Dose-Finding Study of Lyophilized Shigella Sonnei 53G Challenge Strain
Phase: PHASE1
Conditions: Healthy
Sponsor: PATH

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Shigella sonnei 53G Challenge Protocol 
 
 
Dose-Finding Study of Lyophilized Shigella sonnei 53G (Lot 1794) Challenge Strain 
 
IND Sponsor: PATH 
Study Number: WRAIR #2286 
Manufacturer: Walter Reed Army Institute of Research 
Version Number: 7.0 
April 14, 2017 

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Shigella sonnei 53G Challenge Protocol 
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STATEMENT OF COMPLIANCE 
 
This study will be carried out in accordance with the United States (US) Code of Federal 
Regulations (CFR), local regulations, and GCP as required by the following: 
 
US CFR applicable to clinical studies (45 CFR 46; a-d 21 CFR including part 50 and 56 
concerning informed consent and institutional review board [IRB] regulations, 21 CFR 11 
concerning electronic records and 21 CFR 312 for investigational new drug [IND]) 
 
International Conference on Harmonisation (ICH) E6 (R1); 62 Federal Register 25691 
(1997) 
All individuals responsible for the design and conduct of this study have completed Human 
Subjects Protection Training and are qualified to be conducting this research prior to the 
enrollment of any subjects. Curricula vitae for all investigators and sub-investigators 
participating in this trial are on file in a central facility (21 CFR 312.23 [a] [6] [iii] [b] edition) 

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SIGNATURE PAGE 
 
The signature below constitutes approval of this protocol and the attachments and provides the 
required assurances that this trial will be conducted according to all stipulations of the protocol, 
including all statements regarding confidentiality, and according to local legal and regulatory 
requirements, applicable US federal regulations, and (ICH E6 R1) guidelines. 
 
Principal Investigator: 
Robert W. Frenck, Jr., M.D. 
 
 
 
Signed: 
Date: 
Name 
Title 
 
Cincinnati Children’s Hospital Medical Center 
Cincinnati, Ohio, 45229 

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TABLE OF CONTENTS 
 
Page 
STATEMENT OF COMPLIANCE ....................................................................................................... I 
SIGNATURE PAGE ........................................................................................................................ II 
TABLE OF CONTENTS .................................................................................................................. III 
LIST OF ABBREVIATIONS ............................................................................................................ VII 
PROTOCOL SUMMARY ............................................................................................................... IX 
1. KEY ROLES .............................................................................................................................1 
2. BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE .................................................... 2 
2.1. BACKGROUND INFORMATION .............................................................................................................................. 2 
2.2 
SCIENTIFIC INNOVATION....................................................................................................................................... 4 
2.3 
POTENTIAL RISKS AND BENEFITS .......................................................................................................................... 5 
2.3.1 
Potential Risks ........................................................................................................... 5 
2.3.2 
Known Potential Benefits .......................................................................................... 6 
3. OBJECTIVES ............................................................................................................................7 
3.1 
PRIMARY OBJECTIVE ............................................................................................................................................. 7 
3.2 
SECONDARY OBJECTIVES ...................................................................................................................................... 7 
4. STUDY DESIGN .......................................................................................................................7 
4.1 
STUDY OUTCOME MEASURES .............................................................................................................................. 9 
4.1.1 
Primary Outcome Measures ...................................................................................... 9 
4.1.2 
Secondary Outcome Measures ................................................................................ 10 
4.1.3 Exploratory Outcome .................................................................................................. 11 
4.1.3.1 
Stool Polymerase Chain Reaction (PCR) to assess quantitative stool burden of S. sonnei .. 11 
5. STUDY ENROLLMENT AND WITHDRAWAL ............................................................................. 11 
5.1 
SUBJECT INCLUSION CRITERIA ........................................................................................................................... 11 
5.2 
SUBJECT EXCLUSION CRITERIA .......................................................................................................................... 12 
6. TREATMENT ASSIGNMENT PROCEDURES .............................................................................. 13 
6.1 
WITHDRAWAL ................................................................................................................................................... 13 
6.1.1 
Reasons for Withdrawal .......................................................................................... 13 
6.1.2 
Handling of Withdrawals ........................................................................................ 14 
6.2 
TERMINATION OF STUDY ................................................................................................................................... 14 
7. INVESTIGATIONAL PRODUCT ................................................................................................ 14 
7.1 
STUDY PRODUCTS DESCRIPTION ....................................................................................................................... 14 
7.1.1 
Acquisition ............................................................................................................... 15 
7.1.2 
Preparation/Handling ............................................................................................. 17 

