OR 
- 
dysentery: ≥2 loose stools with gross blood (hemoccult positive) in 24 
hours AND fever OR ≥1 moderate constitutional/enteric symptom OR 
≥2 episodes of vomiting in 24 hours 
Secondary 
Objective 
To determine effects of previous challenge when re-challenged with Shigella of a 
different serotype on stool output and clinical symptoms.  
 
Secondary 
Endpoints 
Secondary endpoints for this study following initial challenge and heterologous re-
challenge are: 
- 
Maximum 24-hour stool output 
- 
Percent of participants with severe diarrhea 
- 
Percent of participants with diarrhea of any severity 
- 
Total weight of grade 3-5 stools per participant 
- 
Percent of participants with nausea, vomiting, anorexia, abdominal pain/cramps 
rated as moderate to severe 
- 
Percent of participants who meet the definition of dysentery 
- 
Mean/median time to onset of diarrhea 
- 
Number of participants with more severe diarrhea (defined as ≥ 10 loose [grade 3-5] 
stools within 24h or ≥1000 gr loose [grade 3-5] stools within 24h) 
- 
Number of participants with fever 
- 
Shigella clinical severity score post-challenge 
- 
Number of cfu of the challenge strain per gram of stool 
 
Note: Period of data collection for all secondary endpoints is during the inpatient period.  
Exploratory 
Objectives 
1. To determine IgG and IgA responses to Shigella species specific lipopolysaccharide 
(LPS) upon challenge or rechallenge. 
2. To utilize novel analyses of immune responses, including antigen arrays, to identify 
potentially protective immune responses in individuals protected from shigellosis 
upon challenge or rechallenge. 
3. To identify potential protective antigens on the challenge organisms. 
4. To confirm that the serum IgG to the O-antigen is a correlate of protection for 
shigellosis. 
5. To assist in the development of international standards against Shigella antigens. 
6. To measure mucosal and systemic immune responses to experimental infection. 

Protocol v4.2 
16 JAN 2023 
17
7. To obtain and archive samples for future proteomics, inflammatory marker, 
microbiome, and/or transcriptomics and systems biology efforts based on the 
recently published consensus schedule and events table [2]. 
8. To evaluate the cognitive and sleep impact of acute diarrhea using psychomotor 
vigilance testing (PVT) and actigraphy. 
9. To evaluate serum bactericidal antibody (SBA) titers against S. flexneri 2a 2457T 
and S. sonnei 53G (responders defined as ≥4-fold increase in SBA titer at designated 
timepoints post-initial challenge and heterologous re-challenge.  
10. To evaluate the ability of wearable-collected data to predict clinical and/or 
microbiological endpoints. 
Definitions 
Stool will be graded based on a standard stool grading scale as follows [3]: 
 
Grade 1 = Fully formed (normal)  
 
Grade 2 = Soft (normal)  
 
Grade 3 = Thick liquid (diarrheal)  
 
Grade 4 = Opaque watery (diarrheal)  
 
Grade 5 = Rice-water (diarrheal) 
 
x 
More severe diarrhea: ≥10 loose (grade 3-5) stools within 24 hours (h) or ≥1000 
grams loose (grade 3-5) stools within any 24-hour window 
x 
Severe diarrhea: ≥6 loose (grade 3-5) stools within 24h or >800 grams loose 
(grade 3-5) stools within any 24-hour window 
x 
Moderate diarrhea: 4 to 5 loose (grade 3-5) stools within 24h or 401-800 grams 
loose (grade 3-5) stools within any 24-hour window 
x 
Mild diarrhea: ≥2 loose (grade 3-5) stools weighing ≥200 grams within any 48-
hour window or 1 loose stool weighing ≥300 grams not meeting the definition 
for moderate or severe 
x 
Fever: measured oral temperature ≥ 38°C confirmed within about 20 minutes 
x 
Dysentery: ≥2 loose stools (grade 3-5) with gross blood (hemoccult positive) in 
24 hours AND fever OR ≥1 moderate constitutional/enteric symptom OR ≥2 
episodes of vomiting in 24 hours 
x 
Constitutional/enteric symptom: nausea, abdominal cramps/pain, myalgia, 
arthralgia, malaise 
x 
Shigellosis disease severity will be assessed as previously described [4]. 
Inclusion Criteria  
1. Healthy adults, male or female, aged 18 to 50 years (all inclusive) at the time of 
enrollment. 
2. General good health, without clinically significant medical history, physical 
examination findings, or clinical laboratory abnormalities per judgment of PI. 
3. Willingness to participate in the study after all aspects of the protocol have been 
explained and written informed consent obtained. 
4. Completion of a training session and demonstrated comprehension of the protocol 
procedures and knowledge of Shigella-associated illness by passing a written 
examination (70% passing score). 
5. Availability for the study duration, including all planned follow-up visits. 
6. Female participants must have a negative pregnancy test at screening and prior to 
each challenge. 
7. Female participants must agree to avoid pregnancy for 29 days following the last 
challenge dose by use of an efficacious hormonal or barrier method of birth control 
during the study. Abstinence is acceptable. Female participants unable to bear 
children must have this documented (e.g., tubal ligation or hysterectomy). 

Protocol v4.2 
16 JAN 2023 
18
8. Willingness to refrain from participation in a study of another investigational agent 
for 90 days following the last challenge dose. 
Exclusion 
Criteria  
1. Presence of a significant medical or psychiatric condition that in the opinion of the 
investigator precludes participation in the study. Some medical conditions that are 
adequately treated and stable would not preclude entry into the study. 
2. Clinically significant abnormalities in screening on physical exam or screening 
laboratory results as determined by PI or PI in consultation with the research 
monitor and Sponsor. 
3. Recent receipt of another investigational product (within 30 days before 
enrollment). 
4. Positive enzyme-linked immunosorbent assay (ELISA) and confirmatory tests for 
human immunodeficiency virus (HIV). 
5. Positive hepatitis C (HCV) ELISA and confirmatory test (e.g., HCV ribonucleic 
acid (RNA)). 
6. Positive hepatitis B virus surface antigen (HBsAg) by ELISA. 
7. Use of any medication that affects immune function (e.g., corticosteroids and 
others) within 30 days preceding the first challenge or planned use during the active 
study period (topical and ophthalmologic steroids are allowable). 
8. Evidence of impaired immune function or immune compromise (known 
immunodeficiency syndrome; either congenital, acquired, or iatrogenic; active 
autoimmune disease; repeated serious infections without known cause). 
9. IgA deficiency (serum IgA < 7 mg/dL or below the limit of detection of the assay). 
10. Positive blood test for HLA-B27. 
11. Personal or family history of an inflammatory arthritis. 
12. Currently pregnant or nursing. 
13. Have household contacts who are < 2 years old or > 80 years old or infirm or 
immunocompromised 
14. Employment as a health care worker with direct patient care, in a daycare center (for 
children or the elderly), or direct food handler; includes individuals who work directly 
with food in commercial establishments 
15. Evidence of current alcohol or drug dependence, or history of dependence in the last 
6 months. 
16. Recent vaccination (including licensed vaccines) or receipt of an investigational 
product (within 30 days before challenge through 90 days following the last 
challenge dose). Annual influenza vaccine, an emergency authorized or licensed 
COVID-19 vaccine, or a TdaP or Td booster may be administered beyond 14 days 
before and after each challenge. 
17. Treatment with immunoglobulins or blood products within 3 months of challenge. 
18. Current or prior history of inflammatory bowel disease or irritable bowel syndrome 
or abnormal stool pattern (>3/day or <3/week, or loose or liquid stools). 
19. Chronic use of anti-diarrheal, anti-constipation, or antacid therapy; or use of these 
medications in the 7 days prior to challenge. 
20. Use of proton pump inhibitors or H2 blockers (H2-receptor antagonists) within 48 
hours prior to challenge. 
21. Use of antibiotics within 7 days prior to challenge. 
22. Known allergy to any 2 of the following antibiotics: ciprofloxacin, trimethoprim-
sulfamethoxazole, or a penicillin  
23. Symptoms of travelers’ diarrhea (TD) associated with travel to countries where 
Shigella or other enteric infections are endemic (most of the developing world) 
within 3 years prior to challenge OR planned travel to endemic countries during the 
active study period. 

Protocol v4.2 
16 JAN 2023 
19
24. History of shigellosis, Shigella vaccination or challenge, or a laboratory worker 
with known exposure to Shigella within the last 5 years. 
25. Serum IgG titer > 2500 to either Shigella flexneri 2a or Shigella sonnei LPS. 
26. Any other criteria which, in the investigator’s opinion, would compromise the 
ability of the volunteer to participate in the study, the safety of the study, or the 
results of the study. 
Safety 
Monitoring  
The research monitor and PI will review any safety concerns. 
 
Research Monitor: The research monitor will function as an independent safety advocate 
for participants. This individual will have the following responsibilities: 
x 
Evaluate ongoing safety data and make recommendations in order to ensure 
participants’ safety as required 
x 
Be available for consultation by the clinical investigative team through the 
period of the clinical study in which there is an interaction with human 
participants 
x 
Be available to review all SAEs and other unanticipated problems involving risk 
to participants 
x 
Provide clinical advice, in accordance with the study protocol, on the clinical 
management of participants. This advice may include, but is not limited to: 
• 
Decisions on “borderline” eligibility for enrollment 
• 
Confirmation and discussion of treatment decisions for difficult clinical 
situations 
• 
Must document all clinical decisions including date, time, and signature 
• 
Must communicate all decisions to the study PI and other study 
investigators, which must be stored with participant source documents 
 
All SAEs must be reported, whether or not regarded as possibly attributable to the test 
articles or antibiotic. SAE reports will be provided to the Sponsor, research monitor, and 
IRB(s). The investigator must report SAEs within one calendar day of becoming aware 
of the event by telephone, fax, or e-mail (if appropriate) to the study contact for 
reporting SAEs as described in the protocol. 
 
Study Duration 
The expected duration of the trial is approximately 24 months. 
 
Duration of study participation is approximately 11 months per volunteer from first 
contact, screening, to completion of the last outpatient follow-up visit, 180 days post last 
challenge. Participants will complete 1 – 2 screening visits, a consenting visit, 1 or 2 
inpatient stays of approximately 10 days each, and 5 (if only 1 challenge) or 8 (if 2 
challenges) post-challenge outpatient follow-up visits. 
 
Screening and consenting will occur in the 60 days prior to enrollment. 
Sample Size 
 
A total of up to 54 participants will be challenged in this study (up to 110 will be 
consented to ensure a full complement of volunteers). Based on Table S1, participants 
are delineated as follows: 
x 
S. flexneri 2a 2457T single challenge (n=16; 8 (2 in Group 2A1 and 6 in Group 
2A2) not selected for rechallenge, 8 rechallenge controls in Group 1B1) 
Rechallenge controls are naïve subjects enrolled alongside veteran subjects 
(receiving their second challenge) and both receive the same challenge agent to 
allow a fair comparison for the attack rate across the group.  

Protocol v4.2 
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20
x 
S. sonnei 53G single challenge (n=up to 8; 3 (2 in Group 1A1 and 1 in Group 
1A2) not selected for rechallenge, 5 rechallenge controls in Group 2B1) 
x 
S. flexneri 2a 2457T challenge followed by S. sonnei 53G challenge (n=11 
veterans in Group 2B1) 
x 
S. sonnei 53G challenge followed by S. flexneri 2a 2457T challenge (n=19 
veterans in Groups 1B1 and 1B2) 
 
Presuming a shigellosis rate of 70% in naïve participants, a sample size of 16 
participants will yield 95% confidence intervals (asymptotic estimates) of 48-92%, while 
a sample size of 8 participants will yield 95% confidence interval (asymptotic estimates) 
of 38-100%. Presuming an ability to pool naïve participants, a sample size of 19 
participants in the previously S. sonnei 53G challenged arm and 16 participants in the S. 
flexneri 2457T naïve arm provides a >80% power to detect a shigellosis risk difference 
of 46% presuming a ≥70% attack rate in Shigella naïve participants, while a sample size 
of 11 participants in the previously S. flexneri  2457T challenged arm and 8 participants 
in the S. sonnei 53G naïve arm provides a >80% power to detect a shigellosis risk 
difference of 60% presuming a ≥70% attack rate in Shigella naïve participants (based on 
a Pearson’s chi-squared test for proportional differences and a 2-sided alpha=0.05). 
Statistical 
methods and 
analysis 
All AEs will be summarized and compared between groups. Safety data, including AEs, 
stool information, specified vital signs, and laboratory tests, will be listed by study 
subject. 
 
Descriptive statistics (n, mean, standard deviation, median and ranges for continuous 
variables, percentages for categorical variables) will be compiled for each group. The 
attack rate of shigellosis will be estimated along with 95% confidence intervals (both 
asymptotic estimates and exact) for each study group. Attack rates between Shigella 
naïve participants and previously challenged participants will be compared using a 
Pearson’s chi-squared or Fisher’s exact test, as appropriate. 
 
Serologic and cellular assessments of the immune response will be conducted to assess 
for potentially protective responses in the volunteers resistant to the development of 
shigellosis. Comparisons across groups of single immune parameters reported as 
continuous variables (e.g., reciprocal log10 titers) will be made utilizing t-tests and one-
way analysis of variance, Wilcoxon rank-sum, or Kruskal-Wallis H tests, as appropriate. 
Comparisons of responses within groups over time will be made utilizing appropriate 
paired analyses. Immune parameters that are nominal in nature (e.g., responder status) 
will be compared using chi-squared or exact tests, as appropriate. Additionally, 
comparisons of immune response profiles, assessing multiple immune parameters 
simultaneously, will be explored utilizing multivariable regression models, principal 
component analyses, and/or other multivariable methods, as appropriate. 
 
All hypothesis testing will be conducted using a 2-sided alpha=0.05. 

 
 
Protocol v4.2 
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21
IV. BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE 
ͳǤ ǦShigella
Shigella, a major cause of bacillary dysentery, is an enteric bacterium that can cause 
inflammatory diarrhea. Shigella has long been recognized as a cause of moderate and severe 
diarrhea and dysentery, with a high incidence among children residing in low- and middle-income 
countries [5, 6]. Recent studies highlight the continued burden of disease and provide data for 
burden estimates [7-11]. In addition to an estimated 212,438 annual deaths in people of all ages 
[12], children under 5 years of age experience nearly 75 million cases of shigellosis each year, 
leading to stunting and wasting [12-14]. Shigella also poses a significant enteric disease threat to 
deploying military forces and international travelers [15]. 
 
ʹǤ 
This is a human challenge study designed to assess the cross-protective efficacy and markers of 
protection after challenge and heterologous rechallenge with different Shigella strains in naïve 
volunteers. The study would look for novel, cross-protective Shigella antigens which could 
improve upon the current requirement for serotype-specific O-antigens from the most common 
species, S. flexneri 2a, 3a, and 6, and S. sonnei. Such antigens could be used as carrier proteins in 
third-generation glycoconjugate vaccines or act as stand-alone subunit vaccines to help broaden 
vaccine coverage. 
 
͵Ǥ 
The Shigella Controlled Human Infection Model (CHIM) has been in use to evaluate the efficacy 
of investigational Shigella vaccines since the studies of Shaughnessy and colleagues in 1946 
among prison inmates in Joliet, Illinois [16]. Since then, CHIM trials evaluating Shigella vaccine 
candidates have been conducted at several sites in the United States, mostly with S. flexneri 2a 
(strain 2457T) [17-19] and S. sonnei (strain 53G) [20, 21], but also with wild-type and toxin-
minus mutants of S. dysenteriae type 1 [19, 20, 22-27]. Additionally, trials with 53G have been 
conducted in Thailand [28, 29]. Previous field and CHIMs studies have indicated that infection 
with specific Shigella serotypes will induce protection against of a similar “O” serotype but the 
antigens associated with cross-serotype protection are not as well understood (Levine MM et al. 
2007).  
 
 
S. flexneriʹʹͶͷ͹
S. flexneri 2a has had the greatest range of doses tested for strain 2457T; ranging from 100 to 1 x 
108 colony forming units (cfu) [19].  
 
Experimental S. flexneri 2a challenge was first attempted in the late 1960s in an effort to develop 
a safe and reproducible model for rapid and accurate evaluation of the efficacy of candidate 
Shigella vaccines in humans [13]. The 2457T S. flexneri 2a strain that will be utilized in this 
study as a challenge strain is a well-characterized Shigella strain manufactured under current 
Good Manufacturing Practice (cGMP) conditions at the WRAIR Pilot BioProduction Facility in 
Silver Spring, Maryland. This strain was originally isolated from a clinically ill patient in Japan in 
the early 1950s and has most frequently been given to volunteers participating in IND studies 
evaluating treatment or preventive intervention for the control and clinical management of 
shigellosis [19]. The 2457T strain is also covered by its own manufacturing master file registered 
with the FDA (BB-MF-3408). In all, more than 460 people have been challenged with the strain 
in the published literature, and of the Shigella strains used in challenge studies, this is the least 
likely to cause dysentery. The 2457T strain has been used in 15 published trials at doses ranging 
from 100 to 108 cfu [19], and additional challenge studies carried out by the Center for 

 
 
Protocol v4.2 
16 JAN 2023 
22
Immunization Research (CIR) in recent years that have yet to be published. Diarrhea attack rates 
are somewhat dose-related under 1400 cfu, but plateau at higher doses to approximately 60 to 
80% [19]. The dose of 1500 cfu was confirmed in multiple studies at the University of Maryland 
and the CIR [23, 30, 31].  In total, the CIR has conducted 5 inpatient challenge studies with the 
2457T strain since 2001, with the most recent being in 2015. 1500 cfu is now the standard dose in 
challenge studies. This strain is susceptible to the antibiotics that will be used for treatment 
(ciprofloxacin, trimethoprim-sulfamethoxazole, ampicillin). 
 
 
S. sonneiͷ͵

The second most commonly studied strain (S. sonnei 53G) has historically been used in low 
inoculum doses, ranging between 400 and 500 organisms, with relatively low diarrhea and 
dysentery attack rates. This strain has been utilized at a number of sites in the US (in 296 adults) 
and in Thailand (in 56 adults), including at the CIR by Kirkpatrick and colleagues (Unpublished). 
Recently, a lyophilized preparation of this strain has been manufactured under cGMP conditions 
at the WRAIR Pilot BioProduction Facility (PBF) to simplify the inoculum preparation process. 
A dose-ranging study was conducted at Cincinnati Children’s Hospital under an IND for the 
challenge strain to better characterize and evaluate this challenge strain. The challenge model 
demonstrated an acceptable safety profile, with no signs of early onset or particularly aggressive 
disease. Among subjects receiving the highest dose (1760 cfu), 70% developed moderate to 
severe diarrhea, 50% had dysentery, and 40% had fever. A dose in the range of 1500 to 2000 cfu 
was identified as the optimal dose for a reproducible diarrhea attack rate of ≥60% [32].  This 
strain is susceptible to the antibiotics that will be used for treatment (ciprofloxacin, trimethoprim-
sulfamethoxazole, ampicillin). 
 
ͶǤ 
Multiple studies have been carried out over the last 40 years to refine the challenge models and 
improve reproducibility. Most recently, a convening of the experts from the sites that conduct 
Shigella challenge studies was hosted by the Bill and Melinda Gates Foundation, resulting in a 
series of consensus guidelines on the conduct of these studies [1, 3]. This study is in compliance 
with those guidelines. 
V. STUDY HYPOTHESIS 
Challenge with one species of Shigella (Shigella flexneri 2a or Shigella sonnei) will induce some 
cross-protective immunity such that a subset of volunteers will be protected upon challenge with the 
second strain. 
VI. OBJECTIVES 
ͳǤ ǣ
To evaluate the cross-species protection conferred by a re-challenge with a Shigella species of 
a different serotype. 
 
ʹǤ ǣ
To determine effects of previous challenge when re-challenged with Shigella of a different 
serotype on stool output and clinical symptoms. 
 
͵Ǥ ǣ
1. To determine IgG and IgA responses to Shigella species-specific lipopolysaccharide (LPS) 
upon challenge or rechallenge. 

 
 
Protocol v4.2 
16 JAN 2023 
23
2. To utilize novel analyses of immune responses, including antigen arrays, to identify 
protective immune responses in individuals protected from shigellosis upon challenge or 
rechallenge. 
3. To identify potential protective antigens on the challenge organisms. 
4. To confirm that the serum IgG to the O-antigen is a correlate of protection for shigellosis. 
5. To assist in the development of international standards against Shigella antigens. 
6. To measure mucosal and systemic immune responses to experimental infection. 
7. To obtain and archive samples for future proteomics, inflammatory marker, microbiome, 
and/or transcriptomics and systems biology efforts based on the recently published consensus 
schedule and events table [2]. 
8. To evaluate the cognitive and sleep impact of acute diarrhea using psychomotor vigilance 
testing (PVT) and actigraphy. 
9. To evaluate serum bactericidal antibody (SBA) titers against S. flexneri 2a 2457T and S. 
sonnei 53G (responders defined as ≥4-fold increase in SBA titer at designated timepoints 
post-initial challenge and heterologous re-challenge.  
10. To evaluate the ability of wearable-collected data to predict clinical and/or microbiological 
endpoints. 
VII. 
ENDPOINTS 
ͳǤ 
The primary endpoint for outcomes following initial challenge and heterologous re-challenge is 
the onset of shigellosis, defined as: 
 
x 
Severe diarrhea: ≥6 loose (grade 3-5) stools within 24h or >800gr loose (grade 3-5) stools 
within 24h 
OR 
x 
Moderate diarrhea (4 to 5 loose stools within 24 hr or 401-800gr loose (grade 3-5) stools 
within 24h) with fever OR with one or more moderate constitutional or enteric symptom OR 
≥2 episodes of vomiting in a 24-hour period 
 
OR 
 
x 
Dysentery: ≥2 loose stools with gross blood (hemoccult positive) in 24 hours AND fever OR 
≥1 moderate constitutional/enteric symptom OR ≥2 episodes of vomiting in 24 hours 
 
Fever: oral temperature ≥ 38°C confirmed within about 20 min 
 
Constitutional/Enteric Symptom: nausea, abdominal cramps/pain, myalgia, arthralgia, malaise 
 
ʹǤ 
Secondary endpoints for this study following initial challenge and heterologous re-challenge are: 
- 
Maximum 24-hour stool output 
- 
Percent of participants with severe diarrhea 
- 
Percent of participants with diarrhea of any severity 
- 
Total weight of grade 3 – 5 stools per participant 
- 
Percent of participants with nausea, vomiting, anorexia, abdominal pain/cramps rated as 
moderate to severe 
- 
Percent of participants who meet the definition of dysentery 

 
 
Protocol v4.2 
16 JAN 2023 
24
- 
Mean/median time to onset of diarrhea 
- 
Number of participants with more severe diarrhea (defined as ≥10 loose [grade 3-5] 
stools within 24h or ≥1000 gr loose [grade 3-5] stools within 24h) 
- 
Number of participants with fever 
- 
Shigella clinical severity score post-challenge 
- 
Number of cfu of the challenge strain per gram of stool 
 
Note: Period of data collection for all secondary endpoints is during the inpatient period.  
VIII. 
STUDY DESIGN 
ͳǤ 
This study will be conducted in the isolation unit at the CIR Inpatient Unit, located at the Johns 
Hopkins Bayview Medical Center. This study will be a single-center human challenge study designed 
to assess the cross-protective efficacy and markers of protection after challenge and heterologous 
rechallenge with different Shigella strains in naïve healthy adult volunteers (see Figure 1: Study 
Schema). 
 
Description of study groups as shown in table 1 below:   
Cohort 1, group 1A1: 13 participants will be challenged with 1500 cfu of S. sonnei 53G. At least 
3 months later, group 1B1: 11 of the participants from group 1A1 (who are willing and eligible to 
proceed to another challenge), along with up to 8 naïve participants (group 1B1), will be 
challenged with 1500 cfu of S. flexneri 2a. Up to  9 newly recruited naïve participants will be 
challenged with 1500 cfu of S. sonnei 53G (Group 1A2) and later 8 of these volunteers willing 
and eligible for rechallenge will be rechallenged with 1500 cfu of S. flexneri 2a (group 1B2).  
Cohort 2, group 2A1: 13 naive participants will be challenged with 1500 cfu of S. flexneri 2a. At 
least 3 months later, group 2B1: up to 11 of those volunteers from Group 2A1 (who are willing 
and eligible to proceed to another challenge), along with 5 or more naïve participants (group 
2B1), will be challenged with 1500 cfu of S. sonnei 53G, and another group of up to 6 naive 
participants will be challenged with 1500 cfu of S. flexneri 2a (group 2A2). Each volunteer will 
be followed for 6 months from the time of their last challenge.  
 
 
Table 1: Description of Study Groups 
Group/
cohort 
N 
Sub-
group 
N naïve  
N veterans 
Challenge strain 
(1500 cfu/dose) 
1A 
13 
1 
13 
0 
S. sonnei 53G 
 
9 
2 
9 
0 
S. sonnei 53G 
1B 
19 
1 
Up to 8 
11 from 1A1 
S. flexneri 2a 2457T 
 
8 
2 
0 
8 from 1A2 
S. flexneri 2a 2457T 
2A 
13 
1 
13 
0 
S. flexneri 2a 2457T 
 
Up to 6 
2 
Up to 6 
0 
S. flexneri 2a 2457T 
2B 
Up to 16 
1 
5 
Up to 11 from 2A1 
S. sonnei 53G 
. 
Breakdown of study cohorts/groups: Each cohort is defined by which challenge agent is administered first 
(e.g., Cohort 1, S. sonnei, Cohort 2, S. flexneri 2a).  The groups refer to the admission; Group A is the 
first admission/challenge per cohort, B, the second admission/rechallenge. Within the A groups, all 
volunteers are naïve; within each of the B groups, up to 20 volunteers will be veterans from the A group 
and up to 10 volunteers will be naïve. The numbers after the letter e.g. 1A1, denote subgroups that 
occurred at different admissions. 
 

 
 
Protocol v4.2 
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Figure 1: Study Schema 
 
 
The CIR Inpatient Unit is a 30-bed dorm-like facility for clinical trials. Each group will be 
admitted together to the inpatient unit for up to 10 days or longer. The day after admission, each 
participant will be challenged with approximately 1500 cfu of the fully virulent Shigella strain 
indicated after a 90-minute fast and immediately following ingestion of a bicarbonate solution. 
Volunteers will fast for 90 additional minutes after challenge. 
 
The duration of the active study period for participants who receive one dose of challenge is 
approximately 6 months; for participants who receive both challenges, the active study period is 
about 9 months from enrollment, not including up to 60 days during the screening period. 
 
ʹǤ 
This is a Shigella controlled human infection study with a goal to assess the cross-protective 
efficacy and markers of protection after challenge and heterologous rechallenge with different 
Shigella strains in naïve healthy, 18 to 50 year-old, non-pregnant volunteers. 
 
The study will be divided into 3 parts; about 2/3 of the participants will repeat parts 2 and 3: 
1. 
Screening (usually 2 visits; some visits will be performed under a separate, IRB-approved 
 
screening protocol) (see Section XI for details) 
2. 
Admission and challenge phase 
3. 
Follow-up phase (outpatient visits at Study Days 15, 29, 57, 90, and 180) 
 
All potential volunteers will be screened for titers to S. flexneri and S. sonnei IgG. Those who 
meet eligibility criteria will be admitted to the inpatient unit and receive the challenge strain 
(Investigational Product). Blinding will not be employed in this study as all subjects in each 
cohort will be receiving the same challenge strain.  However, the adjudication of clinical 
outcomes will be blinded to both strain and cohort. Participants are considered enrolled once they 
receive the investigational challenge. Volunteers will be monitored by clinical staff 24/7 while on 
the inpatient unit. They will be assessed for AEs and symptoms of Shigella illness daily post-
challenge. All stools will be collected and assessed for grade, weight, and blood daily post-
challenge until participants are eligible for discharge. Stool samples for culture and research 
54 enrolled
Group 1A:
22 challenged with S. sonnei
Subgroup 1:
13 challenged with 
S. sonnei
Subgroup 2: 
9 challenged with 
S. sonnei
Group 1B: 
27 challenged with 
S. flexneri 2a
Subgroup 1:
11 veterans from Group 1A1 and 8 
veterans from group 1A2
Subgroup 2:
8 naive challenged with
S. flexneri 2a
Group 2A: 19 challenged with S. 
flexneri 2a 
Group 2B:
11 veterans from Group 2A  
challenged with S. sonnei
Up to 5 naive challenged with S. 
sonnei
Up to 6 naive challenged with S 
flexneri

 
 
Protocol v4.2 
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26
testing will be collected daily; blood, nasal swabs, and saliva will be collected at specified time 
points as per the schedule of events. Volunteers will be eligible for discharge once they have 
received at least 2 doses of antibiotics, clinical symptoms are resolved or resolving, and they have 
at least 2 consecutive negative stools for Shigella. 
 
Trial Schedule: 
See Table 9: Schedule of Procedures for Subjects Receiving One Challenge and Table 10: 
Schedule of Procedures for Subjects Receiving Two Challenges. 
IX. STUDY POPULATION 
ͳǤ 
Participants will be recruited from the Baltimore, MD-Washington, DC region and surrounding 
areas via advertisements and word of mouth and screened at the CIR. A sufficient number will be 
screened to provide for the number planned to challenge, with sufficient alternates to allow for 
last-minute dropouts. The total target is 65 participants enrolled. Once challenged, subjects will 
not be replaced. 
 
ʹǤ ǣ
Participants must meet all criteria listed below to be included in the study: 
1. Healthy adults, male or female, aged 18 to 50 years (all inclusive) at the time of 
enrollment. 
2. General good health, without clinically significant medical history, physical examination 
findings, or clinical laboratory abnormalities per judgment of PI. 
3. Willingness to participate in the study after all aspects of the protocol have been explained 
and written informed consent obtained. 
4. Completion of a training session and demonstrated comprehension of the protocol 
procedures and knowledge of Shigella-associated illness by passing a written examination 
(70% passing score). 
5. Availability for the study duration, including all planned follow-up visits. 
6. Female participants must have a negative pregnancy test at screening and prior to each 
challenge.  
7. Female participants must agree to avoid pregnancy for 29 days following the last 
challenge dose by use of an efficacious hormonal or barrier method of birth control during 
the study. Abstinence is acceptable. Female participants unable to bear children must have 
this documented (e.g., tubal ligation or hysterectomy). 
8. Willingness to refrain from participation in a study of another investigational agent for 90 
days following the last challenge dose. 
 
͵Ǥ ǣ
Participants meeting any of the exclusion criteria listed below must be excluded from 
participating in the trial: 
1. Presence of a significant medical or psychiatric condition that in the opinion of the 
investigator precludes participation in the study. Some medical conditions that are 
adequately treated and stable would not preclude entry into the study. 
2. Clinically significant abnormalities in screening on physical exam or screening laboratory 
results as determined by PI or PI in consultation with the research monitor and Sponsor. 
3. Recent receipt of another investigational product (within 30 days before enrollment). 
4. Positive enzyme-linked immunosorbent assay (ELISA) and confirmatory tests for human 
immunodeficiency virus (HIV). 
5. Positive hepatitis C Virus (HCV) ELISA and confirmatory test (e.g., HCV RNA). 
6. Positive hepatitis B virus surface antigen (HBsAg) by ELISA. 

 
 
Protocol v4.2 
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7. Use of any medication that affects immune function (e.g., corticosteroids and others) 
within 30 days preceding the first challenge or planned use during the active study period 
(topical and ophthalmologic steroids are allowable).  
8. Evidence of impaired immune function or immune compromise (known 
immunodeficiency syndrome; either congenital, acquired, or iatrogenic; active 
autoimmune disease; repeated serious infections without known cause). 
9. IgA deficiency (serum IgA < 7 mg/dL or below the limit of detection of the assay). 
10. Positive blood test for HLA-B27. 
11. Personal or family history of an inflammatory arthritis. 
12. Currently pregnant or nursing. 
13. Have household contacts who are < 2 years old or > 80 years old or infirm or 
immunocompromised. 
14. Employment as a health care worker with direct patient care, in a daycare center (for 
children or the elderly), or direct food handler; includes individuals who work directly 
with food in commercial establishments 
15. Evidence of current alcohol or drug dependence, or history of dependence in the last 6 
months. 
16. Recent vaccination (including licensed vaccines) or receipt of an investigational product 
(within 30 days before challenge through 90 days following the last challenge dose). 
Annual influenza vaccine, an emergency authorized or licensed COVID-19 vaccine, or a 
TdaP or Td booster may be administered beyond 14 days before and after each challenge. 
17. Treatment with immunoglobulins or blood products within 3 months of challenge. 
18. Current or prior history of inflammatory bowel disease or irritable bowel syndrome or 
abnormal stool pattern (>3/day or <3/week, or loose or liquid stools). 
19. Chronic use of anti-diarrheal, anti-constipation, or antacid therapy; or use of these 
medications in the 7 days prior to challenge. 
20. Use of proton pump inhibitors or H2 blockers (H2-receptor antagonists) within 48 hours 
prior to challenge. 
21. Use of antibiotics within 7 days prior to challenge. 
22. Known allergy to any 2 of the following antibiotics: ciprofloxacin, trimethoprim-
sulfamethoxazole, or a penicillin. 
23. Symptoms of travelers’ diarrhea (TD) associated with travel to countries where Shigella or 
other enteric infections are endemic (most of the developing world) within 3 years prior to 
challenge OR planned travel to endemic countries during the active study period. 
24. History of shigellosis, Shigella vaccination or challenge, or a laboratory worker with 
known exposure to Shigella within the last 5 years. 
25. Serum IgG titer > 2500 to either Shigella flexneri 2a or Shigella sonnei LPS. 
26. Any other criteria which, in the investigator’s opinion, would compromise the ability of 
the volunteer to participate in the study, the safety of the study, or the results of the study. 
 
ͶǤ ͳʹ
1. Must continue to meet inclusion criteria above. 
2. Must not meet any of exclusion criteria 1-21 and 24 (IgA, HLA-B27, HIV, HCV, and 
HBsAg will not be repeated). 
3. All study subjects with Serious Adverse Events (SAEs) related to the primary challenge 
will be excluded from a repeat (second) challenge. 
 
ͷǤ 
A medical history will be obtained directly from each participant. Medical records will not be 
requested unless there is a need to clarify a question in the participant’s medical history or if the 
participant had an intercurrent illness or injury requiring medical care during the study. Medical 
records will not be requested without a signed medical release and the informed consent of the 

 
 
Protocol v4.2 
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28
participant. Informed consent for the medical release will be obtained from each participant at the 
beginning of the study. 
X. STUDY PROCEDURES 
ͳǤ 
The CIR will use a screening protocol approved by the Johns Hopkins School of Public Health 
(JHSPH) Institutional Review Board (IRB) in recruiting participants for this study. The screening 
protocol is entitled “Screening of adult volunteers for eligibility to participate in clinical studies 
evaluating investigational vaccines, antimicrobial agents, or disease prevention measures or the 
pathogenesis of infectious agents” CIR 200, JHSPH IRB 00010083. Participants will be made 
aware that the screening process may take several visits to complete. Once consented, using this 
screening protocol, a medical history/exam and a series of clinical laboratory tests will be 
completed to rule out occult illness and pregnancy (as per Section XI, 8). Participants who have ≤ 
2 mild (grade 1) non-hematologic abnormalities may be included if the PI determines that their 
participation will not present undue risk to the participant. Participants with > 2 mild 
abnormalities may be included in the study at the discretion of the PI with concurrence from the 
research monitor. Participants with clinical laboratory abnormalities >grade 1 will not participate 
in this clinical trial. 
 
To ensure comprehension of the study, all participants will have to pass a written examination 
before inclusion in the study. Participants who meet all inclusion criteria and none of the 
exclusion criteria, pass the comprehension test, and sign the study Informed Consent Form (ICF) 
may be eligible for the study.  Informed consent is an ongoing process which includes the ICF. 
Participants will watch an audio/visual presentation of the study after which they will be given an 
opportunity to ask any questions. Each prospective participant will be given the written, IRB-
approved informed consent, allowed ample time to read the consent, allowed to ask questions 
about the study, have his/her questions answered, and given time to decide if he/she would like to 
participate in the study. To document participants’ understanding of informed consent, 
immediately before the consent is signed, the person obtaining consent will administer a 
comprehension test. Incorrect answers will be discussed with participants to reinforce the consent. 
A final acceptable test score is 70% or more answered correctly. Participants who fail the 
comprehension test on the first attempt may retake the comprehension test on the same day, or 
they may come back on a separate visit to retake the test. Participants failing after two attempts 
are not eligible for study enrollment. No coercion or influence is allowed in obtaining 
participants’ consent. Before participants participate in the study, consent forms will be signed 
and dated by participants as well as by the PI or designee. Participants will receive copies of the 
signed consent prior to participation. As part of the consent process, participants will also be 
asked to read and sign additional IRB-approved forms including but not limited to, a Medical 
Records/Lab Results Release, alternate information form, and inpatient guidelines, with an 
opportunity to ask questions, if relevant.  
 
As part of the pre-inpatient period, eligible subjects may be asked to perform a 5-minute baseline 
psychomotor vigilance test (PVT). Additionally, subjects will be provided a smartphone and 
actigraphy devices to be worn starting up to 21 days prior to challenge through the first post-
discharge follow-up visit (study day 15 ± 2 days). 
 
ʹǤ 
Ǥ 

Eligible participants will be admitted to the CIR inpatient facility on the Johns Hopkins 
Bayview campus on Day -2 or -1. Once challenged (Day 1), they will be monitored daily 
while in the inpatient unit for the development of shigellosis, including general, 

 
 
Protocol v4.2 
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29
gastrointestinal, and systemic signs and symptoms, have medical conditions reviewed, and 
adverse effects recorded. This will include examination by a study physician/nurse 
practitioner/physician assistant and solicitation of adverse events (as per list in Section XIII, 
5). Additionally, participants will be examined for symptoms and signs of dehydration, 
including thirst, dizziness on standing, decreased skin turgor, and dryness of mucous 
membranes. Vital signs will be recorded three times daily (four times on challenge day), and 
more often when participants are ill.  If participants develop moderate or severe diarrhea, 
postural blood pressure and pulse will be measured as necessary for clinical management 
according to the judgment of the clinician. 
 
A clinician will conduct a daily medical interview and focused physical exam to assess health 
status and treat as indicated. All stools will be collected for weighing and grading [3]. 
Following Shigella challenge, up to 3 stool samples will be collected daily for culture as per 
study specific procedure (SSP) starting the day after challenge. If a participant is unable to 
provide a stool sample by 1300 hours, s/he will be asked to obtain up to 3 rectal swabs. 
Swabs will be used starting the day after challenge. 
 
Ǥ 

An exploratory assessment of the bidirectional relationships between sleep, cognitive 
performance, and shigellosis will be conducted with the use of continuous wrist actigraphy 
and PVT monitoring. Heart rate and skin temperature may also be collected by the wearable 
devices. These outcomes will not be utilized as part of the regulatory safety, immunogenicity, 
or efficacy evaluations of the study products; they are exploratory in nature and will not be 
retained in the regulatory file. Missed PVTs will not be considered protocol deviations. 
Management of symptoms associated with Shigella challenge or other illness will have 
priority over completion of PVTs. 
 
During screening, participants will be issued two wrist-worn devices and a smartphone for 
collection of actigraphy, heart rate, and skin temperature data. (Each device will collect 
different information. There will be no GPS information gathered by the devices). Subjects 
will wear these devices starting up to 21 days prior to challenge through the first post-
discharge follow-up visit (study day 15 ± 2 days). Subjects will be asked to complete a brief 
survey about how they are feeling each day using the smartphone. These surveys will not be 
used to determine medical history or clinical endpoints; they are exploratory in nature and 
will not be retained in the regulatory file. Five-minute PVT tests will be performed by each 
subject up to three times a day. Analyses will explore symptom presence/severity, while 
adjusting for sleep cycles and other confounding variables, as appropriate. Additional 
exploratory assessments will include evaluations of immune activation and cognitive 
performance by use of systems biology parameters. These data will be collected for 
additional exploratory assessments beyond the scope of this protocol. 
 
Ǥ 

Participants passing grade 3-5 stools post-challenge will be offered Oral Rehydration 
Solution (ORS) or Gatorade to prevent dehydration, at the same volume as their stool output. 
For documentation purposes of concomitant medications, ORS will not be considered a 
concomitant medication; however, intravenous (IV) fluids will. 
 
A participant may be administered IV fluids (clinician discretion) for the following reasons:  
• 
Participant experiences abrupt onset of diarrhea defined by passage of an initial 
loose/liquid stool of > 300 grams or passage of > 400 grams of loose/liquid stools over 2 
hours. 
• 
Participant becomes hypovolemic. 

 
 
Protocol v4.2 
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• 
It is determined necessary by the study physician due to diarrhea with nausea/vomiting 
and unable to drink enough to keep up with output, or other reason. 
 
Hypovolemia is a significant decrease in blood volume, characterized by either: 
• 
Orthostatic hypotension, confirmed systolic blood pressure (BP) < 90 mmHg and 
associated symptoms. 
• 
Significant lightheadedness on standing with a confirmed postural change in BP or pulse. 
Postural vital signs will be measured laying and 2 minutes after standing. A significant 
change will be any of the following: decrease in systolic BP of > 20 mmHg or diastolic 
BP of > 10 mmHg, or increase in pulse of > 30 beats/min. 
 
Ǥ 

Treatment for severe nausea or vomiting may be needed. Participants who experience severe 
nausea or vomiting may be given oral or intravenous (IV) ondansetron (Zofran). 
Ǥ 
	
Acetaminophen, ibuprofen, or naproxen may be used to treat fever or pain. 
Ǥ 

 In addition to above medications other medications may be given to participants as needed 
for symptom control (e.g., heartburn), or if a medical problem arises (allergic reaction, 
insomnia). The most commonly utilized medication, including the indication are as follows: 
 
Medication 
Dosage 
Indication 
 
Ondansetron oral dissolving 
tablet 
4 mg, 8 mg once, can be repeated 
if not better in 30 minutes 
Nausea/vomiting 
 
Ondansetron IV 
0.15 mg/kg IV x 1 
Vomiting 
Max 16 mg/dose 
Acetaminophen 
500- 1000 mg q 4-6 hours 
Pain, fever 
Max 4000 mg/day 
Ibuprofen 
400-800 mg q 6-8 hours 
Pain, fever 
Max 2400 mg/day 
Naproxen sodium 
220-440 mg q 12 hours 
Pain, fever 
Max 660 mg/day 
Naproxen 
250-500 mg bid 
Pain, fever 
Max 1000 mg/day 
Benadryl 
25-50 mg oral q 6 hrs prn 
Itching, insomnia 
 
Maalox 
As directed 
Heartburn, indigestion 
 
Tums 
As directed 
Heartburn, indigestion 
 
 
͵Ǥ 
Routine discharge is scheduled for approximately study day 9. Two consecutive negative stool 
cultures for Shigella are required before discharge (can be collected on the same study day). If the 
participant has not completed antibiotics, then the remaining doses of antibiotic will be given to 
the participant for self-administration. Vital signs will be collected. 
 

 
 
Protocol v4.2 
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31
ͶǤ 
Early discharge is permitted in cases where early antibiotic treatment has been initiated. The 
participant needs 2 consecutive stool cultures negative for Shigella and to have taken two doses 
of antibiotic with resolved or resolving clinical symptoms before discharge. Remaining doses of 
antibiotic will be given to the participant for self-administration. Participants discharged before 
study day 8 will return on day 8 and provide the requisite samples (stool, blood) as delineated in 
the Schedule of Procedures. 
 