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7.1.3 
Administration ......................................................................................................... 18 
7.1.4 
Accountability/Final Disposition for the Investigational Products: ......................... 18 
7.1.5 
Ancillary Supplies Description ................................................................................. 19 
8. STUDY SCHEDULE ................................................................................................................. 19 
8.1 
CLINICAL............................................................................................................................................................. 19 
8.1.1 
Screening (day ‐45 to ‐2) (may occur in 2‐3 visits) .................................................. 19 
8.1.2 
Admission day (study day –1) .................................................................................. 20 
8.1.3 
Challenge day (study day 0) .................................................................................... 20 
8.1.4 
Inpatient stay (study days 0‐8). ............................................................................... 21 
8.1.5 
Study day 8 (Planned discharge) ............................................................................. 23 
8.1.6 
Early Discharge ........................................................................................................ 24 
8.1.7 
Study day 14 (+2 day) and 28 (±2 days) .................................................................. 25 
8.1.8 
Study day 56 (± 4 days) ........................................................................................... 25 
8.1.9 
Study day 180 (± 14 days) ....................................................................................... 25 
8.1.10 Unscheduled Visits .................................................................................................. 26 
8.2 
LABORATORY EVALUATIONS AND PROCEDURES .............................................................................................. 26 
8.2.1 
Laboratory Evaluations (blood volumes are estimates only) .................................. 26 
8.2.1.1 
Screening (study days ‐45 to ‐2) .......................................................................................... 26 
8.2.1.2 
Inpatient Baseline (study day ‐1) ......................................................................................... 27 
8.2.1.3 
Inpatient Study Days (study days 0 – 8) ............................................................................... 27 
8.2.1.4 
Study day 14+2 day .............................................................................................................. 28 
8.2.1.5 
Study day 28±2d .................................................................................................................. 29 
8.2.1.6 
Study day 56±4d .................................................................................................................. 29 
8.2.2 
Laboratory Assays ................................................................................................... 29 
8.2.2.1 
Stool Culture ........................................................................................................................ 29 
8.2.2.2 
Microbiology and Molecular Biology ................................................................................... 30 
8.2.2.3 
Antibody secreting cells (ASC) and Antibody in Lymphocyte Supernatant (ALS) ................. 30 
8.2.2.4 
Memory B‐cell Assay: .......................................................................................................... 30 
8.2.2.5 
Serum Immunoglobulins ...................................................................................................... 30 
8.2.2.6 
Fecal Secretory IgA .............................................................................................................. 30 
8.2.2.7 
Salivary IgA .......................................................................................................................... 31 
8.2.2.8 
Systems Biology/Microbiomics/Transcriptomics ................................................................. 31 
8.2.2.9 
Cytokines ............................................................................................................................. 31 
8.3 
CONCOMITANT MEDICATIONS/TREATMENTS ................................................................................................. 31 
8.4 
SPECIMEN PREPARATION, HANDLING, AND SHIPPING .................................................................................... 32 
8.4.1 
Instructions for Specimen Preparation, Handling, and Storage: ............................. 32 
8.4.2 
Study Product Shipment .......................................................................................... 33 
9. SAFETY REPORTING AND SAFETY MONITORING .................................................................... 33 
9.1 
RESPONSIBILITIES............................................................................................................................................... 34 
9.2 
DEFINITIONS ...................................................................................................................................................... 34 
9.2.1 
Adverse Event (AE) .................................................................................................. 34 
9.2.2 
Serious Adverse Event (SAE) .................................................................................... 37 
9.3 
SAFETY REPORTING PROCEDURES .................................................................................................................... 38 
9.3.1 
Reporting Interval ................................................................................................... 38 

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9.3.2 
Notification of the Study Sponsors of Serious Adverse Events ................................ 38 
9.3.3 
Regulatory Reporting for Studies Conducted Under IND ........................................ 41 
9.3.4 
Reporting of Pregnancy ........................................................................................... 42 
9.4 
INVESTIGATOR’S ASSESSMENT OF ADVERSE EVENTS ....................................................................................... 42 
9.4.1 
Assessment of Seriousness ...................................................................................... 42 
9.4.2 
Assessment of Severity ............................................................................................ 42 
9.4.3 
Assessment of Association ...................................................................................... 43 
9.4.4 
Assessment of Reactogenicity ................................................................................. 43 
9.5 
SAFETY MONITORING ........................................................................................................................................ 44 
9.5.1 
Safety Monitoring Committee ................................................................................. 44 
9.5.2 
Research Monitor .................................................................................................... 44 
9.5.3 
Halting Criteria/Rules: ............................................................................................. 45 
10. CLINICAL MANAGEMENT OF EVENTS .................................................................................... 46 
10.1 ADVERSE EVENT MANAGEMENT ...................................................................................................................... 46 
10.1.1 Non‐Administration of Study Product in an Individual Subject ............................... 46 
10.1.2 Pregnancy ................................................................................................................ 47 
11. CLINICAL MONITORING ........................................................................................................ 47 
11.1   SITE MONITORING PLAN ................................................................................................................................... 47 
12. STATISTICAL CONSIDERATIONS ............................................................................................. 48 
12.1 STUDY HYPOTHESES .......................................................................................................................................... 48 
12.2 SAMPLE SIZE CONSIDERATIONS ........................................................................................................................ 48 
12.2.1 Planned Interim Analyses ........................................................................................ 48 
12.2.2 Analysis Plan ............................................................................................................ 49 
13. DATA HANDLING/RECORD KEEPING/SOURCE DOCUMENTS .................................................. 49 
13.1 DATA CAPTURE METHODS ................................................................................................................................ 50 
13.1.1   Types of Data .......................................................................................................... 50 
13.2 STUDY RECORDS RETENTION ............................................................................................................................ 51 
13.3 SOURCE DOCUMENTS ....................................................................................................................................... 51 
13.4 PROTOCOL DEVIATIONS .................................................................................................................................... 52 
14. QUALITY CONTROL AND QUALITY ASSURANCE ..................................................................... 52 
15. ETHICS/PROTECTION OF HUMAN SUBJECTS .......................................................................... 53 
15.1 ETHICAL STANDARD ........................................................................................................................................... 53 
15.2 INSTITUTIONAL REVIEW BOARD ........................................................................................................................ 53 
16. INFORMED CONSENT PROCESS ............................................................................................. 53 
16.1 SUBJECT CONFIDENTIALITY ............................................................................................................................... 54 
16.2 STUDY DISCONTINUATION ................................................................................................................................ 54 
16.3 FUTURE USE OF STORED SPECIMENS................................................................................................................ 55 
17. PUBLICATION POLICY ........................................................................................................... 56 
18. LITERATURE REFERENCES ..................................................................................................... 58 