ͷǤ 
Participants will return to clinic 15, 29, 57, and 90 days after each challenge. They will also have 
a 6-month follow-up after the final challenge. Clinic visits during follow-up will include vital 
signs assessment, clinical checks, including concomitant medications and AEs, and sample 
collection for immunogenicity and exploratory outcome evaluations as per the schedule of events. 
 
͸Ǥ Ȁ
Only concomitant medications approved by the study physician will be used during the study. 
Participants needing to take unapproved or excluded medications will not be eligible for 
enrollment in this study. Any medication given during the trial (e.g., acetaminophen or 
antibiotics) will be documented in the participant’s study chart and on the appropriate page of the 
electronic Case Report Forms (eCRFs). Approved medications that were being taken prior to, as 
well as during, the course of the trial will also be documented in this manner. 
XI. STUDY SCHEDULE 
ͳǤ ǡǡǣ
Healthy participants will be recruited from the Baltimore, MD-Washington, DC region and 
surrounding areas (including other states) via IRB-approved advertisements, word of mouth, and 
contacting previous participants who have expressed interest in studies. Contact methods for this 
protocol include phone, electronic methods (including text and email), and mail. Study staff will 
respond to inquiries by phone, text, or email. Study staff will provide a brief, scripted synopsis 
of the research study that includes basic inclusion and exclusion criteria in compliance with 
the IRB-approved screening protocol. Participants who express interest in participating in the 
study will be asked to complete a telephone pre-screen to assess general health status and basic 
eligibility. Potential volunteers determined to be generally healthy and meeting basic 
eligibility requirements will be scheduled for an in-person screening. Participants will provide 
written informed consent prior to any study procedures. 
 
ʹǤ ʹͲͲǣ
The CIR will use a screening protocol approved by the Johns Hopkins School of Public Health 
(JHSPH) IRB in recruiting participants for this study. The screening protocol is entitled 
“Screening of adult volunteers for eligibility to participate in clinical studies evaluating 
investigational vaccines, antimicrobial agents, or disease prevention measures or the pathogenesis 
of infectious agents,” CIR 200, JHSPH IRB 00010083. Participants will be made aware that the 
screening process may take several visits to complete.  
 
The following procedures will occur: 
a) 
Discuss the study screening process and obtain Screening Informed Consent from the 
participant. 
b) 
Ensure that the participant has a clear understanding of the nature of the screening study by 
independently passing a brief comprehension examination. Then ensure the participant 
signed and received a copy of the ICF.  

 
 
Protocol v4.2 
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32
c) 
Elicit a complete medical history, including menstrual and contraceptive history and/or 
history of surgical sterility for female participants. 
d) 
Pregnancy prevention counseling for females focusing on the importance of preventing 
pregnancy during study participation, potential risks associated with pregnancy while 
taking investigational products, and effective contraceptive options. 
e) 
Vital signs will be collected (heart rate, respiratory rate, blood pressure, and temperature). 
f) 
Obtain blood for Shigella titers specific for S. flexneri 2a and S. sonnei. 
 
Additionally, the following activities may occur or be deferred to a subsequent screening visit: 
a) Administer a complete physical examination. 
b) Provide HIV pre-test counseling, including that the testing is voluntary but necessary for 
study participation, information about HIV testing, transmission and prevention, explanation 
of test results, post-test counseling, and result reporting. 
c) Obtain blood for a complete blood count (CBC) with differential, basic metabolic panel, 
ALT, AST, ABO blood typing, HLA-B27 antigen, IgA, Hepatitis B virus, Hepatitis C virus, 
and HIV testing. Obtain urine sample for toxicology testing, looking for the presence of 
amphetamines, barbiturates, opiates, phencyclidine, benzodiazepine, methadone, or cocaine 
metabolites. Screening CBC or chemistries may be repeated if there are potentially transient 
abnormalities or if there is concern for an error.  
d) Females will have a serum sample taken for E-HCG testing. 
e) Height and weight measurements. 
 
͵Ǥ Ǧǣ
Approximately 110 healthy, potentially eligible and willing participants will be invited back to 
complete study-specific screening to allow for participants who do not continue to meet study 
eligibility criteria or wish to continue in the study. The following procedures will occur during 
this visit: 
 
a) Participants will receive an overview of the study in an individual or group setting and an 
Informed Consent Form (ICF) to read on their own.  
b) Participants will be encouraged to ask questions, and then independently complete a quiz to 
evaluate comprehension of study procedures, requirements, and risks. They must 
independently take and pass with ൒70% understanding. The participant will be able to retake 
the test once if they do not pass the first time. Study staff will meet individually with the 
participant to review the completed comprehension assessment. Incorrect answers will be 
reviewed with the participant.  
c) Both the participant and staff member obtaining consent will sign, date, and time the ICF 
and a signed copy of the consent will be given to the participant.  
d) Participants will read and agree to comply with the Inpatient Unit Guidelines, Alternate 
Agreement, and Handwashing Best Practice. 
e) Volunteers will be asked to complete and sign a HIPAA medical record release form. 
f) Female volunteers will receive pregnancy prevention counseling. 
g) All participants will receive HIV pre-test counseling as described above, if not completed 
prior.  
h) Inclusion and exclusion criteria will be assessed. 
i) Demographics, medical history, including gender, date of birth, race, height, weight, and any 
allergies will be collected and/or reviewed. 
j) Vital signs will be collected (heart rate, blood pressure, temperature). 
k) Research samples will be collected as per schedule of events, if not already completed.  
l) Complete physical exam will be done.  
m) Functional bowel survey will be completed. 

 
 
Protocol v4.2 
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33
n)  All volunteers will be tested for COVID-19 upon admission to the inpatient unit. Depending 
on the level of COVID illness in the community and current CDC and JHU guidelines, 
volunteers may be asked to obtain a COVID-19 test 1-5 days prior to admission. Any 
volunteer who tests positive for COVID-19 will not be eligible for admission. 
o) As part of the pre-inpatient period, eligible subjects may be asked to perform a 5-minute 
baseline psychomotor vigilance test (PVT). Additionally, subjects will be provided a 
smartphone and actigraphy devices to be worn starting up to 21 days prior to challenge 
through the first post-discharge follow-up visit (study day 15 ± 2 days). 
 
Attempts will be made to inform participants of their screening laboratory results either in person 
or over the telephone. Participants with clinically significant abnormalities (as determined by the 
PI or designee) may be asked to have additional blood drawn. If the result(s) is confirmed, 
participants may be referred to their primary care physician. A copy of the laboratory results may 
be provided to the participant at his/her request. 
 
ͶǤ 
Participants will be considered enrolled in the study upon receipt of the Investigational Product. 
 
ͷǤ 
Ǥ 
ǦʹǦͳǡȋͺͻʹȌ
Up to 34 volunteers who have provided informed consent and meet all eligibility criteria will 
be invited and 32 participants will be admitted to the inpatient unit (each admission). This is 
to allow for some dropout of the volunteers due to ineligibility or reversal of consent. Up to 
30 participants each admission will be enrolled into the study and receive challenge as per 
Table 1. Volunteers who are admitted but not enrolled or do not receive challenge at that 
admission will be discharged and will not be followed. 
 
On arrival to the unit, the following will be performed: 
x 
Volunteer will be tested for COVID-19; volunteer must have a negative test on admission 
to be admitted to the inpatient unit. 
x 
Volunteer will be admitted to the unit. Personal items will be reviewed. Unallowed items 
will be stored until discharge.  
x 
Orientation to the unit. 
x 
Vital signs (blood pressure, temperature, and heart rate) at least twice. 
x 
Complete physical examination. 
x 
Assessment for eligibility (inclusion and exclusion criteria). 
x 
Concomitant medications will be reviewed. 
x 
Serum pregnancy testing for females; if results are not available prior to challenge, urine 
pregnancy testing will be performed on Day 1. 
x 
Blood will be collected for research, including immunological assays and serology.  
x 
Blood will be collected for baseline assessments: CBC with diff, BMP, ALT and AST. 
x 
Stool for qualitative and quantitative culture, and immunology (as per schedule of events 
and SSP). In the event a participant is unable to produce a stool on Day -2 or -1, at 
admission, stool may be collected any time on the day of admission or on Day 1, until 4 
hours after challenge. Participant's inability to produce a stool on Day -2 or -1/Day 1 will 
not be exclusionary for challenge. 
x 
Salivary IgA (as per schedule of events and SSP). 
x 
Nasal swab for respiratory metagenomics (as per schedule of events and SSP). 
x 
Pregnancy risk assessment and prevention counseling. 
 

 
 
Protocol v4.2 
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34
Ǥ 
ͳǡȋͻͲʹȌ
x 
On the day of challenge, participants will receive a light breakfast and then initiate at 
least a 90-minute fast. 
x 
Vital signs (BP, heart rate, and temperature) will be collected 4 times: prior to challenge, 
at least 30 minutes post challenge, and two additional times. 
x 
A focused physical exam (symptom focused) and evaluation to assure that there are no 
changes from admission and ensure the volunteer remains eligible for challenge. Solicited 
and unsolicited AEs will be assessed. 
x 
The alternates or a volunteer who does not continue to meet eligibility criteria will be 
discharged without challenge. Participants who never received a challenge dose will not 
be followed. Participants who previously received challenge will complete a Day 90 and 
Day 180 follow-up visit (as described in Section ix below). 
 
Ǥ 

x 
Approximately 1 minute prior to challenge (and within 2 minutes), participants will drink 
120 mL of bicarbonate buffer (buffer formulation: 13.35 grams of sodium bicarbonate in 
1000 mL of sterile water for irrigation). 
x 
For challenge, participants will drink a solution of virulent Shigella bacteria (as per Table 
1 and described in Section XII, 5), approximately 1500 cfu suspended in 30 mL of 
bicarbonate buffer (for 2457T strain) or saline (for 53G strain). 
x 
Participants will be observed for at least 30 minutes for signs of adverse reactions, at 
which point their vital signs will be obtained and recorded 30-60 minutes after challenge. 
x 
Participants will continue fasting for at least an additional 90 minutes post challenge. 
x 
Following receipt of the Shigella challenge inoculum, all stools will be collected for 
weighing, grading and assessment for blood.  
x 
If a participant develops grade 3 – 5 stools on Day 1, a sample of all grade 3 – 5 stools 
produced that day will be collected for possible culture. Up to 3 stools prior to the start of 
antibiotics should be sent for culture. 
 
Ǥ 
ͳȂͺǡȋͻͲǦͻ͹ʹȌ
x 
Vital signs (BP, heart rate, and temperature) at least 3 times a day; once in the morning, 
afternoon, and evening (approximately 6 hours apart). 
x 
Participants will have a focused PE daily, with the assessment of any new or changes in 
solicited and unsolicited AEs. 
x 
All medications will be provided by clinical staff and recorded on an inpatient medication 
administration form. 
x 
All stools will be weighed, graded and assessed for blood. 
x 
A minimum of one stool sample (no more than 3) will be collected daily for culture. If a 
volunteer is unable to provide a stool sample, rectal swabs will be obtained (as per SSP). 
x 
Stool will be processed as per SSP daily while inpatient. (If the participant does not 
produce a stool or enough stool on any day to obtain all samples, this will not be 
considered a protocol deviation.)  
x 
Blood and stool for immunological assessments will be collected as per the Schedule of 
Procedures.  
x 
Saliva IgA collection (as per schedule of events and SSP). 
x 
Nasal swab for respiratory metagenomics (as per schedule of events and SSP). 
x 
Antibiotics will be started on Study Day 6 (5 days post challenge) unless early treatment 
criteria are met. 
x 
Volunteers who have Grade 3 or greater adverse events may get safety labs (chemistries, 
CBC, liver functions tests) if a clinician feels that they are indicated.  

 
 
Protocol v4.2 
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35
x 
If subject develops low blood pressure despite IV hydration, the subject will be 
transferred to a higher level of care. 
 
v. 
ͻǡȋͻͺʹȌ 
Volunteers will be eligible for discharge when symptoms are resolved or are resolving, and 
they have had at least two consecutive stool cultures negative for the challenge strains (can be 
collected on the same study day) and have received at least 2 doses of antibiotics.  
 
Volunteers will have: 
x 
Focused PE prior to discharge 
x 
Review of solicited and unsolicited AEs 
x 
Receive morning dose of antibiotics (if course is not complete) 
x 
Receive their additional doses of antibiotics to take after discharge 
x 
At least one set of vital signs prior to discharge 
 
Volunteers who do not meet criteria for discharge will remain on the unit and continue: 
x 
Daily focused PE 
x 
Vital signs three times a day 
x 
Receive antibiotics (if not complete) 
x 
Review of solicited and unsolicited AEs  
x 
All stools will be weighed, graded and assessed for blood. 
x 
Up to 3 stool samples and/or rectal swabs will be collected for culture  
 
Ǥ 
ͳͷ	ǦȋΪȀǦʹȌȋͳͲͶʹȌ
x 
Interim medical history will be obtained 
x 
AEs and SAEs will be recorded, including assessment of relatedness to challenge or 
antibiotics and severity grade 
x 
Concomitant medications will be recorded 
x 
Vital signs (BP, HR, and temperature) 
x 
Focused PE if participant has any complaints/AEs 
x 
Blood for safety (CBC w/differential and basic metabolic panel, ALT/AST) 
x 
Blood and stool per Schedule of Procedures 
x 
Saliva IgA collection 
x 
Nasal swab for respiratory metagenomics (as per schedule of events and SSP) 
 
Ǥ 
ʹͻ	ǦȋΪȀǦͶȌȋͳͳͺʹȌ
x 
Interim medical history will be obtained 
x 
AEs and SAEs will be recorded, including assessment of relatedness to challenge or 
antibiotics and severity grade 
x 
Concomitant medications will be recorded 
x 
Vital signs (BP, HR, and temperature) 
x 
Focused PE if participant has any complaints/AEs 
x 
Pregnancy test (blood or urine; females only, to ensure that there was not an early 
pregnancy during the challenge period)  
x 
Blood and stool per Schedule of Procedures 
x 
Salivary IgA 
x 
Nasal swab for respiratory metagenomics (as per schedule of events and SSP) 
 
Ǥ 
ͷ͹	ǦȋΪȀǦͶȌȋͳͶ͸ʹȌ
x 
Participants will be questioned about their health status 

 
 
Protocol v4.2 
16 JAN 2023 
36
x 
Adverse Events of Special Interest (AESIs) and SAEs will be assessed 
x 
Vital signs (BP, HR, and temperature) 
x 
Focused PE if participant has any AESIs and SAEs 
x 
Blood and stool per Schedule of Procedures 
x 
Salivary IgA 
x 
Nasal swab for respiratory metagenomics (as per schedule of events and SSP) 
 
Ǥ 
ͻͲȋǦ͹ȀΪʹͺȌ
ǣȋͳͺͲʹȌ
Participants who receive a second challenge will have their day 90 and day 180 after the 
second challenge. Unless the second challenge is >120 days after the first, then participants 
will have a day 90 outpatient visit and a day -2 or -1 visit inpatient. 
 
Volunteers who receive the first challenge and are not selected for the second challenge will 
complete a Day 90 outpatient visit. This visit will include: 
 
x Interim medical history 
x AESIs and SAEs will be assessed 
x Vital signs (BP, HR, and temperature) 
x Focused PE if participant has any AESIs and SAEs 
x Blood for assays as per Schedule of Procedures 
x Nasal swab for respiratory metagenomics (as per schedule of events and SSP) 
 
Ǥ 
Ǧ
The volunteers in groups 1A or 2A will be assessed as to willingness and eligibility to 
participate in a second challenge (based on criteria Section IX, 4). Up to 21 volunteers who 
received the first challenge will be readmitted to the inpatient unit for second challenge, with 
the goal of challenging 20 (1 alternate will be admitted in case of loss of eligibility by a 
volunteer). If more than 21 volunteers from 1A or 2A are willing to be included in 1B or 2B 
21 subjects will be randomly selected from the eligible subjects. Due to isolation unit 
constraints there need to be an even number of males and females to fill the 6 same-gender 
shared rooms.  Additionally, up to 11 naïve volunteers will be admitted to the inpatient unit to 
receive challenge with the goal of challenging 10 participants. 
 
x 
Participants will be questioned about their health status. 
x 
AEs and concomitant medications will be assessed. 
x 
Volunteers will be admitted to the unit. Items from home will be reviewed and items not 
allowed on the unit will be stored in the locked storage area for participant safety. 
x 
Orientation to the unit. 
x 
Vital signs (blood pressure, temperature, and heart rate) at least two times (at least 6 
hours apart). 
x 
Complete physical examination upon admission. 
x 
Assessed for eligibility (inclusion and exclusion criteria). 
x 
Serum pregnancy testing for females; if results are not available prior to challenge, urine 
pregnancy testing will be performed on Day 1. 
x 
Blood will be collected for immunological assays and serology as per the Schedule of 
Procedures. 
x 
Blood will be collected for baseline assessments: CBC with diff, BMP, ALT and AST. 
x 
Stool for assays as per schedule of events and SSP. In the event a participant is unable to 
produce a stool on Day -2 or -1, at admission, stool may be collected at any time on the 

 
 
Protocol v4.2 
16 JAN 2023 
37
day of admission or on Day 1, until 4 hours after challenge. Participants’ inability to 
produce a stool on Day -2 or -1/Day 1 will not be exclusionary for challenge. 
x 
Salivary IgA (as per schedule of events and SSP). 
x 
Nasal swab for respiratory metagenomics (as per schedule of events and SSP). 
x 
Pregnancy risk assessment and prevention counseling. 
 
Prior to challenge, any alternate participants will be discharged from the unit prior to 
challenge. Those who had been previously challenged will receive follow-up as described 
below. The naïve alternates not previously challenged will not be followed-up after 
discharge. Participants who are readmitted and receive the second challenge will repeat the 
challenge schedule as described above in Section XI, 5. 
 
Ǥ 
ͳͺͲȀʹ͹Ͳ	Ǧ
All participants who receive at least one dose of challenge will complete a Day 180/270 
follow-up visit. This visit will be approximately 180 days after their last challenge dose and 
will include: 
 
x 
Functional bowel survey (e.g., Rome IV survey) 
x 
Participants will be questioned about their health status 
x 
AESIs and SAEs will be assessed 
x 
Focused PE if participant has any AESIs and SAEs 
x 
Blood for assays as per Schedule of Procedures 
x 
Nasal swab for respiratory metagenomics (as per schedule of events and SSP) 
 
͸Ǥ 
Routine antibiotic treatment will be given to all participants at about 5 days post-challenge. All 
participants will be treated with trimethoprim 160 mg / sulfamethoxazole 800 mg orally twice 
daily for five days, or ciprofloxacin (500 mg orally twice daily for three days), or, alternatively, 
ampicillin (500 mg) orally four times daily for three days.  
 
Early antibiotic treatment after challenge may commence when any of the following criteria are 
identified and a study physician considers it to be warranted: 
x 
When volunteer meets the primary endpoint (clinical definition of shigellosis) 
x 
Confirmed oral temperature ≥ 39°C (after volunteer has not had anything to drink for about 
20 minutes) 
x 
Any other reason warranting early treatment in the physician’s opinion 
 
If, because of illness, a participant is unable to take oral antibiotics, intravenous antibiotics may 
be given at an appropriate dose based on weight and clinical status. 
 
Once the volunteer has met the criteria for discharge, s/he will be released with the remaining 
antibiotic treatment to be taken at home. Volunteers taking any other non-antibiotic medication 
on a chronic basis may continue with that medication unless directed otherwise by the 
Investigator. These medications must be recorded on the appropriate eCRF. 
 
͹Ǥ 
Participants have the right to withdraw from the study at any time and for any reason withdrawal 
does not affect the responsibility of the investigator to treat the participant for study-related 
conditions. The investigator also has the right to withdraw the participants in the event of 
intercurrent illness, AEs, or for administrative/social reasons. 
 

 
 
Protocol v4.2 
16 JAN 2023 
38
An excessive number of withdrawals can affect the scientific validity of the study; therefore, 
unnecessary withdrawal should be avoided. Should withdrawals occur, efforts will be made to 
ensure participant safety and continued monitoring as thoroughly as possible. In case of 
participant withdrawal, for whatever reason, every attempt to complete a final trial evaluation 
must be made stating the reasons. Withdrawals due to non-attendance must be followed-up by the 
investigator to the extent possible to obtain the reason for non-attendance. 
 
Participants withdrawing from the study after receiving the Shigella challenge will receive 
antibiotics for outpatient treatment and will be educated on the importance of complying with 
treatment and good handwashing.  Attempts will be made to follow the participant for clearance 
of the challenge organism through collection of stool samples and for safety through study day 
29. 
 
ͺǤ 
Blood, stool, saliva and nasal swabs will be collected by trained personnel as per the Schedule of 
Procedures and as per SSPs. They will be labelled with the participant study numbers, processed 
and stored as per SSP on the inpatient unit or the clinic, and then packed and sent to either the 
CIR laboratory, WRAIR, Antigen Discovery Inc (ADi), IAVI or the University of Utah as per the 
SSPs and agreed upon shipment schedule. Research microbiology, including the preparation of 
the live inoculum and the culturing of stool specimens, will be carried out in the laboratory of the 
CIR in the JHSPH.  Immunologic and other research assays (as per study schedule) will be 
carried out at the Bacterial Diseases Branch at WRAIR, the JHSPH CIR Enterics lab, Antigen 
Discovery, Inc (ADi) and the University of Utah.   
 
Samples will be collected based on the Schedule of Procedures. Samples will be stored and 
distributed to various laboratories in compliance with DOT/IATA standards, guidance from the 
JHU Biosafety Review, and based on the SSP. Samples collected under this protocol will be used 
to conduct safety, microbiologic, and immunogenicity evaluations and other research labs (e.g., 
transcriptomics). Study-related samples will be collected and labeled according to the relevant 
SSP. 
 
The following sections provide a listing of procedures and tests to be performed in this protocol at 
designated time points. The maximum volume of blood to be drawn will not exceed 550 mL over 
any 8-week period, which healthy adults should regenerate within this period and which should 
not compromise the health of trial participants. 
 
Clinical Laboratory Tests: 
Standard clinical laboratory tests to determine eligibility of participants and for safety monitoring 
will be carried out at Johns Hopkins Hospital, Johns Hopkins Bayview Medical Center, or Quest 
Diagnostics in Baltimore City, MD:  
 
a) CBC with cell differential and platelets 
b) Basic metabolic panel  
c) Serum E-HCG 
d) ALT/AST 
e) HLA-B27 antigen testing 
f) IgA testing 
g) HIV assay (4rd generation) 
h) HBsAg ELISA 
i) HCV (3rd generation ELISA with immunoblot confirmation) 
j) Urine for drug toxicology screening 
k) ABO typing 

 
 
Protocol v4.2 
16 JAN 2023 
39
 
As per the SSP the JHSPH research lab will perform the following test for screening: 
S. flexneri 2a and S. sonnei 53G anti-LPS serum titer  
 
Urine E-HCG and fecal occult blood tests will be performed by the CIR under a CLIA waiver. 
 
ͻǤ ǣ
All microbiology of stool for Shigella, both quantitative and qualitative cultures, as well as 
possibly PCR, will be done at the JHSPH research lab. 
 
ͳͲǤ ǣ
Blood, stool, saliva, and nasal swabs will be collected for research labs to be completed as per 
SSP at either the SPH research lab or at the Department of Diarrheal Research Laboratory at 
WRAIR, ADi, IAVI/Scripps and the University of Utah and may include, but is not limited to: 
 
1. Blood for Anti-LPS serum IgG and IgA 
2. Blood for serum IgG and IgA against Shigella antigens 
3. Blood for total IgA and possibly IgG 
4. Blood for alpha-4 beta-7 positive and negative cells and antibody from lymphocyte 
supernatant (ALS), memory B-cell assays 
5. Stool for IgA against LPS and potentially Ipa proteins 
6. Stool for inflammatory markers and transcriptomics/proteomics 
7. Stool for microbiome 
8. Stool for qPCR 
9. Serology including bactericidal antibodies 
10. Blood for transcriptomics and proteomics (systems biology) 
11. Blood for antigen arrays (to be sent to ADi) 
12. Blood for identification of potential broadly protective antibodies (IAVI/Scripps) 
13. Saliva for salivary IgA against LPS and potentially Ipa proteins (to be processed in the 
laboratory of Chris Heaney, JHSPH) 
14. Nasal swab for respiratory tract metagenomics (to be sent to the University of Utah) 
 
ͳͳǤ 
Any unused blood, stool, saliva, or nasal swab specimens will be stored once the study is 
complete. Samples and data collected under this protocol may be used only for research purposes. 
No genetic testing will be performed on participant samples. These samples will not be sold or 
used to make commercial products. 
 
Access to research samples will be limited using locked freezers. Samples and data will be stored 
using codes assigned by the investigators or their designee(s). Data will be kept on password-
protected computers. Only investigators or their designee(s) will have access to the samples and 
data. 
 
Any other research or experimental treatments will be conducted under other protocols for which 
separate IRB review and approval will be obtained. 
 
During the study, samples may be sent to various research laboratories as per SSP. At the 
completion of the protocol (termination), samples that remain at JHSPH and data may be 
transferred to a repository (Johns Hopkins Bloomberg School of Public Health [JHSPH] IRB 
protocol R22.05.04.29.A2). Samples remaining at the Department of Defense will be maintained 
as per their SOP. 
 

 
 
Protocol v4.2 
16 JAN 2023 
40
In the future, other investigators (both at the Johns Hopkins University (JHU), the DOD and 
outside) may wish to study these samples and/or data. In that case, IRB approval will be sought 
prior to any sample sharing. Similarly, IRB approval will be obtained prior to sharing any clinical 
information, with or without patient identifiers that may be linked to samples being shared. The 
research use of stored, unlinked, or unidentified samples (for example, as a standard for 
immunological analyses) may be exempt from the need for prospective IRB review and approval. 
Exemption requests will be submitted in writing to the JHU IRB office and to the PATH 
Research Determination Committee, as applicable, which are authorized to determine whether a 
research activity is exempt. 
 
Any loss or unintentional destruction of samples, or data (for example, due to freezer 
malfunction, or missing case report forms [CRFs]) that compromises the scientific integrity of the 
study or poses a potential breach of confidentiality for the participant will be reported to the IRB. 
 
Samples will be stored only with the participant’s permission, which is a requirement for 
participation in the study. The participant may withdraw permission for future use of specimens 
at any time. If a participant withdraws his or her permission for future use of specimens, those 
specimens will be destroyed. In this case, the PI will ensure the destruction of all known 
remaining samples and will report this to both the participant and IRB. This decision may not 
affect the participant’s participation in future protocols. 
 
XII. 
STUDY INTERVENTION/INVESTIGATIONAL PRODUCT 
ͳǤ ShigellaȀ
 

S. flexneriʹʹͶͷ͹
The 2457T S. flexneri 2a strain is one of the strains that will be utilized in this study as a 
challenge. It is a well-characterized Shigella strain manufactured under current Good 
Manufacturing Practice conditions at the WRAIR Pilot BioProduction Facility (PBF) in Silver 
Spring, Maryland. 
 
S. flexneri 2a strain 2457T was originally isolated from a patient with diarrhea in Tokyo in the fall 
of 1954 [17]. The organism was transferred to the WRAIR where it was used to produce a 
Production Cell Bank of strain 2457T under current Good Manufacturing Practices (cGMP) at 
WRAIR PBF. Validation and characterization of the 2457T MCB (lot 0589, BPR-321-00) is 
described in the BB-MF 3408 – Type II Master File – Shigella Species for Vaccine and Challenge 
(Live, Oral) (Serial No. 11 and Serial No. 14) held by the Office of the Surgeon General of the 
Army. The 2457T strain has since been used in several studies at the CIR [19], most recently in 
2015 when 59 volunteers were challenged [26].  The Shigella flexneri 2a 2457T strain is 
susceptible to ciprofloxacin, trimethoprim-sulfamethoxazole and ampicillin. 
 
Strain dose-finding studies have been completed for the 2457T S. flexneri 2a, and it will be given 
as approximately 1,500 cfu of 2457T S. flexneri 2a with a 90-minute fast in this study. 
 
 
S. sonneiͷ͵
 
The 53G strain of S. sonnei was isolated in August 1954 in Tokyo, Japan, from a 5-yr-old male 
with diarrhea lasting for 2 days. The strain was described by Dupont et al in 1989 [21], 
confirming and establishing the low-dose virulence of 53G in humans. A master cell bank (MCB) 
(Lot 0593) was manufactured by the WRAIR PBF. The lyophilized 53G strain (Lot 1794) of S. 
sonnei was manufactured under current good manufacturing practices (cGMP) conditions at the 
WRAIR PBF. 

 
 
Protocol v4.2 
16 JAN 2023 
41
 
Strain dose-finding studies have been completed for the 53G S. sonnei strain under BB IND 
17015, held by PATH. The optimal dose and fasting times were determined from the outcome of 
a challenge development study conducted in Cincinnati by investigators at the Cincinnati 
Children’s Medical Center. In that study, various doses of lyophilized bacteria were given to a 
total of 56 adult volunteers. A dose of approximately 1500 cfu resulted in 70% of the volunteers 
having diarrhea [32]. Based on those data, participants will be given approximately 1500 cfu of 
53G S. sonnei with a 90-minute fast in this study. S. sonnei is sensitive to ciprofloxacin, 
trimethoprim-sulfamethoxazole, and ampicillin.  
 
The disease manifestation of infection with orally administered wild-type S. flexneri 2a strain and 
S. sonnei 53G strain will be evaluated.  
 
ʹǤ 
The challenge strains are stored as 1.0 ml aliquots (S. flexneri 2457T) with 1.8 x 108 cfu/mL or 
2.0 mL aliquots of S. sonnei 53G with 2 x109 cfu/mL. The tubes are in cryostorage tubes held at -
80qC +/- 10qC under controlled conditions at the WRAIR PBF. Labels of the strains are in 
Figures 2 and 3 below. 
 
Figure 2: Production Cell Bank for 2457T S. flexneri 2a 
Production Cell Bank for 2457T S. flexneri 2a 
BPR No.: BPR-322-01 
Lot No. : __1617___________ 
Contents: 1.0 ml 
Cautions: For Manufacturing Use Only; Viable 
organism 
Date of Mfg: 27 Jan 10 
Manufactured By: WRAIR, Silver Spring, MD 20910 
 
 
 
Figure 3: Shigella sonnei strain 53G 
Shigella sonnei strain 53G 
BPR No.: BPR-1094-00 
Lot No. : ___1794__________ 
Contents: 2.0 ml 
Cautions: New Drug – Limited by Federal (or United 
States) law to investigational use 
Date of Mfg: 25 Feb 13 
Manufactured By: WRAIR, Silver Spring, MD 20910 
 
͵Ǥ ǡ
 
 
S. flexneriʹʹͶͷ͹
The Shigella flexneri 2a 2457T challenge vials are stored at -80°C ± 10°C for short-term storage 
and in liquid nitrogen for long-term storage. The challenge strain will be transferred on dry ice from 
the WRAIR PBF to the CIR Enterics Research Laboratory at JHSPH, utilizing a temperature 
monitoring device, logged in and stored at -80°C ± 10°C in a locked and temperature-monitored 
freezer.  Any use of these vials will be done under the supervision of the CIR Enterics Research 
Laboratory, JHSPH and tracked in an accountability log.  Any vials remaining at the end of the 
study will be disposed of (via autoclaving) or returned to WRAIR for use in non-clinical research 
studies at the direction of study Sponsor and after all IP monitoring is completed. 
 

 
 
Protocol v4.2 
16 JAN 2023 
42
Colony forming units (cfu) are not routinely monitored during frozen storage because the master 
cell bank is subcultured twice during inoculum preparation, and the total number of surviving 
organisms in the original frozen ampule is not critical. Nonetheless, strain stability is confirmed 
by subculture on tryptic soy broth (TSB)-containing Congo red dye since virulent Shigella bind 
this dye and grow as red colonies. In addition, the identity of isolated colonies used as a source of 
the inoculum for volunteers is confirmed as S. flexneri 2a by slide agglutination in type 2 
antiserum. 
 
 
S. sonneiͷ͵

The S. sonnei 53G vials are stored at -80°C (±10°C) freezer at the WRAIR PBF. Prior to 
administration, the requisite number of vials will be transferred to the research lab at the CIR 
Enterics Research Laboratory at JHSPH, logged in and stored at -80°C ± 10°C in a locked and 
temperature-monitored freezer. Any use of these vials will be done under the supervision of the 
CIR Enterics Research Laboratory, JHSPH and tracked in an accountability log. Any vials 
remaining at the end of the study will be disposed of (via autoclaving) or returned to WRAIR for 
use in non-clinical research studies at the direction of study Sponsor and after all IP monitoring is 
completed. 
 
ͶǤ 
The details of challenge strain preparation will be in the SSP.  The challenge strains will be 
prepared by an experienced laboratory scientist under the supervision of an Investigational Drug 
Service Pharmacist. 
 
S. flexneriʹʹͶͷ͹
For the 2457T S. flexneri 2a strain, fresh, plate grown organisms will be used for challenge 
inocula. Approximately 48 hours before challenge, a vial of the cGMP Master Cell Bank will be 
thawed. The GMP cell bank is plated onto 5 plates containing trypticase soy agar (TSA), to which 
Congo red (CR) dye (0.01%) has been added.  The plates are streaked for isolation to yield well-
separated colonies and then incubated at 37°C r 1o C for 22 ± 2 hours. After incubation, six 
small, smooth, CR-positive colonies are tested for agglutination with commercial S. flexneri Type 
2 antiserum (Denka Seiken Ltd., Japan). If these colonies give strong and rapid agglutination 
reactions, 10 well-isolated CR-positive colonies are picked and used to prepare a bacterial 
suspension in 3 mL of phosphate buffered saline (PBS).  This suspension is used to inoculate 
multiple TSA plates to yield confluent growth (~6 plates for 30 volunteers, plus 2-3 extra plates 
as back-up in case of individual plate contamination).  After growth for 22 hours at 37°C r 1o C, 
the TSA plates are visually screened for purity, growth is re-checked for agglutination in 
antiserum and cells are harvested in sterile PBS (pH 7.4). For this step, 10 mL of PBS is added to 
each TSA plate and a sterile cotton swab is used to gently loosen the bacterial growth from the 
surface of the TSA plate. The bacterial suspension from each plate is collected with a sterile 
pipet, pooled and diluted in PBS to the target dose (1.5x103 CFU/ml) based on target OD600 
values. The diluted preparation is administered to challenge study volunteers within 4 hours of 
preparation. 
 
 
S. sonneiͷ͵

The day of challenge, vials will be removed from the freezer and placed on ice to thaw for 30 
minutes. Then 2 mL of cold (2-8 ˚C) sterile water for injection (SWI) are added to each vial. To 
ensure complete rehydration of the challenge strain along with homogeneous mixing of the vial’s 
contents, the vials remain on ice for another 15 minutes with intermittent swirling of the 
suspension. Vials will then be combined and diluted in cold, 0.9% USP sterile saline to arrive at 
the desired concentration. The diluted challenge material is kept on ice until administration. 
During reconstitution, an aliquot is removed for testing by colony count to be able to document 
the actual dose administered to the participants. The CFU total in the challenge inoculum that are 

 
 
Protocol v4.2 
16 JAN 2023 
43
Form I is calculated according to study specific procedures and the reconstitution time is 
documented.  
 
ͷǤ 
Participants will be challenged with approximately 1.5x103 cfu of S. flexneri 2a strain 2457T or 
1.5x103 cfu of S. sonnei strain 53G. 
 
Participants will fast for 90 minutes prior to ingesting the challenge inoculum. A sodium 
bicarbonate (USP-grade) solution of 2 g/150 ml water will be prepared. Each participant will 
drink 120 ml of this buffer one minute prior to ingesting the challenge inoculum, to neutralize 
gastric acidity. The bicarbonate buffer is prepared from USP-grade sodium bicarbonate by 
dissolving 13.35 grams of sodium bicarbonate in 1000 mL of sterile water for irrigation.  Within 
2 minutes, participants will drink the challenge inoculum (1.5 x 103 cfu) dissolved in either the 
remaining 30 mL of buffer (for the S. flexneri 2457T strain) or saline (for the S. sonnei 53G 
strain). 
 
Participants will fast for an additional 90 minutes before resuming a normal eating pattern. 
Participants will be monitored for at least 30 minutes post challenge for any signs or symptoms of 
a reaction, and for any AEs including nausea and vomiting. If a participant vomits or does not 
ingest the whole dose, this will be documented; however, the participant will not be rechallenged 
or replaced. After at least 30 minutes, participants will have vital signs taken and will be free to 
move about the unit. 
 
͸Ǥ 
The investigator must ensure that the investigational product is stored as specified in the protocol 
and in a secured area, with access limited to authorized study personnel. The investigator must 
maintain accurate records of the receipt of all investigational product, including date received, 
amount received and disposition. A record will be maintained that includes the dispensation date, 
amount of investigational product dispensed, initials and identification number. 
 
Any use of the challenge vials will be done under the supervision and direct oversight of the 
Research Pharmacy and JHU Laboratory. The laboratory staff or Research Pharmacist will 
maintain a strain accountability log which tracks the status of all vials received. Any vials 
remaining at the end of the study will be returned to WRAIR or destroyed by autoclaving at the 
direction of study Sponsor and after all IP monitoring is completed. If destroyed, the investigator, 
in accordance with Sponsor’s specifications, will document the destruction of any investigational 
product. 
XIII. 
ASSESSMENT OF SAFETY   
Safety monitoring will be conducted throughout the study; therefore, safety concerns will be 
identified by continuous review of the data by the PI, clinic staff, clinical monitor, research monitor, 
and the Sponsor.  
 
Study Safety Management: The research monitor and PI will review any safety concern. A data 
safety monitoring board (DSMB) is not required for this study. 
 
Research Monitor: A qualified physician with previous experience in vaccine biology and infectious 
diseases will be appointed as Research Monitor. Contact information, references and responsibilities 
of the Research Monitor will be included in the Safety Management Plan (SMP). Briefly, the 
Research Monitor will review any SAEs that occurred during the trial and any additional safety data 

 
 
Protocol v4.2 
16 JAN 2023 
44
if necessary. In addition, the Research Monitor will provide guidance to the Principal Investigator on 
questions of eligibility and any question of participant safety. 
 
The research monitor, in accordance with JHSPH guidelines, will have the following responsibilities: 
x Evaluate ongoing safety data and make recommendations in order to ensure participants’ safety as 
required 
x Be available for consultation by the clinical investigative team through the period of the clinical 
study in which there is an interaction with human participants 
x Be available to review all SAEs and other unanticipated problems involving risk to participants  
x Provide clinical advice, in accordance with the study protocol, on the clinical management of 
participants. This advice may include, but is not limited to: 
o Decisions on participants who have “borderline” eligibility for enrollment  
o Confirmation and discussion of treatment decisions for difficult clinical situations 
 
Protocol Safety Review Team (PSRT): The Protocol Safety Review Team (PSRT) will consist of 
the PATH Medical Officer, the PI and the Research Monitor and will be responsible for conducting 
periodic reviews of accumulating safety data and of trial conduct throughout the trial. Details on the 
responsibilities and processes of the PSRT, including periodicity of meetings and content of reports, 
will be provided in the Safety Management Plan. In addition to safety review, the PSRT may elect to 
discuss trial conduct issues that impact study integrity and/or participant safety. These may include, 
but are not limited to, data quality, critical monitoring findings, and issues with research specimens. 
 
All clinical decisions must be documented, including date, time, and signature. All decisions must be 
communicated to the study PI and other study investigators, which must be stored with participant 
source documents. 
 
All safety reports (i.e., SAEs, deviations, unanticipated problems involving risk and participant 
deaths) will be submitted to the JHSPH IRB and NMRC IRB per reporting requirements of each 
regulatory body. 
 
ͳǤ Ǧ
Vital signs (temperature, blood pressure, and heart rate) will be obtained throughout the inpatient 
period and at study visits after discharge as per the study schedule (see Table 2 for applicable AE 
coding). 
 
Table 2: Reference Ranges and Adverse Event Coding for Vital Signs Parameters 
Vital Signs 
Mild  
 (Grade 1) 
Moderate 
 (Grade 2) 
Severe  
 (Grade 3) 
Potentially Life 
Threatening (Grade 4) 
Heart rate 
 
Tachycardia 
101–115 
116-130 
>130 
ER visit or hospitalization for 
arrhythmia 
 
Bradycardia 
50-54a 
45–49 
<45 
ER visit or hospitalization for 
arrhythmia 
Fever (°C)  
 (°F)  
38.0–38.4 
100.4–101.1 
38.5–38.9 
101.2–102.0 
>39.0 
>102.0 
 
Life threatening hyperthermia 
Blood Pressure 
 
Hypertension  
 (systolic, mm Hg) 
141–150 
151 - 155 
>155 
ER visit/hospitalization for 
malignant hypertension 
Hypertension  
(diastolic, mm Hg) 
91–95 
96 – 100 
>100 
ER visit/hospitalization for 
malignant hypertension 

 
 
Protocol v4.2 
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45
Vital Signs 
Mild  
 (Grade 1) 
Moderate 
 (Grade 2) 
Severe  
 (Grade 3) 
Potentially Life 
Threatening (Grade 4) 
Hypotension  
(systolic, mm 
Hg)b 
85–89 
80 – 84 
<80 
ER visit/hospitalization for 
hypotensive shock 
aGrade 1 bradycardia will not be considered an abnormality for this study unless judged to be clinically significant by the PI or the PI 
in consultation with the Research Monitor and Sponsor. 
 bIf a participant has a baseline systolic BP in the 90’s then a decrease in BP < 10 without associated clinical symptoms will not be 
considered an abnormality for this study unless judged to be clinically significant by the PI. 
Based on the FDA Guidance for Industry: Toxicity grading scale for healthy adult and adolescent volunteers enrolled in Preventative 
Vaccine Clinical Trials https://www.fda.gov/regulatory-information/search-fda-guidance-documents/toxicity-grading-scale-healthy-
adult-and-adolescent-volunteers-enrolled-preventive-vaccine-clinical 
 
ʹǤ 
A complete physical exam will be conducted during the screening visit and on Day -2 or -1 as 
part of the screening process. PE to include HEENT (head, ears, eyes, nose, throat), skin, 
respiratory, cardiovascular, abdomen/GI, neurological, and musculoskeletal. Focused physical 
exams (symptom focused), will be conducted prior to receipt of any challenge product, daily 
during participants’ inpatient stay, and as needed during outpatient visits with specific attention to 
the identification of AEs, AESIs, and SAEs. 
 
͵Ǥ 
Participants will be asked to collect all stools passed on the unit and bring them to the lab for 
assessment and processing. Stools will be weighed and graded based on the following scale [3]: 
x 
Grade 1, firm formed;  
x 
Grade 2, soft formed;  
x 
Grade 3, viscous opaque liquid or semiliquid which assumed the shape of the container;  
x 
Grade 4, watery opaque liquid;  
x 
Grade 5, clear watery or mucoid liquid.  
 
Stools defined as grade 3, 4, or 5 are considered to be loose. 
Stools will be observed for presence of gross blood; if a stool appears grossly bloody, it will be 
tested for blood using a hemoccult card. Stool that is not grossly bloody will not be tested for 
occult blood. 
 
An episode of diarrhea will be considered complete after 24 hours without a loose stool.  
 
During the outpatient period, loose stools will only be graded and assessed by study team based 
on the subject’s description. No stools will be weighed during the outpatient period. 
 
ͶǤ 
Venous blood samples will be collected for chemistry, hematology, and immunological 
parameters during the screening phase of this study, to provide a baseline sample, and after 
challenge as per the Schedule of Procedures.  Hematology and chemistry analyses will be 
performed by a commercial laboratory. Additional specimens may be collected to confirm and 
evaluate any abnormal values and may be obtained as part of the clinical care of an individual 
participant.  Targeted urine drug screenings are planned for this study at screening and urine or 
serological screening (including for surreptitious use of antibiotics) may be performed later at the 
discretion of the study clinician. 
 