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19. APPENDICES ........................................................................................................................ 60 
19.1 APPENDIX A: SCHEDULE OF EVENTS – FOR EACH COHORT .............................................................................. 60 
19.2 APPENDIX B: TOXICITY GRADING ..................................................................................................................... 63 
19.3 APPENDIX C: INFORMATION FORM IF A SUBJECT PREMATURELY WITHDRAWS FROM THE TRIAL ................ 65 

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LIST OF ABBREVIATIONS 
 
AE 
Adverse Event/Adverse Experience 
ALS 
Antibody Lymphocyte Supernatant 
ASC 
Antibody-Secreting Cells 
BGS 
Buffered Glycerol Saline 
BMGF 
Bill and Melinda Gates Foundation 
CCHMC 
Cincinnati Children’s Hospital Medical Center 
CFR 
Code of Federal Regulations 
CRF 
Case Report Form 
cfu 
Colony Forming Units 
CRADA 
Cooperative Research and Development Agreement 
CRO 
Contract Research Organization 
DoD 
Department of Defense 
eCRF 
ELISPOT 
Electronic Case Report Form 
Enzyme-Linked ImmunoSpot 
FDA 
Food and Drug Administration 
FIH 
First-in-human 
GCP 
Good Clinical Practice 
HEA 
Hektoen Enteric Agar 
HLA 
Human Leukocyte Antigen 
IB 
Investigator’s Brochure 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation 
ICMJE 
International Committee of Medical Journal Editors 
ICS 
Inventory Control System 
ID 
Identification 
IEC 
Independent or Institutional Ethics Committee 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
LPS 
Lipopolysaccharide 
MOP 
Manual of Procedures 
USAMRMC 
US Army Medical Research and Materiel Command 
n 
Number (typically refers to subjects) 
PBF 
Pilot Bioproduction Facility 
PCB 
Production Cell Bank 
PCR 
Polymerase Chain Reaction 
PI 
Principal Investigator 
PI-IBS 
Post-Infectious Irritable Bowel Syndrome 
QA 
Quality Assurance 
QC 
Quality Control 
SAE 
Serious Adverse Event/Serious Adverse Experience 
SMC 
Safety Monitoring Committee 

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LIST OF ABBREVIATIONS 
 
SOP 
Standard Operating Procedure 
SSP 
SWI 
Study Specific Procedure 
Sterile Water for Injection 
US 
United States 
WRAIR 
Walter Reed Army Institute for Research 

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PROTOCOL SUMMARY 
 
Title: 
Dose-Finding Study of Lyophilized Shigella sonnei 53G (Lot 
1794) Challenge Strain 
Abbreviated Title: 
Shigella sonnei 53G Challenge 
Phase: 
Phase 1; First-in-human (FIH) study. 
Population: 
Inpatient study of approximately 60 healthy adults (aged 18 to 
49, inclusive at time of participation) who live in the recruiting 
range of the Cincinnati Children’s Hospital Medical Center 
(CCHMC). 
Number of Sites: 
1 
Cincinnati Children’s Hospital Medical Center (CCHMC), 
Cincinnati, Ohio 
Clinical: CCHMC 
Laboratory: CCHMC and WRAIR 
IND Sponsor: 
PATH 
Study Sponsor/Funding 
Mechanism: 
Department of Defense (DoD) and PATH 
Study Duration: 
1-1/2 years 
Subject Participation 
Duration: 
6-8 months 
Screening: 1 to 45 days 
Inpatient participation: 8 days 
Follow-up: 180 days (approximately three 1-day follow-up 
visits and one phone call) 
 
 
Test Article and Dosing: 
The challenge product will be lyophilized Shigella sonnei 53G 
strain: The starting dose is 500 cfu 
(Doses may be increased or lowered based on results) 
After a 90 minute fast, subjects will drink 120 ml of sodium 
bicarbonate just prior to ingesting 30 ml of sterile normal 
saline 0.9% containing the Shigella inoculums. 

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Study Objectives: 
Primary Objective: 
1. Establish a human challenge model of S. sonnei 
53G infection using a lyophilized formulation of the 
challenge strain. 
2. Identify a dose of lyophilized S. sonnei 53G that 
induces the primary outcome in approximately 60% 
of subjects with no adverse safety concerns. 
Secondary Objective: 
1. Estimate 
quantitative 
shedding 
and 
basic 
immunogenicity of the challenge strain. 
2. Collect and archive blood and fecal samples for 
systems biology, microbiome, and other omics- 
based work to be conducted under a separate 
research protocol in future studies. 
 
Description of Study 
Design: 
Healthy subjects 18-49 years of age (up to 50th birthday) will 
be admitted to an inpatient unit at CCHMC. Subjects will 
remain in the inpatient unit for approximately 8 days post- 
challenge to allow close clinical observation as well as 
collection of blood, stool, and saliva (cohorts 2-5 only) 
samples at multiple time points during the inpatient period. On 
day 
5 
post-challenge; 
subjects 
will 
be 
administered 
ciprofloxacin to eradicate the Shigella from the stool of the 
subject. Subjects will return to the outpatient clinic on 2-3 
occasions post-challenge for clinical check and additional lab 
collection. The trial will consist of five cohorts; four dose 
ranging, and one confirmatory cohort. Ten subjects will be 
enrolled in each dose ranging cohort while up to 20 subjects 
will be enrolled in the confirmatory cohort. 
 
An adaptive design will be used to determine the dose of 
Shigella sonnei 53G that induces the primary outcome in 
approximately 60% of subjects. In the first cohort, about 10 
subjects will be administered 500 cfu (range 400-600) of S. 
sonnei 53G. Depending on the percentage of subjects who 
met clinical endpoints, defined as the attack rate (AR), the 
dose for the second cohort will be adjusted. If the AR is less 
than 60%, the next cohort (cohort 2) will receive a higher 
inoculum, not to exceed 1000 cfu, based on deliberations 
between the investigative team, the research monitor and the 
Safety Monitoring Committee (SMC). ARs at or above 60% 

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may result in verification of the attack rate at the same 
inoculum dose or a decrease in the inoculum to as low as 100 
cfu based on deliberations between the investigative team, the 
research monitor and the SMC. Similar to the second cohort, 
the dosing for the third and fourth cohorts will be based on the 
AR in the previous cohort. The results from the four dose 
ranging cohorts will be reviewed to select the dose to be 
administered to the confirmatory cohort of up to 20 subjects. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Estimated Time to 
Complete Enrollment: 
If the targeted AR is not achieved (either too high an AR at the 
low inoculum or too low an AR at the high inoculum), the 
results will be evaluated to determine if the dose should be 
increased or decreased. An iterative process will be used to 
select the optimal dose with each step reviewed and approved 
by the safety monitoring committee. An interim safety report 
will compile findings from the preceding dose group with the 
PI’s interpretation and plan to go forward. The safety 
monitoring committee must concur with the plan before 
proceeding. The decision to advance to the next dose level 
will be based solely on the disease AR and the observed 
safety profile at the tested dose level. 
Approximately 8 months 

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Schematic of Study Design 
Dose escalation will occur in a stepwise fashion as described in the table below. For the initial 
dose cohort, enrollment will be limited initially to one subject per day for the first three days. If no 
unexpected symptoms occur within 24 hours of dosing the third subject, concurrent enrollment 
of the remaining subjects in the cohort may proceed. 
 
A research monitor, in conjunction with an SMC, established as an advisory committee to the 
study sponsors, will review the inpatient safety data and any reported SAEs through Day 14 
from each dose level prior to making a recommendation for advancement to the next dose level. 
One of the following recommendations will be made: advancing to the next dose level, halting, 
additional subjects at the current dose, or decreasing the current dose. The decision will be 
dependent upon the assessment of safety parameters and by meeting criteria for advancement 
to the next dose. The safety data to be reviewed by the research monitor and the SMC will 
include inpatient safety data and all reported serious adverse events (SAEs). Subjects 
participating in one dose level will not be permitted to participate in a subsequent dose level. 
 