The clinical toxicity grading scale that will be used as a guideline is based on the FDA Guidance 
for Industry: Toxicity Grading Scale for Healthy Adult and Adolescent Participants enrolled in 
Preventive Vaccine Clinical Trials and the DAIDS Table for Grading the Severity of Adult and 

 
 
Protocol v4.2 
16 JAN 2023 
46
Pediatric Adverse Events. Final grading determination will be made by the PI and according to 
Table 3 and Table 4.  
 
In the event of a clinically significant abnormal laboratory value, the test will be repeated and 
followed up, if clinically relevant.  Slightly abnormal laboratory values that remain consistent 
from the time of screening throughout the study will not be recorded as AEs. 
 
Table 3: Reference Ranges and Adverse Event Coding for Clinical Hematology Parameters 
Test 
Normal 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially Life 
Threatening 
(Grade 4) 
Hemoglobin (g/dL) 
(for screening purposes only) 
M: LLN = 11.0 
F: LLN = 10.5 
  
 
 
 
Hemoglobin, low 
 
M: 10.0 to 10.9 
F:9.5 to 10.4 
M: 9.0 to 
<10.0 
F: 8.5 to <9.5 
M: 7.0 to <9.0 
F:6.5 to <8.5 
M: <7.0 
F: <6.5 
Eosinophils (cells/mm3) 
15-500 
551-1,500 
1,501-5,000 
> 5,000 
Hypereosinophilic 
Leukocytes (white blood cells) 
(cells/mm3) 
2,500 to 10,800 
 
 
 
 
Leukopenia 
 
2,000 to 2,499 
1,500 to 1,999 1,000 to 1,499 
<1,000 
Leukocytosis 
 
10,801-15,000 
15,001- 
20,000 
20,001- 
25,000 
> 25,000 
Lymphocytes, low (cells/mm3) 
>650 
600 to <650 
500 to <600 
350 to <500 
<350 
Neutrophils, low (cells/mm3) 
>1,000 
800 to <1,000 
600to 799 
400to 599 
<400 
Platelets decreased 
(cells/mm3) 
≥125,000 
100,000 to 124,999 50,000 to 
<100,000 
25,000 to 
<50,000 
<25,000 
 
 
Table 4: Reference Ranges and Adverse Event Coding for Blood Chemistry Parameters 
Test 
Normal 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially Life 
Threatening 
(Grade 4) 
BUN (elevation) 
7-25 
26-28 
29-31 
> 31 
Requires dialysis 
Creatinine (elevation) 
M: 0.7-1.4 
F: 0.5-1.1 
1.1 to 1.3 x ULN 
 
> 1.3 to 1.8 x ULN OR 
Increase of > 0.3 mg/dL 
above baseline 
> 1.8 to < 3.5 x ULN 
OR Increase of 1.5 to 
< 2.0 x above 
baseline 
≥ 3.5 x ULN OR 
Increase of ≥ 2.0 x 
above baseline 
Glucose, Random (mg/dL)  
65 to115 
 
Hypoglycemia 
 
55 to 64 
40 to <55 
30 to <40 
< 30 
Hyperglycemia 
 
116 to 160 
>160 to 250 
>250 to 500 
> 500 
Potassium (mEq/L; mmol/L) 
3.4 to 5.6 
 
Hypokalemia 
 
3.0 to < 3.4 
2.5 to <3.0 
2.0 to <2.5 
< 2.0 
Hyperkalemia 
 
>5.6 to <6.0 
6.0 to <6.5 
6.5 to <7.0 
≥ 7.0 
SGOT/AST (elevation) 
M: 10 to 40 
F: 10 to 35 
1.25 to <2.5 x 
ULN 
2.5 to <5.0 x ULN 
5.0 to <10.0 x ULN 
≥ 10.0 x ULN 
SGPT/ALT (elevation) 
M:9 to 46 
F: 6 to 29 
1.25 to <2.5 x 
ULN 
2.5 to <5.0 x ULN 
5.0 to <10.0 x ULN 
≥ 10.0 x ULN 

 
 
Protocol v4.2 
16 JAN 2023 
47
Sodium (mEq/L; mmol/L) 
136 to145 
 
Hyponatremia 
 
130 to <135 
125 to <130 
121 to <125 
≤ 120 
Hypernatremia 
 
146 to <150 
150 to <154 
154 to <160 
≥ 160 
 
ͷǤ Ǧ
The following terms, as defined by 21 CFR 312.32, apply to IND safety reporting. 
 

An adverse event is any untoward, undesired, or unplanned event in the form of signs, 
symptoms, disease, or laboratory or psychological/physiologic observations occurring in a 
subject enrolled in the clinical trial. This includes an exacerbation or worsening of pre-
existing conditions or events, intercurrent illnesses, injuries, or vaccine or drug interaction, or 
worsening of abnormal clinical laboratory values.  Anticipated day-to-day fluctuations of pre-
existing conditions that do not represent a clinically significant exacerbation need not be 
considered AEs. Discrete episodes or worsening of chronic conditions occurring during a 
study period will be reported as AEs to assess changes in frequency or severity.  Stable, pre-
existing conditions and/or elective procedures are not AEs (including any not recognized or 
not reported prior to study entry) and will be recorded in the Medical History. 
 
AEs will be documented in terms of signs and symptoms observed by the investigator or 
designee or reported by the participants at each study encounter, with a medical diagnosis 
stated. AEs occurring after informed consent is obtained, but prior to IP receipt, will be 
documented in the Medical History form within the participant's eCRF.  
 

A “suspected” adverse reaction means any adverse event for which there is a reasonable 
possibility that the investigational product caused the adverse event. For the purposes 
of IND safety reporting, “reasonable possibility” means there is evidence to suggest a 
causal relationship between the investigational product and the adverse event. 
Suspected adverse reaction implies a lesser degree of certainty about causality than 
adverse reaction, which means any adverse event caused by an investigational product. 
 
ȋȌ
A solicited AE is a predetermined event, which may reflect safety concerns related to the IP.  
This study includes challenge with live Shigella bacteria, therefore all the symptoms of 
Shigella illness are expected.  The most common effects of Shigella infection are listed 
below. These potential Shigella-associated AEs will be solicited daily during the challenge 
phase: 
 
1. Abdominal cramps* 
2. Abdominal pain* 
3. Anorexia (poor appetite) 
4. Arthralgias* 
5. Bloating 
6. Chills 
7. Constipation 
8. Excessive flatulence 
9. Generalized myalgia* 
10. Headache 

 
 
Protocol v4.2 
16 JAN 2023 
48
11. Lightheadedness  
12. Malaise* 
13. Nausea* 
14. Tenesmus 
15. Urgency 
 
*Constitutional and enteric symptoms that are in the definition of shigellosis 
 
The following anticipated AEs will be documented via clinical assessments during 
the inpatient challenge phase: 
 
1. Diarrhea of any severity (via stool logs) 
2. Dysentery 
3. Hypovolemia 
4. Fever (oral temperature > 100.4° F / 38.0qC) 
5. Vomiting 
 

An AE of special interest is an AE of scientific and medical concern specific to the Sponsor’s 
product or program, for which ongoing monitoring and rapid communication by the 
investigator to the Sponsor can be appropriate. Such an event might warrant further 
investigation in order to characterize and understand it. 
 
AEs that are considered AESI are the following: 
x 
Development of a new chronic illness 
x 
Development of Reactive Arthritis 
x 
Development of Irritable Bowel Syndrome or other continuing gastrointestinal condition 
 

An adverse event or suspected adverse reaction is considered “serious” if, in the view of 
either the investigator or Sponsor, it results in any of the following outcomes:  
x 
Death 
x 
Life-threatening adverse event 
x 
Inpatient hospitalization or prolongation of existing hospitalization 
x 
Persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions 
x 
Congenital anomaly/birth defect (abortion, stillbirth and any malformation/disease must 
be reported as an SAE). 
 
Although not considered SAEs, cancers will be reported in the same way as SAEs.  
 
Pertinent definitions include: 
x 
Life threatening - An AE is life threatening if the participant was at risk of death at the 
time of the event; it does not refer to an event that hypothetically might have caused 
death if it were more severe. 
x 
Disabling/incapacitating - An AE is incapacitating or disabling if it results in a substantial 
disruption of the participants' ability to carry out normal life functions. This definition is 
not intended to include experiences of relatively minor medical significance such as 
headache, nausea, vomiting, diarrhea injection site reactions and minor accidental trauma 
(e.g., sprained ankle). 

 
 
Protocol v4.2 
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49
x 
Hospitalization: In general, hospitalization signifies that the participant has spent 
significant time (usually involving at least an overnight stay) at the hospital or emergency 
ward for treatment that would not have been appropriate in the physician's office or 
outpatient setting. Hospitalization for either elective surgery related to a pre-existing 
condition which did not increase in severity or frequency following initiation of the study 
or for routine clinical procedures (including hospitalization for "social" reasons) that are 
not the result of an adverse event need not be considered as AEs and are therefore not 
SAEs.  
x 
Routine Clinical Procedure: A procedure which takes place during the study and does not 
interfere with the test article administration or any of the ongoing protocol specific 
procedures. 
 
Note:  If anything untoward is reported during an elective procedure, that occurrence must be 
reported as an adverse event, either `serious' or non-serious according to the usual criteria. 
When in doubt as to whether ‘hospitalization’ occurred or was necessary, the AE will be 
considered serious. Important medical events that may not result in death, be life-threatening, 
or require hospitalization may be considered serious when, based upon appropriate medical 
judgment, they may jeopardize the patient or participant and may require medical or surgical 
intervention to prevent one of the outcomes listed in this definition. Examples of such 
medical events include allergic bronchospasm requiring intensive treatment in an emergency 
room or at home, blood dyscrasias or convulsions that do not result in inpatient 
hospitalization, or the development of drug dependency or drug abuse. 
 
͸Ǥ ȋȌ
The investigator or designee must assign a relationship of each AE to the receipt of the IP. The 
investigator or designee will use clinical judgment in conjunction with the assessment of a 
plausible biologic mechanism, a temporal relationship between the onset of the event in relation 
to receipt of the IP, and identification of possible alternate etiologies including underlying 
disease, concurrent illness or concomitant medications. Every effort will be made to explain AEs 
and assess causal relationships, if any, to administration of the challenge, or antibiotic treatment.   
 
AEs occurring after receipt of the challenge (Day 1) will be assessed as to their relationship with 
the Shigella challenge strain or the antibiotic, if applicable. The degree of certainty with which an 
AE can be attributed to these products (or alternative causes, e.g., natural history of the 
underlying diseases, concomitant therapy, etc.) will be determined by how well the event can be 
understood in terms of one or more of the following: 
 
x 
Reaction of similar nature having previously been observed with Shigella challenge strains or 
antibiotic administration 
x 
Published literature accounts supporting causality 
x 
Temporal relationship with administration 
 
The following guidelines should be used by investigators to assess the relationship of an AE to 
study product administration.  Only a physician or nurse practitioner can make this determination. 
The investigator will assess causality of all AEs. Non-serious and serious adverse events will be 
evaluated as two distinct types of events given their different medical nature. If an event meets 
the criteria to be determined ‘serious’ it will be examined by the investigator to the extent 
possible to determine ALL contributing factors applicable to the event.  
 
Other possible contributors include: 
x 
Underlying disease 
x 
Other medication 

 
 
Protocol v4.2 
16 JAN 2023 
50
x 
Protocol-required procedure 
x 
Other cause (specify) 
 
Causality of all AEs will be assessed by the investigator using the following criteria: 
In the investigator’s opinion, the likelihood of the relationship of the AE to challenge 
administration is to be recorded as follows: 
x 
Related: There is a reasonable causal relationship between the challenge strain administered 
and the AE. 
x 
Not Related: There is no reasonable causal relationship between the challenge strain 
administered and the AE. 
 
All AEs will be recorded on the appropriate AE form of the participant’s eCRF and recorded 
irrespective of severity or whether or not they are considered related to the challenge inoculum. 
AEs will be tabulated separately for pre-and post-challenge data.  
 
͹Ǥ ȂȀ
AEs occurring from the time of consenting through 28 days after administration of Investigational 
Product will be recorded on the appropriate eCRF (AEs reported prior to challenge will be 
reported in the medical history eCRF). All AEs observed by the investigator or one of the clinical 
staff or reported by participants spontaneously or in response to a direct question will be 
evaluated by a study investigator or designee and recorded in the participant eCRF. The nature of 
each event, date of onset, outcome, severity and causality will be established. Details of any 
symptomatic/corrective treatment will be recorded on the source documents and entered in the 
appropriate page of the eCRF. AEs will be assessed as to their relationship with the challenge 
strain, the antibiotic (if treatment has started) or the protocol.  
 
During the inpatient phase, both solicited and unsolicited adverse events will be assessed, 
whereas at outpatient visits only unsolicited AEs will be assessed. AEs already documented in the 
eCRF, i.e., at a previous assessment, and designated as ‘ongoing’ will be reviewed at subsequent 
follow-up assessments. If resolved, documentation in the eCRF will be completed. Changes in 
AE severity for solicited events during the inpatient period will be documented as a new event. 
Unsolicited AEs during the inpatient period, and any reported AEs with onset during the 
outpatient period, will be recorded as single events with the highest severity for the duration of 
the event entered in the eCRF. 
 
Challenge-specific solicited AEs will be assessed during the inpatient phase of the study. Adverse 
events which occur after informed consent is obtained, but prior to challenge, will not be 
documented as AEs but as medical history, irrespective of severity. All AEs will be assessed 
through 28 days after each challenge, documented in the source records, and recorded on the 
eCRFs using accepted medical terms and/or the diagnoses that accurately characterize the event. 
AESIs and SAEs will be assessed through study completion (approximately 180 days from last 
challenge). Solicited AEs (listed above) will be recorded as individual events. Unsolicited AEs 
may be recorded as a diagnosis. When a diagnosis is known, the AE term recorded on the eCRF 
will be the diagnosis rather than a constellation of symptoms. The investigator will assess all AEs 
for causality, severity, and outcome.  When an event has not resolved by the prescribed reporting 
period, it will be left open/without an end date on the AE eCRF and will be updated with end date 
or ongoing at the final visit.  
 
ͺǤ 	Ǧ
Any SAE deemed related to IP that is ongoing at the time of last study visit will continue to be 
followed until it is resolved, assessed to be resolved with sequelae, or assessed to be 

 
 
Protocol v4.2 
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51
stable/chronic. SAEs deemed not related to IP that are unresolved at the time of the last study will 
be classified as ongoing. Investigators are required to follow related SAEs to resolution, even if 
this extends beyond the prescribed reporting period. Resolution is the return to baseline status or 
stabilization of the condition with the probability that it will become chronic. The SAE outcomes 
will be reported to the Sponsor. 
 
Investigators are not obligated to actively seek SAEs in former participants; however, if a SAE 
considered to be related to the IP is brought to the attention of the investigator at any time until 
closure of the study, the event will be reported. 
 
Investigators should follow-up adverse events at least until the final study visit. This may include 
repeat safety laboratory analysis. Outcome should be assessed as: 
x 
Resolved 
x 
Resolved with sequelae 
x 
Severity change (the highest severity in a day will be recorded in the source documents; for 
solicited events during the inpatient period, if the severity on day 1 is moderate, then mild 
and then moderate, it will be entered as moderate for day 1 only, then if on day 2 the ongoing 
event is mild, the moderate AE will stop and the AE will be reentered as mild; unsolicited 
events that occur during both the inpatient and outpatient periods will be entered as single 
events to document the entire duration in the eCRFs, with the highest severity recorded for 
the event duration) 
x 
Ongoing  
x 
Died 
x 
Lost to follow-up 
 
ͻǤ 
All AEs will be assessed for severity by the investigator/designee. Inherent in this assessment is 
the medical and clinical consideration of all information surrounding the event including any 
medical intervention required. Each event will be assigned one of the following categories: mild, 
moderate, severe, or life-threatening. The criteria below may be used for any symptom not 
included in the grading scale. Any grade 4 (life-threatening) AE must be reported as an SAE. 
 
For solicited AEs during the inpatient period, the eCRF will document daily changes for ongoing 
events. For any solicited AEs with onset during the outpatient period, the eCRF will reflect the 
highest severity for the entire duration of the event. Severity changes will be documented as 
described above. 
 
Mild 
Grade 1 
Does not interfere with routine activities; minimal level 
of discomfort 
Moderate 
Grade 2 
Interferes with routine activities; moderate level of 
discomfort 
Severe 
Grade 3 
Unable to perform routine activities; significant level 
of discomfort 
Potentially life-threatening 
Grade 4 
Hospitalization or ER visit for potentially life-
threatening event 
 
As defined by the International Conference on Harmonisation (ICH) guideline for Good Clinical 
Practice (GCP), the term “severe” is often used to describe intensity (severity) of a specific event 
(as in mild, moderate, or severe myocardial infarction); the event itself however, may be of 
relatively minor medical significance (such as severe headache). This is not the same as 
“serious”, which is based on participant/event outcome or action criteria usually associated with 

 
 
Protocol v4.2 
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52
events that pose a threat to a participant’s life or functioning. Seriousness (not severity) serves as 
a guide for defining regulatory reporting obligations. 
 
During the challenge phase of the study, Shigella disease-specific adverse events will be graded 
in accordance with the table below. 
 
Table 5: Challenge Inpatient Phase Shigella Infection Solicited Adverse Event/Endpoint 
Reactogenicity/Adverse Event 
Severity 
Parameter 
Diarrhea based on highest output 
of loose/liquid stools in any 24-
hour period. (A diarrhea episode 
ends when there is a 48 hour 
window with no grade 3-5 
stools.) 
1- Mild 
t 2 loose / liquid stools weighing t 200g within 48 hrs. 
or 1 stool weighing ≥300g, not meeting the definition for 
moderate or severe 
2- Moderate 
4 to 5 loose/liquid stools within 24 hours or 401 - 800g of 
loose/liquid stool within 24 hours 
3- Severe^ 
 
≥6 loose/liquid stools within 24 hrs. or >800g of 
loose/liquid stool within 24 hours 
4 
Potentially life threatening  
Oral temperature* (t) 
1- Mild 
>100.4oF and <101.1oF (38.0-38.4oC) 
2- Moderate 
>101.2oF and <102.0oF (38.5-38.9oC) 
3- Severe 
>102.0oF and < 104 oF (39.0 - 40oC) 
4 
> 104 oF or 40oC Life threatening hyperthermia 
Vomiting 
1- Mild 
One episode within a 24-hour period 
2- Moderate 
Two episodes within a 24-hour period 
3- Severe 
Three or more episodes within a 24-hour period 
4 
Potentially life threatening consequence of emesis 
Other solicited and non-solicited 
adverse events 
1- Mild 
Discomfort noted, but no disruption of normal daily 
activities; slightly bothersome; relieved with or without 
symptomatic treatment. 
2- Moderate 
Discomfort sufficient to reduce or affect normal daily 
activity to some degree; bothersome; interferes with 
activities, only partially relieved with symptomatic 
treatment. 
3- Severe 
Discomfort sufficient to reduce or affect normal daily 
activity considerably; prevents regular activities; not 
relieved with symptomatic treatment. 
4 
Potentially life threatening 
*Oral temperature; no recent hot or cold beverages, eating or physical activity. If temperature is ≥ 100.4 °F every attempt should be made to 
repeat the temperature in about 20 minutes after the participant has rested with no eating or drinking. If the repeat temperature is WNL and 
the investigator feels the repeated temperature is a more accurate reflection of the participant's real temperature a fever will not be entered 
into the eCRF. ^A subset of more severe diarrhea is ≥10 loose (grade 3-5) stools within 24h or ≥1000 gr loose (grade 3-5) stools within 24h. 
 
 
Table 6: Classification of Intensity of Diarrhea during the Outpatient Phase 
 
Diarrhea 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially Life 
Threatening (Grade 4) 
Loose stools are stools to 
be determined to be grade 
3 - 5 by study team 
3 loose stools in 24 
hours 
4-5 loose stools 
in 24 hours 
6 or more loose 
stools in 24 
hours 
Requires hospitalization  
 
ͳͲǤ 
The PI will report all AEs as per regulatory requirements. 
 
ͳͳǤ 
To obtain an unbiased determination of the efficacy outcomes, an independent outcome 
adjudication committee will blindly evaluate challenge outcome data after completion of all 

 
 
Protocol v4.2 
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53
inpatient phases of the study. To ensure a blinded review, new subject IDs that differ from those 
at assigned at enrollment will be utilized. 
The committee will be comprised of at least 3 individuals, independent of the study Sponsor and 
investigative team, who are experts on diarrheal illness case identification and pathogen 
diagnosis. The committee will also include a statistician data analyst who will lead and coordinate 
the committee but will have a non-voting role in deliberations. 
The committee voting members will review all potential efficacy-related cases and endpoint data. 
Among the committee’s responsibilities, they will review and confirm all primary endpoint cases 
and provide guidance regarding secondary and other endpoint classifications to include 
agreement on objective criteria for classification of endpoints. Specific duties and responsibilities 
will be outlined in the manual of procedures (MOP) prior to the start of the study. 
ͳʹǤ 
SAE reports will be provided to the Sponsor, research monitor, and IRB(s).  The investigator 
must report SAEs within one calendar day of becoming aware of the event by telephone, fax or e-
mail (if appropriate) to the contacts in Table 7 below for reporting SAEs as described in the 
protocol. This initial notification will include minimal, but sufficient information to permit 
identification of the reporter, the participant, the test articles, SAE(s), and date of onset. The 
investigator will not wait for additional information to fully document the event before notifying. 
The first notification will be confirmed by an acknowledgement by email from the Sponsor, 
research monitor, and IRB. The report is then to be followed by submission of a completed SAE 
Report Form as soon as possible but not more than 3 calendar days past the initial report, 
detailing relevant aspects of the SAE in question. All actions, if any, taken by the investigator 
regarding the SAE and event outcomes must also be reported immediately. SAE Report Forms 
are to be used for documentation of these various aspects regarding the event. Hospital records 
and autopsy reports will be obtained if applicable. 
The research monitor is required to review all unanticipated problems involving risk to 
participants or others, SAEs, and provide an unbiased written report of the event.  At a minimum, 
the research monitor will comment on the event outcomes, and in the case of a SAE or death, 
comment on the relationship to participation in the study.  The research monitor will indicate 
concurrence or nonconcurrence with the details of the report provided by the investigator.  
Reports for events determined by either the investigator or research monitor to be related to 
participation and reports of events resulting in death will be based on regulatory guidance. 
Unanticipated problems involving risk to participants or others, SAEs related to participation in 
the study and all participant deaths will be reported by phone, by email, or by facsimile. A 
complete written report will follow the initial notification. 
ͳ͵Ǥ 
All SAEs must be reported promptly to the Sponsor as per 21 CFR 312.64, whether or not the 
event is considered related to study product. Further, the investigator should comply with relevant 
study site SOPs on reporting SAEs. The minimum information that the investigator will provide 
to the Sponsor is specified in Table 8.  
 