Cohort 
Product 
Inoculum dose 
N 
A 
S. sonnei 53G 
500 cfu 
10 
B 
S. sonnei 53G 
250 cfu or 1000 
cfu (based on 
results of cohort A) 
10 
C 
S. sonnei 53G 
TBD based on 
cohort A and B 
10 
D 
S. sonnei 53G 
TBD based on 
cohorts A-C 
10 
E 
S. sonnei 53G 
Based on results 
of previous cohorts 
Up to 
20 

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1. KEY ROLES 
 
 
Principal Investigator 
Dr. Robert Frenck, MD 
Cincinnati Children’s Hospital Medical Center 
3333 Burnet Ave, MLC 6014 
Cincinnati, OH 45229 
Phone: 513-636-4463 
Robert.Frenck@cchmc.org 
Research Monitor 
Grant Paulsen, M.D. 
3333 Burnet Ave, MLC 7017 
Cincinnati, OH 45229 
Phone: 513-803-7468 
 
Manufacturer’s Contact: 
Dr. Akamol E. Suvarnapunya 
Product Manager 
Department of Enteric Infections 
Pilot Bioproduction Facility 
Walter Reed Army Institute of Research (WRAIR) 
503 Robert Grant Ave 
Bldg 503 
Silver Spring, MD 20910 

2 
 
 
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2. BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE 
 
2.1. Background Information 
Diarrheal diseases continue to be major public health problems throughout the world. In 1999, 
Kotloff et al. conducted a systematic literature review and estimated the global incidence of 
shigellosis, a major bacterial diarrheal pathogen [1]. This review estimated there to be 164.7 
million cases of Shigella per year resulting in approximately 1.1 million shigellosis-related 
deaths [1]. Shigella is comprised of four species (S. dysenteriae, S. flexneri, S. boydii and S. 
sonnei) with S. sonnei being the most common species of Shigella-associated diarrhea in 
developed countries and S. flexneri being the most common species of Shigella-associated 
diarrhea in the developing world [2]. More recent data has estimated mortality rates to be lower; 
however epidemiological information on incidence rates of Shigella-mediated diarrhea from 
much of Asia and Africa is missing [3]. 
Shigella is a low-inoculum infection transmitted by the fecal-oral route with as few as 10 colony 
forming units (cfu) sufficient to cause disease in highly susceptible people [4]. Due to ease of 
transmission, multiple secondary cases from an initial case are common [4]. Infections caused 
by Shigella can range from mild watery diarrhea to severe disease characterized by fever, 
headaches, abdominal cramps, and dysentery (frequent small volume stools containing blood 
and mucus). Although Shigella-associated diarrhea typically is self-limiting, infection frequently 
is debilitating for 5-7 days if untreated [2, 5]. 
Currently, the mainstays against Shigella are prevention and treatment of infections. Improved 
sanitation and education on safe eating practices are the principal means of prevention while 
treatment typically requires administration of antimicrobial therapy [1, 6]. Improving sanitation is 
difficult, particularly in resource limited areas thus limiting the effectiveness of prevention. 
Additionally, the emergence of multidrug-resistant Shigella strains has complicated the 
treatment of shigellosis [7-9]. Thus, alternative methods to prevent infection, such as vaccines, 
are critically needed. 
Humans are the only natural host for Shigella making it more difficult to study the prevention 
and/or treatment of infections with the organism. While non-human primate models have been 
developed, inoculums multiple logs higher than needed to cause an infection in humans are 
needed to reproducibly induce infection. Islam et al. demonstrated that for rhesus monkeys, an 
intragastric challenge of 2 x109 cfu of Shigella dysenteriae 1 1617 strain was required to induce 
disease in 4 of 5 monkeys with no previous exposure to Shigella (80% attack rate)[10]. 
Additionally, of the 5 monkeys who previously received the 2 x109 cfu dose, only 2 developed 
symptoms after administration of a 2 x1010 cfu dose (60% protection). In comparison, when a 2 
x1010 cfu dose was administered to monkeys without previous exposure to Shigella; all 
developed severe dysentery and one monkey died within 24 hours of challenge [10]. 
Gregory et al. have developed a challenge model of infection for S. flexneri in the Aotus 
nancymaae monkey [11]. An oral dose of 1x1011 cfu of Shigella flexneri 2a strain 2457T induced 
infection in 75% of the monkeys. However, unlike the rhesus model; a previous infection with 
the challenge organism did not protect against infection when the animals were administered a 

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second challenge dose 9 weeks after the first [11]. The limitations of the currently available 
animal models reinforce the need for a safe and reproducible human challenge model. 
Two strains of Shigella, S. flexneri 2457T and S. sonnei 53G, have been most commonly used 
in human challenge models. As part of a study of a bi-valent Shigella vaccine; subjects from the 
United States were given 3 doses of vaccine or placebo followed by challenge with 500 cfu of S. 
sonnei 53G 1 month after the third dose of vaccine [12]. Of the 38 healthy control subjects; 20 
(53%) developed diarrhea and fever. In a follow-up of the previously described study; 16 
placebo recipients were challenged with 500 cfu of S. sonnei 53G 1 month after the third dose 
of placebo. Ten of the 16 subjects (62%) became ill [13]. A third study from the same group 
compiled results from previous studies to determine if there were a correlation between peak 
temperature of the subject and severity of the infection [14]. A total of 85 control subjects 
received 500 cfu of S. sonnei 53G and 40 (48%) developed fever and diarrhea [14]. The 
investigators noted that fever did correlate with more severe illness. However, the duration of 
symptoms was shorter in the group that developed the higher fevers. In a trial evaluating the 
cytokine response to Shigella infection; 11 subjects were administered 500 cfu of S. sonnei 53G 
and 6 (55%) became ill [15]. The summary of the above studies, all conducted in the United 
States, is that approximately 50% of subjects develop diarrhea and/or dysentery (range 40- 
60%) after ingesting 500 cfu of S. sonnei 53G. 
A critical step to increase the likelihood of inducing an infection with an enteric organism is 
neutralization of stomach acid. All of the above studies used skim milk to neutralize stomach 
acidity. A finding of all the above studies was that the rate of infection plateaued at about 60% of 
subjects regardless of the dose of the challenge strain administered. In an attempt to increase 
the attack rate; milk was substituted with sodium bicarbonate [16]. When subjects were 
administered 103 cfu of S. flexneri 2457T mixed with sodium bicarbonate; 10 of 14 subjects 
(71%) developed fever and 9 of 14 (64%) developed diarrhea and/or dysentery [16]. In a second 
study, subjects were administered 1.4 x 103 cfu of S. flexneri 2457T mixed with sodium 
bicarbonate [17]. Of the 12 subjects, 10 (83%) developed fever along with diarrhea and/or 
dysentery. Lowering the dose to 1.4 x 102 cfu of S. flexneri 2457T lowered the infection rate to 
43% (3/7 subjects). A third study confirmed the results of the previous work when 8 of 10 (80%) 
subjects administered 1.5 x 103 cfu of S. flexneri 2457T mixed with sodium bicarbonate 
developed diarrhea [18]. These studies thus demonstrated that substituting skim milk with 
sodium bicarbonate could markedly increase the rate of infection caused by S. flexneri 2457T. 
Additionally, the studies suggested a steep dose response curve between administration of 140 
and 1400 cfu of the challenge strain. 
A recent study evaluated the infectivity of S. sonnei 53G when administered to people in 
Thailand [19]. In ascending cohorts of 12; subjects were administered either 100, 400 or 1600 
cfu of S. sonnei 53G after drinking 150 mL of a sodium bicarbonate solution. The two lower 
doses resulted in 6 (50%) subjects in each cohort meeting clinical endpoints while 9 (75%) 
subjects receiving 1600 cfu met clinical endpoints [19]. These data closely mirror the results of 
the studies discussed above when subjects were challenged with S. flexneri 2457T mixed with 
sodium bicarbonate and again suggest a rather steep dose response curve for infection 
between 500 and 1500 cfu as a challenge dose. 

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While the human challenge studies of Shigella have produced important data, many concerns 
have been raised about these data including; lack of standard inoculum; lack of reproducibility; 
small sample size of study subjects; varying outcome definitions and minimal understanding of 
the immune response to infection [20]. Additionally, the method of preparation of the Shigella 
inoculum from a bacterial suspension with colony count estimated from a fixed optical density 
has been cited as a barrier to standardization and reproducibility [20]. The current proposal will 
address these deficiencies and establish a well characterized model of infection with S. sonnei 
53G. 
 
2.2 Scientific Innovation 
The project will employ many innovative approaches and technology in the development of the 
challenge model. The first is the use of a lyophilized strain of S. sonnei 53G. In previous 
challenge studies; doses of 53G typically have been grown from thawed glycerol stock or starter 
culture from one or more earlier passages [17-19]. The confluent bacterial lawns on agar plates 
are then scraped into some diluent and the dense suspension is adjusted over several iterations 
to the desired concentration of bacteria. This ad hoc practice is vulnerable to many sources of 
potential variation, including; the use of different reagent lots between preparations; different 
bacterial starter cultures; different research personnel, and pipetting and instrument error when 
handling viscous and; incompletely homogenous suspensions. 
In contrast to growing the bacteria from a stock or starter culture; the lyophilized preparation of 
53G, grown under GMP conditions, produces a reliable, consistent concentration of bacteria 
which are fresh and with a low percentage of Form II colonies (See Table 2). S. sonnei vaccine 
candidates have been successfully produced and lyophilized by our team and demonstrated 
highly reproducible colony counts in a recently completed series of S. sonnei vaccine trials at 
Cincinnati Children’s Hospital Medical Center (CCHMC) (unpublished data). The successful 
evaluation of the S. sonnei 53G product will validate the importance of having the lyophilized 
strain and possibly encourage the manufacture of a lyophilized S. flexneri 2a challenge product. 
The availability of standardized cGMP challenge strains of both S. sonnei and S. flexneri, the 2 
most common circulating Shigella serotypes, will accelerate the development of a bivalent or 
trivalent Shigella vaccine that has the potential to control 70-80 percent of shigellosis worldwide. 
An adaptive design will be used in this study. Rather than a priori determining the dose in all 
study groups; subsequent doses are based on the response to previous doses. This approach 
has been shown to more rapidly identify the dose of interest which results in minimizing 
exposure of subjects to the risks of the study as well as maximizing utilization of time and 
resources. We have successfully used such a design in a study to determine the dose of 
norovirus required to induce infection in at least 50% of subjects administered the virus 
(unpublished data). By using the adaptive design, we also will maximize the number of subjects 
receiving the dose of interest which will improve the power of the study. 
Previous human challenge studies have demonstrated that some individuals (immunologically 
naïve) are resistant to enteric infection. One possible explanation is the microbiome of the host. 
The human gut is composed of trillions of symbiotic bacteria which contribute to the normal host 
physiology including host metabolic signaling and immune pathways. Alterations in the gut flora 