 
 
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Table 7: Study Contacts for Reporting Serious Adverse Events 
Sponsor 
 
PATH 
Patricia Njuguna, MD 
 
ACS Plaza, 4th floor 
Lenana Road 
P. O. Box 76634-00508 
Nairobi, Kenya 
 
Institutional Review Board  
JHSPH IRB Office 
615 N. Wolfe Street, Suite E1100 
Baltimore, Maryland 21205 
Phone: 410-955-3193  Toll-Free: 1-888-262-3242 
Fax: 410-502-0584 
Email: JHSPH.irboffice@jhu.edu 
Research Monitor 
Anna P. Durbin, MD 
Professor, International Health 
Johns Hopkins Bloomberg School of Public Health 
624 N. Broadway, Room 251 
 
 
Table 8: SAE Information to be Reported to the Sponsor 
Notification 
Method 
Information to be Provided 
Email or 
Telephone (within 
72 hours) 
IND number, Sponsor study number, name of the IP, investigator name and contact 
number 
 
Participant identification number 
 
SAE, onset date, date of IP administration, severity, relationship, and participant’s 
current status 
AND 
 
Email or Fax 
Cover sheet or letter 
 
Serious adverse event report form 
 
Concomitant medication case report form or a list of concomitant medications 
 
Medical record progress notes including pertinent laboratory/diagnostic test results 
NOTE: When submitting SAE reports via email, the subject line of each email notification will read as 
follows: 
SAFETY REPORT – IND # _____, Study #_____, Participant# _____, Event term: _____ 
 
In order to comply with regulations mandating Sponsor notification of specified SAEs to the FDA 
within 7 calendar days, investigators must submit additional information as soon as it is available. 
The sponsor will report unexpected SAEs associated with the use of the challenge strain to the 
FDA as specified at 21 CFR 312.32 (c).  
 
Investigators must follow all relevant regulatory requirements as well as specific policy at each 
institution regarding the timely reporting of SAEs to the local IRB and research monitor.  
Reporting to the Sponsor does not fulfill the investigator’s duty to report all unanticipated 
problems involving risk to human participants or others to the IRB. The PI will notify the local 
IRB and the research monitor. 
 
ͳͶǤ 
Unanticipated problems involving risk to participants or others, SAEs related to participation in 
the study and all participant deaths should be promptly reported by phone, email, or fax to the 

 
 
Protocol v4.2 
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local JHSPH IRB and protocol associated IRBs as needed. A written report will follow the initial 
notification. 
Investigators are required to forward safety information provided by the Sponsor’s representative 
to the IRB. All SAEs will be reported to the JHSPH IRB according to IRB guidelines. 
JHSPH IRB contact: Phone 410-955-3193; Fax 410-502-0584; e-mail: jhsph.irboffice@jhu.edu. 
Investigators are required to promptly report adverse events that fit the following criteria using 
the Problem/Event Report Form: 
Event (including adverse event reports, injuries, side effects, breaches of confidentiality, or other 
problems) that occurs any time during or after the research study, which in the opinion of the 
principal investigator: 
1. Involved harm to one or more participants or others, or placed one or more participants or 
others at increased risk of harm; 
2. Is unexpected (an event is “unexpected” when it is not described with specificity in the 
protocol and informed consent document; or if described with specificity, it occurs beyond 
the expected frequency and/or severity identified); and 
3. Is related to the research procedures (an event is “related to the research procedures” if in 
the opinion of the principal investigator, it was more likely than not to be caused by the 
research procedures.) 
 
ͳͷǤ Ǧ
A serum sample for pregnancy testing (female participants) will be collected at the screening visit 
and on Day -2 or -1.  A urine pregnancy test will be collected (female participants) on Day 1 only 
if serum pregnancy test results are not available prior to challenge, and Day 29. Any participant 
with a positive pregnancy test prior to any challenge will not be enrolled in the study. Any 
participants who become pregnant during the study will be removed from the study and  followed 
until the end of their pregnancy.  
 
Each pregnancy must be reported within 72 hours of identification by email or fax to the 
Sponsor and the IRB. The investigator must report any pregnancy in study participants to the 
Research Monitor within 14 calendar days of learning of this occurrence. 
Participants who become pregnant after Day 1 through 29 days after each challenge dose will be 
followed to term, and the following information will be gathered for outcome: date of delivery; 
health status of the mother and child, including the child’s gender, height, and weight. 
Complications and/or abnormalities should be reported, including any premature terminations. A 
pregnancy is reported as an AE or SAE only when there is suspicion that the IP may have 
interfered with the effectiveness of contraception or there was a serious complication in the 
pregnancy, including a spontaneous abortion or an elective termination for medical rationale. 
Pregnancies that occur after 29 days after each challenge will not be followed. 
ͳ͸Ǥ Ǧ
Any AE-related withdrawal of consent during the study must be reported immediately (within 
24 hours of identification) by email or fax to the Sponsor and the IRB. 

 
 
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ͳ͹Ǥ Ȁ
The knowledge of any pending compliance inspection/visit by the FDA, Office for Human 
Research Protections (Department of Health and Human Services), or other government agency 
concerning clinical investigation or research, the issuance of Inspection Reports, FDA Form 483, 
warning letters, or actions taken by any Regulatory Agencies including legal or medical actions 
and any instances of serious or continuing noncompliance with the regulations or requirements 
will be reported immediately to IRBs and the Sponsor. 
The investigator will notify the Sponsor within 24 hours following contact by a regulatory agency. 
The investigator and study coordinator will be available to respond to reasonable requests and audit 
queries made by authorized representatives of regulatory agencies. The investigator will provide 
the Sponsor with copies of all correspondence that may affect the review of the current study or 
his/her qualification as an investigator in clinical studies conducted by the Sponsor. The Sponsor 
will provide any needed assistance in responding to regulatory audits or correspondence. The 
investigator will permit independent auditors (employees of the Sponsor or an external company 
designated by the Sponsor) to verify source data validation of the regularly monitored clinical trial. 
The auditors will compare the entries in the eCRFs with the source data and evaluate the study site 
for its adherence to the clinical study protocol and GCP guidelines and applicable regulatory 
requirements.  
ͳͺǤ 

The lead investigator will be responsible for the preparation of a detailed annual synopsis of 
clinical activity, including adverse events, for submission to the Sponsor. Each annual report will 
summarize IND activity for 1 year beginning approximately 3 months before the IND FDA 
anniversary date.  
	
A final study report will be prepared in accordance with “Guidance for Industry: Submission of 
Abbreviated Reports and Synopses in Support of Marketing Applications” and ICH E3 Guideline 
“Structure and Content of Clinical Study Reports” and provided to the Sponsor for review and 
approval. The Sponsor representative will use this report to prepare the final clinical study report 
for submission to the FDA.  The investigative team will report all AEs to the Sponsor and the 
local IRB in the appropriate safety, annual, and/or final reports.  
ͳͻǤ 	
The PI, along with the research monitor, may determine if certain events warrant discontinuation 
of challenge for all participants in a cohort. If any of the following events occur, administration of 
the challenge will be discontinued for all participants in that cohort, and the PI and the research 
monitor will undertake a thorough review of the events: 
x 
The occurrence of one or more serious adverse events (SAEs) determined to be related to the 
challenge. 
x 
Systemic allergic reaction, including but not limited to generalized urticaria, generalized 
petechiae, or erythema multiforme, occurring in two or more participants in a group.  
Bronchospasm or anaphylaxis occurring in any participant.  
 
Based on prior experience with Shigella challenge studies, it is expected that some participants 
will have severe AEs (such as severe diarrhea). 
 
AEs which will prompt stopping the challenge for an individual participant include: 
x 
The investigator deems that stopping test article administration is in the best interest of the 
participant. 

 
 
Protocol v4.2 
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Further challenge, in accordance with the protocol, may be resumed with the concurrence of 
the research monitor, Sponsor, and PI. 
 
ʹͲǤ 
The PI, Research Monitor, IRB, Sponsor or FDA may stop or suspend the use of either or both 
challenges at any time. Suspension or termination of research must be reported to the PATH 
Office of Research Affairs (ORA) within 72 hours. 
XIV. 
STUDY MONITORING 
ͳǤ 
Monitoring shall be conducted by a designated contract research organization according to an 
approved monitoring plan that will be finalized prior to the start of the study. Local monitoring 
will commence at study initiation, during the study, and at closeout. 
The study monitor shall be available for consultation with the investigator. The study monitor or 
other authorized representatives of the Sponsor may inspect all documents and records 
maintained by the investigator, including, but not limited to, medical records (office, clinic or 
hospital) and pharmacy records for the participant in this study. The clinical study site will permit 
access to such records. The investigator will obtain, as part of informed consent, permission for 
authorized representatives of the Sponsor, or regulatory authorities, to review, in confidence, any 
records identifying individuals in this clinical study. 
ʹǤ 
Source documents will be maintained in real-time. Data from the source documents will be 
entered into the database as per the Data Management Plan. Data entry will commence when the 
last participant is discharged from the inpatient unit. Priority data points may be entered for all 
participants first before remaining data. Outpatient visits will be completed, and data will be 
entered into the database within about 3 working days. 
 
͵Ǥ ȀȀ
The investigator will maintain complete and accurate documentation of the study, including 
records of medical treatments external to the research received during the study, records detailing 
the progress of the study for participants, laboratory reports, source documents, signed informed 
consent forms, investigational product disposition records, correspondence with the IRB, the 
study monitor and the Sponsor, AE reports, and information regarding participant discontinuation 
and completion of the study. All required data will be clearly and accurately recorded by 
authorized study personnel in the eCRFs. Only designated study site personnel will record or 
change data in an eCRF.  The investigator will be responsible for procuring data and for quality 
of data recorded in the eCRFs.  Complete source documentation (the documents in which data 
collected for the clinical trial are first recorded) is kept for each participant in his/her individual 
study chart.  All laboratory specimens, reports, study data collection, and administrative forms 
will be identified by coded number only to maintain participant confidentiality.  eCRFs using 
coded identifiers will be used to record data for participants enrolled in the study.  Forms, lists, 
logbooks, appointment books, and any other listings that link participant ID numbers to other 
identifying information will be stored in a separate, locked file in an area with limited access. All 
information regarding study participants is kept in password-protected computer files or in locked 
file cabinets that can be accessed only by authorized study personnel.  Samples are identified by 
coded participant numbers only.  Chart information and information from study records is not 
released without written permission of the volunteer. 

 
 
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The source documents will be retained at the site. All study-related documents will be kept in 
locked cabinets in locked rooms with limited access.  Information in the electronic database is 
password-protected and access is available only to authorized research team members.  
Additionally, each authorized research team member is assigned a level of security clearance 
(also password-protected, with mandatory password changes) for the purpose of limiting access 
to certain areas or functions of the database.  Any information printed from this database is stored 
in locked files until its use is complete and then shredded. 
For this study, an electronic data capture (EDC) database system will be used for the collection of 
the study data. The EDC and the eCRFs will be created by the CRO Emmes with input from the 
site and the Sponsor. The EDC database system will be designed based on the protocol 
requirements, the approved eCRF layouts and specifications, and in accordance with 21 CFR Part 
11. The eCRF layouts and specifications define and identify the applicable source data that will 
be collected and captured into the EDC database system. The applicable source data will be 
electronically transcribed by the site designee into the eCRF in the EDC database system. The 
investigator is ultimately responsible for the accuracy of the data transcribed on the eCRF. Data 
monitoring and management will be performed in the EDC database system by the study monitor 
and the designated Data Management group. 
A detailed data management plan (DMP) will be developed by the designated contract 
research organization and approved by the study team, the PI, and the Sponsor.  The DMP 
will explain how all AEs will be coded. Additionally, a data dictionary and metadata report will 
be compiled to explain the values that can be entered into the study database. 
XV. 
STATISTICAL CONSIDERATIONS 
ͳǤ 
Safety, efficacy, and clinical outcome data will be entered into the eCRFs using standard software 
for data management.  Data will be reviewed and errors corrected with standard strategies for 
range and consistency checks.  AEs for all participants, regardless of the number of challenge 
doses they have received, will be included in the safety analysis.  The null hypothesis for this 
study is that there is no protection provided by the initial challenge upon rechallenge.  
Immunology data will be imported after appropriate immunological testing has been completed. 
ʹǤ 
Volunteers will be recruited from the community as per Sections XI and XX. Volunteers will be 
recruited sequentially into each Cohort/Group. Cohort 1 is defined as the collection of individuals 
who are challenged with S. sonnei first followed by S. flexneri 2a challenge; Cohort 2 is the 
collection of individuals who are challenged with S. flexneri 2a first followed by S. sonnei. A 
group is the subset of the cohort that is admitted to the unit at any given time (4 groups, 1A, 1B, 
2A, and 2B). If more than 21 volunteers from 1A or 2A are willing to be included in 1B or 2B, 21 
subjects will be randomly selected from the eligible subjects. Due to isolation unit constraints, 
there needs to be an even number of males and females to fill the 6 same-gender shared rooms. 
 
͵Ǥ 
 
Safety population 
All subjects who have been screened, consented, and proceeded to receive the challenge strain.  
This population will serve as the primary analysis population for the safety endpoints. 
 
Per protocol population 

 
 
Protocol v4.2 
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All participants in the Safety population who have no major protocol violations that are 
determined to potentially interfere with the immunogenicity assessment.  This population will 
serve as the primary analysis population for the immunogenicity endpoints.  The population will 
be adapted by time point to include all eligible subjects’ data up to the time of the disqualifying 
protocol deviation. Membership in this study population will be determined in a blinded fashion. 
 
ͶǤ 
A statistical analysis plan (SAP) for this study is in development and will be finalized prior to 
database lock. Safety and immunogenicity populations will both be comprised of subjects who 
have been screened, consented, and proceeded to receive the challenge strain. Missing data are 
expected to be minimal, no imputation will be performed for missing values and data will be 
assumed to be missing at random. SAS version 9.4 (Cary, NC) will be used to complete study 
analyses. Due to the descriptive nature of this study no adjustments for multiple testing will be 
performed. 
 

Baseline data such as demographics, medical history, as well as disposition of the volunteers 
will be summarized in tables or text in the final study report, as per the details in the SAP.  
 

Each day of the inpatient period, subjects will be monitored for loose stools (grade 3-5 based 
on assessment by the clinical team that do not meet the study’s diarrhea definition), diarrhea, 
nausea, vomiting, abdominal pain or cramps, fever, headache, myalgia, arthralgia, and 
malaise along with the other solicited symptoms listed above.  
 
Attack rates of shigellosis will be calculated for all study groups using the standard definition 
as follows: (# with endpoint / # receiving inoculum) x 100%. These attack rates will be 
estimated along with 95% confidence intervals (both asymptotic estimates and exact) for each 
study group. Attack rates between Shigella naïve participants and previously challenged 
participants will be compared using a Pearson’s chi-squared or Fisher’s exact test, as 
appropriate. 
 

All AEs will be summarized and compared between groups. Safety data, including 
reactogenicity, AEs, stool information, specified vital signs, abnormal physical exam findings 
and laboratory tests will be listed by study subject. This information will be summarized in 
data tables as per the SAP. Additionally, tables will be prepared to list each commonly 
observed AE, the number of subjects who experienced an event at least once, and the rate of 
subjects with AEs. All AEs will be divided into defined severity grades (mild, moderate, 
severe, and life-threatening).  There will be separate summaries of SAEs, as well as severe 
AEs, AEs of special interest, SUSARs, etc. should they occur.  
 
Descriptive statistics (n, mean, standard deviation, median and ranges for continuous 
variables, percentages for categorical variables) will be compiled for each group. 
 

Immunological outcomes will be summarized in a tabular format and graphed to demonstrate 
kinetics of response. Qualitative (responder rates) and quantitative assessments (log 
transformed values) will be analyzed. Median increases (fold rises) of antibody 
concentrations and seroconversion rates will be calculated along with their 95% confidence 
intervals. Geometric mean titers will also be determined and presented with their 95% 

 
 
Protocol v4.2 
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60
confidence intervals.  For each assay, the fold-increase will be considered a response, or the 
threshold for responders or non-responders will be pre-determined if possible. This will be 
described in greater detail in the SAP. 
 
Comparisons across groups of single immune parameters reported as continuous variables 
(e.g., reciprocal log10 titers) will be made utilizing t-tests and one-way analysis of variance, 
Wilcoxon rank-sum, or Kruskal-Wallis H tests, as appropriate. Comparisons of responses 
within groups over time will be made utilizing appropriate paired analyses. Immune 
parameters that are nominal in nature (e.g., responder status) will be compared using chi-
squared or exact tests, as appropriate. Additionally, comparisons of immune response 
profiles, assessing multiple immune parameters simultaneously, will be explored utilizing 
multivariable regression models, principal component analyses, and/or other multivariable 
methods, as appropriate. 
 
All hypothesis testing will be conducted using a 2-sided alpha=0.05. 
 
Further details of the statistical analysis plan for secondary and tertiary endpoints, if relevant, 
will be included in the SAP.  
 
ͷǤ 
Presuming a shigellosis rate of 70% in naïve participants, a sample size of 16 participants will 
yield 95% confidence intervals (asymptotic estimates) of 48-92%, while a sample size of 8 
participants will yield 95% confidence interval (asymptotic estimates) of 38-100%. 
Presuming an ability to pool naïve participants (if the cohorts have a comparable attack rate), 
a sample size of 19 participants in the previously S. sonnei 53G  challenged arm and 16 
participants in the S. flexneri 2457T naïve arm provides a >80% power to detect a shigellosis 
risk difference of 46% presuming a ≥70% attack rate in Shigella naïve participants, while a 
sample size of 11 participants in the previously S. flexneri  2457T  challenged arm and 8 
participants in the S. sonnei 53G naïve arm provides a >80% power to detect a shigellosis risk 
difference of 60% presuming a ≥70% attack rate in Shigella naïve participants  (based on a 
Pearson’s chi-squared test for proportional differences and a 2-sided alpha=0.05). A power 
curve, corresponding to a sample size of 19 participants in the previously S. sonnei 53G 
challenged arm and 16 participants in the S. flexneri 2457T naïve arm and an attack rate of 
70% in Shigella naïve participants is provided below.  
 
 
0
20
40
60
80
100
0.1
0.2
0.3
0.4
0.5
Power (%)
Attack Rate in Previously Shigella Challenged Participants
Power (%) vs. Attack Rate in Previously Shigella
Challenged Participants

 
 
Protocol v4.2 
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61
 
 
 
XVI. 
OBLIGATIONS AND ROLES OF THE SPONSOR, INVESTIGATOR, AND 
STUDY PERSONNEL 
This study will be conducted using GCP and in accordance with all federal regulations regarding the 
protection of human participants in research including The Nuremberg Code, The Belmont Report, 
US 21 CFR Part 50 – Protection of Human Subjects, 32 CFR 219 (The Common Rule) and all 
regulations pertinent to the Department of Defense. 
The investigators agree to conduct the research in strict accordance with this protocol, the ICH 
Guideline for GCP (CPMP/ICH/135/95), as well as in conformity with any federal, provincial or local 
regulations regarding the conduct of clinical studies. The Sponsor and investigator must comply with 
all applicable regulations. In addition, the investigator must follow local and institutional 
requirements including, but not limited to, IP, clinical research, informed consent and IRB 
regulations. The Sponsor will provide notification to the investigator of protocol and amendment 
approvals by regulatory authorities when applicable. Except where the investigator's signature is 
specifically required, it is understood that the term "investigator" as used in this protocol and on 
source documents refers to the investigator or appropriate study personnel that the investigator 
designates to perform a certain duty. The investigator is ultimately responsible for the conduct of all 
aspects of the study. Sub-investigators or other appropriate study personnel are eligible to sign for the 
investigator on designated source documents. 
All amendments to the protocol, consent form and/or any IRB approved documents, including a 
change of PI, will be submitted to the Sponsor and IRB(s) for review and approval prior to 
implementation. 
XVII. 
QUALITY CONTROL AND ASSURANCE 
During the study, the investigator will maintain complete and accurate documentation of the study, 
including records detailing the progress of the study for each participant, laboratory reports, eCRFs, 
signed informed consent forms for each study participant, investigational product disposition records, 
correspondence with the IRB, the study monitor and the Sponsor, adverse event reports and 
information regarding participant discontinuation and completion of the study. All required study data 
will be clearly and accurately recorded by authorized study personnel in the eCRFs. Only designated 
study site personnel shall record or change data in an eCRF. During the study, the investigator will be 
responsible for the procurement of data and for quality of data recorded in the eCRFs. The study 
monitor will ensure accuracy of the eCRFs. 
XVIII. 
PROTOCOL DEVIATIONS 
Major protocol deviations are a subset of protocol deviations that may significantly impact the 
completeness, accuracy, and/or reliability of the study data or that may significantly affect a subject's 
rights, safety, or well-being.  Non-emergent/minor deviations are routine departures that typically 
involve a participant’s failure to comply with the protocol. Examples: missing scheduled visits; 
failing to complete required questionnaire.  
Other-than-minimal-risk changes and all unanticipated major problems involving human subjects or 
others will be reported promptly to the IRBs, and no such changes will be made to the research 
without IRB approval unless necessary to eliminate apparent immediate hazards to human subjects. 

 
 
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Minor minimal-risk deviations necessitated during the course of the trial will be made on site as 
needed and documented for subsequent review within a reasonable time period. Deviations from the 
protocol that potentially impact participant safety will be promptly reported to the Research Monitor, 
IRB, and PATH ORA. Other deviations will be reported at the time of continuing review. 
XIX. 
PROTECTION OF HUMAN SUBJECTS 
ͳǤ 
There are limited funds available to the study for care of study-related injury. Although the 
investigator and Sponsor will make every effort to cover the costs of any study-related injury, full 
coverage cannot be guaranteed, and uncovered costs may fall on participants or their insurers. 
Therefore, participants will be informed that there are limited funds to cover study-related injury 
care costs and that there is the possibility that they may ultimately be responsible for at least some 
of the cost of care. 
 
The Sponsor will obtain and maintain appropriate and commercially reasonable amounts of 
insurance, including commercial general liability insurance, and product and completed 
operations liability insurance, including coverage for the clinical trial. 
 
ʹǤ Ȁ
Risks Associated with Shigella Infection: Naturally-acquired illness caused by S. sonnei and S. 
flexneri 2a ranges from mild-to-severe watery diarrhea that may contain mucus or blood. Nausea, 
vomiting, abdominal cramping, headache, abdominal gurgling or gas, anorexia, fever, muscle 
and/or joint aches, and malaise may occur. For most adults, the illness is not life threatening but 
often leads to mild-to-moderate dehydration and significant inconvenience associated with loss of 
sleep and activity. Study facilities will have personnel and resources capable to manage diarrheal 
illness and potential complications. 
Potential risks associated with Shigella infection/challenge are not limited to the acute effects of 
disease, but might extend to post-infectious conditions, like reactive arthritis (characterized by 
symptoms including inflamed joints and inflammation of the genital, urinary, or gastrointestinal 
systems) or irritable bowel syndrome (IBS) (23). In order to limit the risk of post-infection 
conditions, history of autoimmune disease or inflammatory arthritis and positive tests for HLA-
B27 (a marker associated with reactive arthritis) are checked at screening as exclusion criteria. In 
addition, a functional bowel disorder survey will be conducted at screening and at Day 180/270. 
Participants with reported prior history of abnormal bowel patterns who might be at higher risk of 
this post-infectious sequelae will be excluded. 
Risks Associated with Transmission of Challenge: The Shigella strains have the potential for 
risks to both the environment and to the research personnel; however, the risk to the environment 
in regard to potential transmission outside of the CIR facility is low. There is a minimal risk of 
acquiring Shigella infection associated with participant inoculum administration, patient care 
activities on the ward, or processing Shigella-infected stool. The risk to the environment will be 
reduced by ensuring that all human waste products from inpatients are disinfected with bleach 
prior to disposal, ensuring all participants comply with discharge criteria (two consecutive 
negative stool cultures for Shigella), emphasizing the importance of handwashing for participants 
and staff, ensuring proper disposal/cleaning of linen, and cohorting participants in the CIR while 
shedding Shigella. Participants will be instructed as to the importance of completing the 3- or 5-
day course of antibiotics and this instruction will be documented. 
 
Risks Associated with Antibiotics: Therapeutic antibiotics for use in this study are licensed, 
approved medications that have been used extensively, and shown to be very safe with only rare 

 
 
Protocol v4.2 
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side effects. The most commonly reported side effects for ciprofloxacin are gastrointestinal 
symptoms (nausea, vomiting, and diarrhea) in as many as 5 persons in 100. Other reported 
symptoms in less than 1 person in 100 include rash, dizziness, and headache. Rarely, allergic 
reactions to these medications have been observed. Ciprofloxacin is not recommended for use in 
pregnancy due to concerns of joint damage to the unborn child (based on studies in young 
animals). Pregnancy is exclusionary for study participation and is documented through testing 
prior to study interventions and provided discussion on methods to prevent pregnancy during 
study. Fluoroquinolones, including ciprofloxacin, are associated with an increased risk of 
tendonitis and tendon rupture in all ages. The risk of developing fluoroquinolone-associated 
tendonitis and tendon rupture is further increased in older patients, usually over 60 years of age, 
in patients taking corticosteroid drugs, and in patients with kidney, heart, or lung transplants, all 
of whom are excluded from this study. Clostridium difficile-associated diarrhea (CDAD/ 
pseudomembranous colitis) has been reported with use of nearly all antibacterial agents. 
The most common adverse effects of trimethoprim-sulfamethoxazole are gastrointestinal (nausea, 
vomiting, anorexia), and allergic skin reactions (rash and urticaria). Rare effects can be severe 
allergic reactions, such as Stevens-Johnson syndrome, toxic epidermal necrolysis, hepatitis, and 
hematologic abnormalities. 
The most common adverse reactions (>1%) of ampicillin are diarrhea, vomiting, and nausea, 
rash. 
Risks Associated with Saliva Collection: Risks occasionally associated with saliva collection 
include discomfort and (theoretically) choking. 
 
Risks Associated with Nasal Swab: Risks occasionally associated with nasal swabs may include 
discomfort with procedure, nasal irritation, or bleeding. 
 
Risks Associated with Venipuncture or IVs: Risks occasionally associated with venipuncture 
include pain and bruising at the site of venipuncture, bleeding, infection (occasionally), 
lightheadedness, and syncope (rarely). IVs may also infiltrate and cause swelling and discomfort. 
 
Risks Associated with Wearable Data Collection: Risks may include feeling uncomfortable about 
wearing a wrist device in public. The wrist band may also cause some minor wrist irritation.  
 
Risks with Isolation: Volunteers may feel bored or anxious about not being able to leave the unit 
or being separated from family, friends, and community while they are on the unit. The unit will 
be staffed 24/7 with medical and security staff. There will be planned activities to participate in or 
not. There are big screen TVs throughout the unit, games, and a kitchen and dining area. 
 
Risks Associated with Laboratory Results: There may be physical, psychological, and social risks 
if participants test positive for hepatitis B, hepatitis C, and/or HIV. Participants testing positive 
will be counseled and referred for treatment. 
 
Risks Associated with Collection of Stool: There is no risk associated with collecting stool 
samples; however, risks of using rectal swabs could include mild discomfort, embarrassment, and 
(very rarely) bleeding or irritation. 
 
Risks Associated with Post-Infectious Irritable Bowel Syndrome (PI-IBS): Recent studies also 
suggest an increased risk of PI-IBS following bacterial enteritis. PI-IBS, a functional bowel 
disorder characterized by unexplained abdominal discomfort or pain associated with changes in 
normal bowel patterns, has been described in a recent systematic review to occur 6-7 times more 
frequently after an acute enteric infection compared to similar matched controls without such a 

 
 
Protocol v4.2 
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history [33]. 
 
Risk of Breach of Confidentiality: A breach of confidentiality in which private health information 
is made public is possible.  Medical records associated with this protocol are subject to provisions 
of the Privacy Act of 1974, 5 U.S.C., Section 552A, and AR 340-21.  All data and medical 
information obtained about participants will be considered privileged and held in confidence.  
Participants will not be identified by name in any published report/presentation of the results.  
The Sponsor and the FDA may inspect the records of this research as part of their responsibility 
to oversee research and ensure protection of participants.  Study results and data may be 
published in scientific/medical journals; the identity of individual participants will not be 
disclosed. 
Risk of SARS-CoV-2 Exposure and Risk Mitigation: SARS-CoV-2, the virus that causes 
COVID-19 infections and disease, is the cause of the current global pandemic that started in 
2020. COVID-19 can cause respiratory symptoms and pulmonary disease.  Symptoms include 
fever, increased cough, shortness of breath, trouble breathing, fatigue, chills, body aches, 
headache, sore throat, congestion/runny nose, loss of taste or smell, nausea, and diarrhea. The 
virus can lead to more severe illness including pneumonia, respiratory failure, heart problems, 
liver problems, blood clots, septic shock, and death. 
 
It is the intent of the Center for Immunization Research to mitigate risk and provide a safe 
environment for participants while they are inpatient on the unit during the study. The CIR will 
educate and encourage all potential study volunteers to be fully vaccinated with one of the 
COVID licensed/EUA vaccines prior to enrolling in this study. Volunteers will not be required to 
be vaccinated against SARS-CoV-2 however vaccinated volunteers will be prioritized for 
enrollment. 
 
Pandemic restrictions for vaccinated and unvaccinated persons continue to change based on 
positivity rates and CDC recommendations. The CIR will follow recommendations from the CDC 
when implementing clinical trials. The CIR will also follow institutional guidelines and policies. 
Current recommendations include vaccination with one of the licensed/EUA vaccines for all 
adults.  
 
Unvaccinated volunteers will be educated on the risks of not being vaccinated. They will sign a 
disclaimer regarding vaccination status and possible exposure to the virus. Unvaccinated 
volunteers will be strongly encouraged to wear a mask and maintain social distance from others.  
Depending on the level of COVID illness in the community and current CDC and JHU 
guidelines, volunteers may be asked to obtain a COVID-19 test 1-5 days prior to admission.  
 
Every attempt will be made to enroll cohorts of exclusively vaccinated or unvaccinated 
volunteers. All volunteers will be screened for symptoms and tested on arrival to the unit on the 
day of inpatient admission. Potential volunteers who test positive for SARS-CoV-2 will not be 
eligible for enrollment. 
 
For staff working on the unit, vaccination will be required.  
 
As CDC recommendations evolve and change, we will continue to update our plan to incorporate 
updated CDC recommendations. The addition of precautions or reduced precautions may be 
instituted throughout the study, updated recommendations will be discussed with volunteers and 
posted in the unit. 
 

 
 
Protocol v4.2 
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65
͵Ǥ 
Participants will be questioned and examined daily for evidence of infection and diarrhea 
complications. Vital signs will be recorded at least three times per day. Based on prior studies, 
infected participants tend to develop illness with incubation periods of approximately 1-3 days. 
The risk of diarrhea complications will be minimized by a conservative approach to timing of 
antibiotic administration, well within an interval that has been shown to be efficacious, as well as 
daily clinical monitoring. Stool output will be closely monitored. All participants will be treated 
no later than day 5 post-dosing. 
Aggressive fluid management will be undertaken to ensure the most common complication, 
dehydration, does not occur. The procedures to institute early oral and/or intravenous rehydration 
therapy are detailed above. In addition to rehydration therapy, prospectively defined criteria and 
procedures to institute early antibiotic therapy are also fully described above. In order to ensure 
clinical resolution and limit the potential for secondary spread upon discharge, predefined 
discharge criteria have been established. Participants will be discharged from the inpatient phase 
of the study when clinical symptoms are resolved or resolving, they have received at least 2 doses 
of antibiotics, AND they have two consecutive stool cultures negative for Shigella. 
Systemic or severe gastrointestinal complications rarely occur with Shigella infection. The 
following clinical findings necessitate immediate consideration and management of complicated 
enteritis: 
x Physical examination compatible with an acute abdomen 
x Severe GI bleeding (any evidence of GI blood loss other than hemoccult positivity only, with 
evidence of hemodynamic instability, decrease in hemoglobin, hypovolemia) 
x Sepsis (high fever: temperature >102ºF [39ºC], rigors, hemodynamic instability) 
 
Any of these findings require prompt clinical management and discussion with the independent 
Research Monitor and the Sponsor Medical Officer. 
 
The Shigella strain has the potential for risk to both the environment and to the research 
personnel; however, the risk to the environment in regard to potential transmission outside of the 
CIR facility is low. The risk to the environment will be reduced by: 
x ensuring that all human waste products from inpatients are disinfected with bleach prior to 
disposal 
x ensuring all participants comply with discharge criteria, emphasizing the importance of 
handwashing for participants and staff 
x ensuring proper disposal/cleaning of linen, and cohorting participants in the CIR while 
shedding 
x ensuring participants will not be discharged until they are no longer shedding the challenge 
strain, as per procedures outlined in the protocol 
 
Participants with prior history of abnormal bowel patterns who might be at higher risk of post-
infectious sequelae are excluded. Predefined criteria to assure early treatment as appropriate also 
may further reduce risk of post-infectious sequelae and is likely to reduce the risk associated with 
PI-IBS given the positive association between diarrheal illness duration and PI-IBS risk [34]. 
 
It is important for this study that volunteers return for the second challenge if they are invited to 
do so. We will work with each volunteer to stress this importance, make sure that they are 
available for both challenges, and address any questions or concerns that they have.  It is also 
important for participants to return for the outpatient visits, and this will be stressed repeatedly 
with volunteers. We also have some flexibility if volunteers need to alter the dates of outpatient 
visits. 

 
 
Protocol v4.2 
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ͶǤ 
There is no benefit that can be guaranteed to participants for participating in this research study. 
However, there is a potential societal benefit in the development of a vaccine to prevent Shigella. 
XX. 
INFORMED CONSENT 
The informed consent process and document(s) will be reviewed and approved by the JHSPH IRB 
prior to initiation of the study. The consent document(s) will contain a full explanation of the possible 
risks, advantages, and alternate treatment options, and availability of treatment in the case of injury, 
in accordance with 21 CFR 50. Participants will receive an oral presentation of the study in language 
(i.e., using lay terms as appropriate) they can understand. Questions on the purpose of the protocol, 
protocol procedures, and risks to the participants will then be solicited. Any question that cannot be 
answered will be referred to the PI. The participant will be allowed to take the consent document 
home to consider and discuss it with others. The participant should understand that the study products 
are investigational and are not licensed by the FDA for commercial use but are permitted to be used 
in this clinical research. Informed consent includes the principle that it is critical the participant be 
informed about the principal potential risks and benefits. This information will allow the participant 
to make a personal risk versus benefit decision and understand the following: 
 
x Participation is entirely voluntary. 
x Participants may withdraw from participation at any time. 
x Refusal to participate involves no penalty. 
x The individual is free to ask any questions that will allow him/her to understand the nature of the 
protocol. 
 
Participants will be given the written, IRB-approved informed consent, allowed ample time to read 
the consent, allowed to ask questions about the study, have the questions answered, and given time to 
decide if he/she would like to participate in the study. To document participants’ understanding of 
informed consent, immediately before the consent is signed, the person obtaining consent will 
administer a brief comprehension test. A participant must achieve ≥70% correct to be eligible for 
inclusion in the study. Incorrect answers will be discussed with participants to reinforce the consent. 
Participants who fail the comprehension test on the first attempt will be given one additional 
opportunity, either on the same day or another day, to take the test after reviewing the quiz, and re-
reading the consent. Participants failing the comprehension test on the second attempt are not eligible 
for study enrollment. No coercion or influence is allowed in obtaining participants’ consent. 
 
A written informed consent document, in compliance with 21 CFR Part 50, 32 CFR Part 219, the 
Belmont Principles will be signed and dated by participants as well as by the PI or designee before 
any study-related procedures are initiated. This consent document must be retained by the investigator 
as part of the study records. Participants will receive copies of the signed consent prior to 
participation.  As part of the consent process, participants will also be asked to read and sign a 
Medical Records/Lab Results Release, with an opportunity to ask questions, if relevant. Participants 
will also be asked to sign a separate information form for HIV-1 testing. The consent document 
indicates that by signature, the participant, or where appropriate, legal guardian, permits access to 
relevant medical records by the Sponsor’s representative and by representatives of the FDA. 
 
The Sponsor’s representative will submit a copy of the initial IRB- and Sponsor’s representative-
approved consent form to the FDA and will maintain copies of revised consent documents that have 
been reviewed and approved by the IRB/ethics committee. 
 

 
 
Protocol v4.2 
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A description of this clinical trial will be available on http://www.ClinicalTrials.gov, as required by 
US law. 
 
All non-exempt research involving human participants shall, at a minimum, meet the requirement of 
32 CFR 219.116(a)(6) in the Code of Federal Regulations. 
 
ͳǤ ǣ
This study will not include individuals less than 18 years of age, incarcerated, or unable to meet 
the requirements to sign the informed consent form. 
ʹǤ 
A multimedia approach to recruitment will be taken, including the use of social media. 
Additionally, participants in previous studies that have expressed interest in participating in future 
trials will be contacted about the proposed study. All study-related advertisements will be 
reviewed and approved by the JHSPH IRB, NMRC IRB and Human Research Protections Office 
-Office of Research Protection, if applicable. Participants responding to the advertisements by a 
phone call to the center will be screened for eligibility based on a standard screening 
questionnaire administered by the CIR recruiter. Some elements of the inclusion/exclusion 
criteria will be discussed with the participant at that time and a preliminary determination will be 
made regarding the individual’s eligibility for study participation. Active duty military members 
will not specifically be recruited for this study. 
XXI. 
COMPENSATION 
ͳǤ 
Compensation will occur as detailed below. Compensation will be provided only for completed 
study procedures designated for compensatory payment. If a participant is eligible to 
participate in the investigational protocol after screening, and s/he completes all study 
visits, procedures and follows all the rules s/he will receive the following compensation: 
 
For participants who complete one inpatient phase: 
x 
$100 total for screening (only if enrolled in the study or presents as an alternate) 
x 
$3,150 for each inpatient period (as long as all study requirements are met) 
x 
$300 for each of the outpatient study visits 
x 
$200 bonus for those who complete only 1 challenge including outpatient visits, and  
x 
$300 bonus for those who complete 2 challenges including outpatient visits 
If a participant completes the study according to the protocol and does not stay additional days on 
the inpatient unit, total compensation is $4,950 for participation in one challenge, and $9,100 for 
participation in 2 challenges. 
Volunteers that present to the unit as an alternate but are not admitted will receive $100 for 
screening (if not already) and $200 for presenting to the unit (a total of $300). Volunteers who are 
admitted to the unit either for the first or second challenge and are discharged prior to challenge 
will receive $100 for screening (if not already received) and $250 for each full day on the 
inpatient unit.  
If a participant is not eligible for discharge on day 9 because of illness or not having 2 
consecutive negative stool culture results s/he will receive $350 per additional inpatient day. 

 
 
Protocol v4.2 
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68
Participants will not be paid for missed outpatient visits and may forfeit some or all of their bonus 
as a result of missed/late visits or non-compliance. 
ʹǤ 
There is no financial compensation for investigators in this study. All investigators will be 
required to complete a form for the disclosure of significant financial interest. 
͵Ǥ 	
Participants will not be discriminated against on the basis of race, sex, or religion. Due to the 
nature of the study, we have excluded individuals under 18 and women who are pregnant or 
nursing and we have excluded individuals who are over the age of 50 due to the frequency of 
exclusionary medical conditions. Any individual who is unable to consent due to any reason will 
not be included in this study. 
XXII. 
PRIVACY AND CONFIDENTIALITY 
ͳǤ 
All records pertaining to this protocol will be stored in a locked filing cabinet at JHU or at an 
offsite, locked storage facility per regulations for a minimum of 5 years.  Access to these records 
will be limited to researchers in the Translational and Clinical Research Department at NMRC, 
the JHU CIR and the Sponsor as well as those responsible for regulatory monitoring of data to 
include representatives of the Sponsor, DoD and JHU. The investigator will obtain permission 
from the Sponsor in writing before destroying any study records and the Sponsor will notify the 
investigator in writing when records can be destroyed. Relevant IRBs will be notified in writing 
prior to destruction of any research records.  Specimens will be stored indefinitely in the JHU or 
the Shigella laboratory at WRAIR. 
ʹǤ 
Individual participant medical information obtained as a result of this study is considered 
confidential and disclosure to third parties, other than those cited below, is prohibited. 
Participant confidentiality will be further ensured by utilizing participant identification 
code numbers and participant initials. Neither the Sponsor, NMRC nor the JHSPH are 
HIPAA-covered entities. 
Confidentiality agreements may be developed with other clinical trials groups (e.g., at the 
University of Maryland Vaccine Research Center or Walter Reed Clinical Trials Center), and the 
investigative team may check verbally with these sites to see if participants have participated 
in studies that would preclude their participation in this study. No written list will be 
exchanged with these sites. 
XXIII. 
PROTOCOL REVIEW PROCESS 
The protocol will undergo scientific review at PATH and NMRC, and ethical review at the CIR. In 
addition to these reviews, the JHU Biosafety Committee and Pharmacy and Therapeutics Committee 
will review the protocol. The protocol will also require FDA review as part of the IND application. 
The IND Sponsor will be PATH. Continuing review will be undertaken in accordance with existing 
regulations. 
XXIV. 
PUBLICATION POLICY 
Results of this study will be presented and published after review by the Sponsor in an open-access 

 
 
Protocol v4.2 
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69
journal as per the guidelines of PATH and the Bill and Melinda Gates Foundation. 
XXV. 
CONFLICT OF INTEREST POLICY 
The independence of this study from any actual or perceived influence, such as by the pharmaceutical 
industry, is critical. Therefore, any actual conflict of interest of persons who have a role in the design, 
conduct, analysis, publication, or any aspect of this trial will be disclosed and managed. Furthermore, 
persons who have a perceived conflict of interest will be required to have such conflicts managed in a 
way that is appropriate to their participation in the design and conduct of this trial. The study 
leadership in conjunction with JHU has established policies and procedures for all study group 
members to disclose all conflicts of interest and will establish a mechanism for the management of all 
reported dualities of interest. 