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in response to enteric pathogens have also been noted; but the complexity of the microbiome 
and its role in both protection against and response to infection remains to be characterized. A 
better understanding of the changes in the microbiome associated with Shigella infections and 
treatment of the infection may allow us to elucidate the biological mechanisms that underlie 
pathogenesis and point to potential opportunities for prevention and treatment of Shigella. 
Another critical area to explore is the “systems biology” to an infection with Shigella. In contrast 
to previous “reductionist” methods of study; systems biology is a field of study that focuses on 
complex interactions within biological systems, using a holistic approach. Studying the 
“signature of infections”; it may be possible to predict people immune to a specific 
microorganism as well as who may be at increased risk for developing severe infection. Also, 
this approach may allow us to better understand the immune response to natural infections 
which could result in vaccines targeted to stimulate those portions of the immune system. We 
have discussed the above proposal with Dr. Bali Pulendran of Emory, and member of the 
Systems Biology/Systems Immunology Consortium of the Vaccine Accelerator Platform. 
 
2.3 Potential Risks and Benefits 
 
2.3.1 
Potential Risks 
The principal risk is also the primary objective of the study; shigellosis. Illness caused by 
Shigella ranges from mild (watery diarrhea being the main symptom) to severe (fever and 
abdominal cramping with frequent small volume blood streaked stools containing mucus). 
Complaints may include; nausea, vomiting, headache, abdominal gurgling or gas, anorexia, 
muscle and/or joint aches and malaise. Symptoms typically are self-resolving in 5-7 days. 
However, administration of an effective antibiotic will significantly reduce, or eliminate, 
symptoms within 24 hours of initiation. Unlike infections with Enterotoxigenic E. coli (ETEC), a 
common cause of Travelers Diarrhea; Shigella is not commonly associated with significant 
dehydration as stool volume typically is not large. The S. sonnei 53G challenge strain is known 
to be susceptible to multiple antimicrobial agents including TMP-SMX and quinolones. There is 
a risk of bacteremia, although Shigella bacteremia rarely occurs in adult populations with wild- 
type Shigella [2]. 
Reiter’s syndrome or post-dysenteric reactive arthritis, with ocular and/or urethral inflammation, 
which has an onset typically 1 to 3 weeks after the onset of diarrhea, has been reported to occur 
in approximately 2 to 3 percent of Caucasian patients with shigellosis, where the risk of arthritis 
was proportional to the severity of the disease [21]. Some data suggest that the risk of reactive 
arthritis following shigellosis may be higher in persons carrying the Human Leukocyte Antigen 
(HLA)-B27 antigen [22]. Therefore, subjects who were HLA-B27 positive will be excluded from 
participation. 
Recent studies suggest an increased risk of post-infectious irritable bowel syndrome (PI-IBS) 
following bacterial enteritis, with limited studies associating Shigella infection specifically with 
these sequelae [23, 24]. 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 

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enteric infection compared to similar matched controls without such a history [23]. To minimize 
study risks, subjects with a history of abnormal bowel patterns who might be at higher risk for 
these post-infectious sequelae will be excluded. Pre-defined criteria to ensure early treatment 
as appropriate also may reduce further the risk of post-infectious sequelae as has been 
observed with reactive arthritis [22] and possibly would reduce the risk associated with PI-IBS 
given the positive association between diarrheal illness duration and PI-IBS risk. Mild 
gastrointestinal symptoms may occur due to ingestion of the bicarbonate solution. 
To avoid potential risks; pregnant or nursing women will be excluded from the study and women 
of childbearing potential will be counseled against becoming pregnant during their participation 
in the study. 
 