 
 
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XXVI. 
REFERENCES 
1. 
MacLennan, C.A., et al., Consensus Report on Shigella Controlled Human Infection 
Model: Clinical Endpoints. Clin Infect Dis., 2019. 69(Supplement_8): p. S591-S595. doi: 
10.1093/cid/ciz891. 
2. 
Kaminski, R.W., et al., Consensus Report on Shigella Controlled Human Infection 
Model: Immunological Assays. Clin Infect Dis., 2019. 69(Supplement_8): p. S596-S601. 
doi: 10.1093/cid/ciz909. 
3. 
Talaat, K.R., et al., Consensus Report on Shigella Controlled Human Infection Model: 
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4. 
Porter, C.K., et al., Clinical endpoints in the controlled human challenge model for 
Shigella: A call for standardization and the development of a disease severity score. 
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2018. 
5. 
Hardy, A.V. and J. Watt, Studies of the acute diarrheal diseases; etiology. Public Health 
Reports, 1945. 60(3): p. 57-84. 
6. 
Kotloff, K.L., et al., Global burden of Shigella infections: implications for vaccine 
development and implementation of control strategies. Bull World Health Organ, 1999. 
77(8): p. 651-66. 
7. 
Kotloff, K.L., et al., Burden and aetiology of diarrhoeal disease in infants and young 
children in developing countries (the Global Enteric Multicenter Study, GEMS): a 
prospective, case-control study. Lancet, 2013. 382(9888): p. 209-22. 
8. 
Liu, J., et al., Use of quantitative molecular diagnostic methods to identify causes of 
diarrhoea in children: a reanalysis of the GEMS case-control study. Lancet., 2016. 
388(10051): p. 1291-301. doi: 10.1016/S0140-6736(16)31529-X. 
9. 
Platts-Mills, J.A., et al., Pathogen-specific burdens of community diarrhoea in developing 
countries: a multisite birth cohort study (MAL-ED). Lancet Glob Health., 2015. 3(9): p. 
e564-75. doi: 10.1016/S2214-109X(15)00151-5. Epub 2015 Jul 19. 
10. 
Estimates of global, regional, and national morbidity, mortality, and aetiologies of 
diarrhoeal diseases: a systematic analysis for the Global Burden of Disease Study 2015. 
Lancet Infect Dis., 2017. 17(9): p. 909-948. doi: 10.1016/S1473-3099(17)30276-1. Epub 
2017 Jun 1. 
11. 
Kotloff, K.L., et al., Shigellosis. Lancet., 2018. 391(10122): p. 801-812. doi: 
10.1016/S0140-6736(17)33296-8. Epub 2017 Dec 16. 
12. 
Khalil, I.A., et al., Morbidity and mortality due to shigella and enterotoxigenic 
Escherichia coli diarrhoea: the Global Burden of Disease Study 1990-2016. Lancet 
Infect Dis, 2018. 18(11): p. 1229-40. 
13. 
Black, R.E., K.H. Brown, and S. Becker, Effects of diarrhea associated with specific 
enteropathogens on the growth of children in rural Bangladesh. Pediatrics., 1984. 73(6): 
p. 799-805. 
14. 
Lee, G., et al., Effects of Shigella-, Campylobacter- and ETEC-associated Diarrhea on 
Childhood Growth. Pediatr Infect Dis J, 2014. 33(10): p. 1004-1009. 
15. 
Olson, S., et al., Travelers' diarrhea: update on the incidence, etiology and risk in 
military and similar populations - 1990-2005 versus 2005-2015, does a decade make a 
difference? Trop Dis Travel Med Vaccines., 2019. 5:1.(doi): p. 10.1186/s40794-018-
0077-1. eCollection 2019. 

 
 
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16. 
Shaughnessy, H.J., R.C. Olsson, and et al., Experimental human bacillary dysentery; 
polyvalent dysentery vaccine in its prevention. J Am Med Assoc., 1946. 132: p. 362-8. 
17. 
DuPont, H.L., et al., The response of man to virulent Shigella flexneri 2a. J Infect Dis., 
1969. 119(3): p. 296-9. 
18. 
DuPont, H.L., et al., Immunity in shigellosis. II. Protection induced by oral live vaccine 
or primary infection. J Infect Dis., 1972. 125(1): p. 12-16. 
19. 
Porter, C.K., et al., The Shigella human challenge model. Epidemiol Infect., 2013. 
141(2): p. 223-32. doi: 10.1017/S0950268812001677. Epub 2012 Aug 21. 
20. 
Black, R.E., et al., Prevention of shigellosis by a Salmonella typhi-Shigella sonnei 
bivalent vaccine. J Infect Dis, 1987. 155(6): p. 1260-5. 
21. 
DuPont, H.L., et al., Inoculum size in shigellosis and implications for expected mode of 
transmission. Journal of Infectious Disease, 1989. 159(6): p. 1126-8. 
22. 
Coster, T.S., et al., Vaccination against shigellosis with attenuated Shigella flexneri 2a 
strain SC602. Infect Immun, 1999. 67(7): p. 3437-43. 
23. 
Kotloff, K.L., et al., Evaluation of the safety, immunogenicity, and efficacy in healthy 
adults of four doses of live oral hybrid Escherichia coli-Shigella flexneri 2a vaccine 
strain EcSf2a-2. Vaccine, 1995. 13(5): p. 495-502. 
24. 
Levine, M.M., et al., Pathogenesis of Shigella dysenteriae 1 (Shiga) dysentery. Journal of 
Infectious Diseases, 1973. 127(3): p. 261-70. 
25. 
Harro, C., et al. Shigella flexneri 2a Invaplex 50 intranasal vaccine phase 2b challenge 
study. in 5th International Vaccines for Enteric Diseases Conference. 2009. Malaga, 
Spain. 
26. 
Talaat, K.R., et al. Flexyn2a, a candidate bioconjugate vaccine against Shigella flexneri 
2a induces protective immune response in a controlled human infection model. in 
Vaccines for Enteric Diseases. 2017. Albufeira, Portugal. 
27. 
Samandari, T., et al., Production of IFN-gamma and IL-10 to Shigella invasins by 
mononuclear cells from volunteers orally inoculated with a Shiga toxin-deleted Shigella 
dysenteriae type 1 strain. (0022-1767 (Print)). 
28. 
Bodhidatta, L., et al., Establishment of a Shigella sonnei human challenge model in 
Thailand. Vaccine., 2012. 30(49): p. 7040-5. doi: 10.1016/j.vaccine.2012.09.061. Epub 
2012 Oct 12. 
29. 
Pitisuttithum, P., et al., Clinical Trial of an Oral Live Shigella sonnei Vaccine Candidate, 
WRSS1, in Thai Adults. Clin Vaccine Immunol., 2016. 23(7): p. 564-75. doi: 
10.1128/CVI.00665-15. Print 2016 Jul. 
30. 
Kotloff, K.L., et al., A modified Shigella volunteer challenge model in which the 
inoculum is administered with bicarbonate buffer: clinical experience and implications 
for Shigella infectivity. Vaccine, 1995. 13(16): p. 1488-94. 
31. 
Taylor, D.N., et al., Rifaximin, a nonabsorbed oral antibiotic, prevents shigellosis after 
experimental challenge. Clin Infect Dis, 2006. 42(9): p. 1283-8. 
32. 
Frenck, R.W., Jr., et al., Establishment of a Controlled Human Infection Model with a 
Lyophilized Strain of Shigella sonnei 53G. mSphere, 2020. 5(5). 
33. 
Thabane, M., D.T. Kottachchi, and J.K. Marshall, Systematic review and meta-analysis: 
The incidence and prognosis of post-infectious irritable bowel syndrome. Aliment 
Pharmacol Ther, 2007. 26(4): p. 535-44. 
34. 
Spiller, R. and K. Garsed, Postinfectious irritable bowel syndrome. Gastroenterology, 
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35.  
Levine MM, Kotloff KL, Barry EM, Pasetti MF, Sztein MB. 2007. Clinical trials of 
Shigella vaccines: two steps forward and one step back on a long, hard road. Nat. Rev. 
Microbiol. 5(7):540-554. doi:10.1038/nrmicro1662. 

 
 
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Table 9: Schedule of Procedures for Subjects Receiving One Challenge 
Study Phase 
Outpatient 
Inpatient 
Outpatient 
Study Day 
Screening 
Screening 
-2 to -1 
1 
2 
3 
4 
5 
6 
7 
8 
9 
15 
29 
57 
90 
180 
Compliance Range 
(Days) 
Day -60 to 
Day -2 
Day -30 
to Day -2 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
±2 
±4 
±4 
-7/ 
+28 
±28 
Review Inclusion 
/Exclusion Criteria 
X 
 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
JH 200 screening 
consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Study 
briefing/Informed 
Consent 
(X) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Comprehension 
assessment 
(X) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HIV testing 
information sheet 
(X) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Functional Bowel 
Survey 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Medical History 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical 
examination/Focused 
PE 
(X) 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
(X) 
(X) 
(X) 
(X) 
(X) 
Vital Signs (BP, HR, 
Temp) 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Screening Titers to S. 
flexneri and S. sonnei 
IgG levels 
X3 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Hematology/CBC 
w/diff 
 
X 
X1 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
Basic Metabolic panel, 
ALT, AST 
 
X 
X1 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
Urine for toxicology 
testing 
X 
(X) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Hepatitis B, C, and 
HIV  
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Serum/Urine 
Pregnancy Test 
(X) 
X 
X 
(X) 
 
 
 
 
 
 
 
 
 
X 
 
 
 
IgA  
X 
(X) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HLA-B27 
antigen/ABO typing 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
COVID-19 testing 
 
X2 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Concomitant 
Medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Clinical 
Check/Medical 
Interview 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Collection of AEs4 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 

 
 
Protocol v4.2 
16 JAN 2023 
74 
Study Phase 
Outpatient 
Inpatient 
Outpatient 
Study Day 
Screening 
Screening 
-2 to -1 
1 
2 
3 
4 
5 
6 
7 
8 
9 
15 
29 
57 
90 
180 
Compliance Range 
(Days) 
Day -60 to 
Day -2 
Day -30 
to Day -2 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
±2 
±4 
±4 
-7/ 
+28 
±28 
Collections of SAEs or 
AESIs 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Serology including 
bactericidal antibodies 
 
X 
X 
 
 
 
 
 
 
 
X 
 
X 
X 
X 
X 
X 
Challenge 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Stool weighing, 
grading, assessed for 
blood 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
Stool culture for 
Shigella 
 
 
X 
(X) 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
Stool for IgA 
 
 
X 
(X) 
 
 
X 
 
 
 
X 
 
X 
X 
 
 
 
Stool for PCR (RNA 
later) 
 
 
X 
(X) 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
Stool for Microbiome 
(RNA Later) 
 
 
X 
(X) 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Stool for Inflammatory 
Markers 
 
 
X 
(X) 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
Stool for 
transcriptomics (RNA 
later) 
 
 
X 
(X) 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
Blood for 
transcriptomics 
(systems biology) 
(whole blood) 
(X) 
X 
X 
(X) 
X 
 
 
 
 
X 
 
 
X 
X 
 
 
 
Blood for Antigen 
Arrays (ADI) 
 
 
X 
(X) 
X 
 
X 
 
 
X 
X 
 
X 
X 
 
 
X 
Antibody lymphocyte 
supernatant (ALS) 
 
X 
 
 
 
 
X 
 
 
X 
X 
 
 
 
 
 
 
Memory B-Cell 
 
 
X 
 
 
 
 
 
 
 
 
 
 
X 
X 
X 
X 
Serum cytokines 
 
 
X 
(X) 
X 
 
X 
 
 
X 
 
 
 
 
 
 
 
Cytokines stool 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Salivary IgA 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
X 
 
 
Nasal swab for 
metagenomics 
 
 
X 
 
X 
 
 
X 
 
 
X 
 
X 
X 
X 
X 
X 
Start antibiotic therapy 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
Planned discharge 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
Study completion (1 
challenge only) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Post-study safety 
assessment 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Approximate blood volume 
(mL)# 
5 
119 
63 
 
12.5 
 
74 
 
76.5 
 
84 
 
31 
54.5 
42 
42 
52 
(X) event may occur on this day in place of another day. 

 
 
Protocol v4.2 
16 JAN 2023 
75 
1Day -2 to -1 labs are for baseline labs not for determination of eligibility, Day -2 to -1 labs will be obtained on admission to the inpatient unit. Subjects who are completing D90 visit and 
not being admitted to the inpatient unit will not have safety bloods obtained.  
2Volunteers will have a COVID-19 test (may be done at local testing site or home) 1 – 5 days prior to admission and will have a COVID-19 test on admission day. If at home antigen-
based COVID-19 testing is done, volunteers should be in compliance with current CDC guidelines which include a repeat antigen test 48 hours after a negative first test.  
3Screening titers may be drawn within 90 days of enrollment.  
4During the inpatient phase, both solicited and unsolicited adverse events will be assessed, whereas at outpatient visits only unsolicited AEs will be assessed. 
# Blood volumes are approximate and may change somewhat from these numbers; the total volume will not exceed 550 mL in any 8-week period. 
 
 
 
Table 10: Schedule of Procedures for Subjects Receiving Two Challenges 
Study Phase 
Outpatient 
Inpatient 
Outpatient 
Inpatient 
Outpatient 
Study Day 
Screening 
Screening 
-2 or-1 
1 
2 
3 
4 
5 
6 
7 
8 
9 
15 
29 
57 
901 
-2to 
-1 
1 
2 
3 
4 
5 
6 
7 
8 
9 
15 
29/ 
118 
57/ 
14
6 
90/ 
1801 
180/
2701 
Compliance 
Range (Days) 
Day -60 
to Day -2 
Day -30 
to Day -2 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
±2 
±4 
±4 
-7/ 
+28 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
±2 
±4 
±4 
-7/ 
+28 
±28 
Review Inclusion 
/Exclusion Criteria 
X 
 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
JH 200 screening 
consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Study 
briefing/Informed 
Consent 
(X) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Comprehension 
assessment 
(X) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HIV testing 
information sheet 
(X) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Functional Bowel 
Survey 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Medical History 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical 
examination/Focus
ed PE 
(X) 
X 
X 
X 
X X X X X X X X (X) 
(X) 
(X) 
(X) 
X 
X 
X X X X X X X 
X 
(X) 
(X) 
(X) 
(X) 
(X) 
Vital Signs (BP, 
HR, Temp) 
X 
X 
X 
X 
X X X X X X X X 
X 
X 
X 
X 
X 
X 
X X X X X X X 
X 
X 
X 
X 
X 
 
Screening Titers to 
S. flexneri and S. 
sonnei IgG levels 
X6 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Hematology/CBC 
w/diff 
 
X 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
 
X3 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
Basic Metabolic 
panel, ALT, AST 
 
X 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
 
X3 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
Urine for 
toxicology testing 
X 
(X) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Hepatitis B, C, 
and HIV  
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
Protocol v4.2 
16 JAN 2023 
76 
Study Phase 
Outpatient 
Inpatient 
Outpatient 
Inpatient 
Outpatient 
Study Day 
Screening 
Screening 
-2 or-1 
1 
2 
3 
4 
5 
6 
7 
8 
9 
15 
29 
57 
901 
-2to 
-1 
1 
2 
3 
4 
5 
6 
7 
8 
9 
15 
29/ 
118 
57/ 
14
6 
90/ 
1801 
180/
2701 
Compliance 
Range (Days) 
Day -60 
to Day -2 
Day -30 
to Day -2 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
±2 
±4 
±4 
-7/ 
+28 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
±2 
±4 
±4 
-7/ 
+28 
±28 
Serum/Urine 
Pregnancy Test 
(X) 
X 
X 
(X) 
 
 
 
 
 
 
 
 
 
X 
 
 
X 
(X) 
 
 
 
 
 
 
 
 
 
X 
 
 
 
IgA  
X 
(X) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HLA-B27 
antigen/ABO 
typing 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
COVID-19 test 
 
X4 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Concomitant 
Medications 
X 
X 
X 
X 
X X X X X X X X 
X 
X 
 
 
X 
X 
X X X X X X X 
X 
X 
X 
 
 
 
Clinical 
Check/Medical 
Interview 
X 
X 
X 
X 
X X X X X X X X 
X 
X 
X 
X 
X 
X 
X X X X X X X 
X 
X 
X 
X 
X 
X 
Collection of AEs2 
 
 
X 
X 
X X X X X X X X 
X 
X 
 
 
 
X 
X X X X X X X 
X 
X 
X 
 
 
 
Collections of 
SAEs or AESIs 
 
 
 
X 
X X X X X X X X 
X 
X 
X 
X 
X 
X 
X X X X X X X 
X 
X 
X 
X 
X 
X 
Serology 
including 
bactericidal 
antibodies 
 
X 
X 
 
 
 
 
 
 
 
X 
 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 
X 
 
X 
X 
X 
X 
X 
Challenge 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Stool weighing, 
grading, assessed 
for blood 
 
 
 
X 
X X X X X X X X 
 
 
 
 
 
X 
X X X X X X X 
X 
 
 
 
 
 
Stool culture for 
Shigella 
 
 
X 
(X) 
X X X X X X X X 
 
 
 
 
X 
(X) 
X X X X X X X 
X 
 
 
 
 
 
Stool for IgA 
 
 
X 
(X) 
 
 
X 
 
 
 
X 
 
X 
X 
 
 
X 
(X) 
 
 
X 
 
 
 
X 
 
X 
X 
 
 
 
Stool for PCR 
(RNA later) 
 
 
X 
(X) 
X X X X X X X X 
 
 
 
 
X 
(X) 
X X X X X X X 
X 
 
 
 
 
 
Stool for 
Microbiome 
(RNA Later) 
 
 
X 
(X) 
X X X X X X X X 
X 
X 
X 
 
X 
(X) 
X X X X X X X 
X 
X 
X 
X 
 
 
Stool for 
Inflammatory 
Markers 
 
 
X 
(X) 
X X X X X X X X 
 
 
 
 
X 
(X) 
X X X X X X X 
X 
 
 
 
 
 
Stool for 
transcriptomics 
(RNA later) 
 
 
X 
(X) 
X X X X X X X X 
X 
 
 
 
X 
(X) 
X X X X X X X 
X 
X 
 
 
 
 
Blood for 
transcriptomics 
(systems biology) 
(whole blood) 
(X) 
X 
X 
(X) 
X 
 
 
 
X 
 
 
 
X 
X 
 
 
X 
(X) 
X 
 
 
 
 
X 
 
 
X 
X 
 
 
 
Blood for Antigen 
Arrays (ADI) 
 
 
X 
(X) 
X 
 
X 
 
X 
 
X 
 
X 
X 
 
 
X 
(X) 
X 
 
X 
 
 
X X 
 
X 
X 
 
 
X 

 
 
Protocol v4.2 
16 JAN 2023 
77 
Study Phase 
Outpatient 
Inpatient 
Outpatient 
Inpatient 
Outpatient 
Study Day 
Screening 
Screening 
-2 or-1 
1 
2 
3 
4 
5 
6 
7 
8 
9 
15 
29 
57 
901 
-2to 
-1 
1 
2 
3 
4 
5 
6 
7 
8 
9 
15 
29/ 
118 
57/ 
14
6 
90/ 
1801 
180/
2701 
Compliance 
Range (Days) 
Day -60 
to Day -2 
Day -30 
to Day -2 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
±2 
±4 
±4 
-7/ 
+28 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
±2 
±4 
±4 
-7/ 
+28 
±28 
Antibody 
lymphocyte 
supernatant (ALS) 
 
X 
 
 
 
 
X 
 
X 
 
X 
 
 
 
 
 
X5 
 
 
 
X 
 
 
X X 
 
 
 
 
 
 
Memory B-Cell 
 
 
X 
 
 
 
 
 
 
 
 
 
 
X 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
X 
X 
X 
X 
Serum cytokines 
 
 
X 
(X) 
X 
 
X 
 
X 
 
 
 
 
 
 
 
X 
(X) 
X 
 
X 
 
 
X 
 
 
 
 
 
 
 
Cytokines stool 
 
 
X 
X 
X X X X X X X X 
X 
X 
 
 
X 
X 
X X X X X X X 
X 
X 
X 
 
 
 
Salivary IgA 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
X 
 
 
Nasal swab for 
metagenomics 
 
 
X 
 
X 
 
 
X 
 
 
X 
 
X 
X 
X 
X 
X 
 
X 
 
 
X 
 
 
X 
 
X 
X 
X 
X 
X 
Start antibiotic 
therapy 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
Planned discharge 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
Study completion 
(RE-challenge 
only) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Post-study safety 
assessment 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Approximate Blood 
volumes (mL)# 
5  
113.5 
63  
 
1
2
.
5 
 
7
4  
 
7
6
.
5 
 
8
4 
 
31 
54.5 
42 
42 
63 
naïve 
127 
veter
ans 
 
1
2
.
5 
 
7
4 
 
7
6
.
5 
 
84 
 
31 
54.5 
42 
42 
52 
1Participants who receive the second challenge will have their day 90 and day 180 after the second challenge. If the second challenge is >120 days after the first, the participants will have 
a day 90 and a day-2 or -1 visit. 
2During the inpatient phase, both solicited and unsolicited adverse events will be assessed, whereas at outpatient visits only unsolicited AEs will be assessed. 
# Blood volumes are approximate and may change somewhat from these numbers; the total volume will not exceed 550 mL in any 8-week period.   
3Second admission, Day -2 or -1 safety labs are for baseline labs not for determination of eligibility, Day -2 or -1 labs will be obtained on admission to the inpatient unit. Subjects who are 
completing D90 visit and not being admitted to the inpatient unit will not require safety bloods.  
4Volunteers will have a COVID-19 test (may be done at local testing site or home) 1 – 5 days prior to admission and will have a COVID-19 test on admission day. Volunteers for 2nd 
admission will have a COVID-19 test (may be done at local testing site or home) 1 – 5 days prior to admission and will have a COVID-19 test on admission day. If at home antigen-based 
COVID-19 testing is done, volunteers should be in compliance with current guidelines which includes a repeat antigen test 48 hours after a negative first test.  
5Only subjects returning for second challenge will need ALS drawn on day -2 or -1 second admission (naïve subjects will have pre-study ALS collected during screening). 
6Screening titers may be drawn within 90 days of enrollment.  
(X) Event may occur on this day in place of another day.