Potential risk from Ciprofloxacin and Bactrim (sulfamethoxazole and trimethoprim): 
Ciprofloxacin, a broad spectrum antibacterial agent belonging to the fluoroquinolone family of 
antibiotics, is the first-line treatment agent for all subjects on day 5 of the clinical trial. 
Ciprofloxacin is generally well-tolerated. The most frequently reported drug related events for all 
indications of ciprofloxacin therapy have been nausea, diarrhea, abnormal liver function tests, 
vomiting, and rash. Most of the AEs reported were described as only mild or moderate in 
severity, abated soon after the drug was discontinued, and required no treatment. 
Fluoroquinolones, including ciprofloxacin, are associated with an increased risk of tendonitis 
and tendon rupture in all ages. Other symptoms rarely seen include hypersensitivity, dizziness, 
pseudomembranous colitis and peripheral neuropathy. 
The most common AEs of Bactrim (alternate treatment agent) are gastrointestinal disturbances 
(nausea, vomiting, and anorexia) and allergic skin reactions (such as rash and urticaria). 
 
Other risks: 
There is the risk of pain, hematoma, or infection at the site of venipuncture. There is also a risk 
of the study staff acquiring the infection from subjects who were administered the S. sonnei 
53G. Therefore, proper personal protective equipment will be worn by staff, and good personal 
hygiene such as frequent hand washing will be followed by staff and subjects. 
 
2.3.2 Known Potential Benefits 
Study subjects will have no direct benefit from study participation. However, a successful 
outcome of the trial could lead to the development of a model that will allow easier and more 
reproducible evaluation of preventive and therapeutic measures against disease due to S. 
sonnei. 

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3. OBJECTIVES 
 
3.1 Primary Objective 
1. Establish a human challenge model of S. sonnei 53G infection using a lyophilized 
formulation of the challenge strain. 
2. Identify a dose of lyophilized S. sonnei 53G that induces the primary outcome in 
approximately 60% of subjects with no adverse safety concerns. 
 
3.2 Secondary Objectives 
1. Estimate quantitative shedding and basic immunogenicity of the challenge strain. 
2. Collect and archive blood and fecal samples for systems biology, microbiome, and 
other omics-based work to be conducted under a separate research protocol in 
future studies. 
 
4. STUDY DESIGN 
 
An adaptive design will be used to determine the dose of Shigella sonnei 53G that induces the 
primary outcome in approximately 60% of subjects. 
 
An unexpected shock-like illness of unknown etiology occurred in one of six subjects at Johns 
Hopkins Center for Immunization Research at 10 hours after inoculation with Shigella sonnei 
strain 53G prepared from frozen stock. Therefore, for the initial dose cohort, enrollment will be 
limited initially to one subject per day for the first three days. If no unexpected symptoms occur 
within 24 hours of dosing the third subject, concurrent enrollment of the remaining subjects in 
the cohort may proceed. 
In the first cohort, about 10 subjects (minimum 8) will be administered 500 cfu (range 400-600) 
of S. sonnei 53G. Depending on the attack rate (AR) of the starting inoculum (defined as the 
percentage of subjects who met clinical endpoints); the dose for the second cohort will be 
adjusted. If the AR is less than 60%, the next cohort (cohort 2) will receive a higher inoculum, 
not to exceed 1000 cfu, based on deliberations between the investigative team, the research 
monitor and the SMC. ARs at or above 60% may result in verification of the attack rate at the 
same inoculum dose or a decrease in the inoculum to as low as 100 cfu based on deliberations 
between the investigative team, the research monitor and the SMC. Using the process 
described above; the dosing for the third and fourth cohorts will be based on the AR in the 
previous cohort. The results from the four dose ranging cohorts will be reviewed to select the 
dose to be administered to the confirmatory cohort of up to 20 subjects. 
If the targeted AR is not achieved (either too high an AR at the low inoculum or too low an AR at 
the high inoculum); the results will be evaluated to determine if the dose should be increased or 

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decreased. An iterative process will be used to select the optimal strain and dose with each step 
reviewed and approved by the SMC. 
An interim safety report will compile findings from the preceding dose group with the PI’s 
interpretation and plan to go forward. The research monitor, in collaboration with the SMC, must 
concur with the plan before proceeding. The decision to advance to the next dose level will be 
based solely on the disease attack rate and the observed safety profile at the tested dose level. 
Screening will be done on days -45 to -2, with admission to the inpatient unit on day -1. 
Challenge will occur on day 0 and discharge is planned on day 8 but the exact date will be when 
the subject meets discharge criteria. Two to three additional subjects per cohort will be 
screened and deemed “alternates”. The “alternates” will be admitted to the inpatient unit along 
with the “primary” cohort. Prior to dosing, the eligibility of subjects in the “primary” cohort will be 
re-confirmed. In the event a subject in the “primary” cohort is unavailable, or becomes ineligible; 
that subject will be replaced by an alternate. Any alternate not needed to become a member of 
the “primary” cohort will be discharged from the unit prior to administration of the challenge 
dose. For subjects receiving a challenge dose, following discharge from the inpatient unit, there 
will be clinic visits on days 14±2, 28±2, and 56±4, and a safety follow-up call will occur at day 
180±14 after challenge. 
During the inpatient stay and post challenge administration, subjects will be evaluated at least 
daily to assess for any symptoms or adverse events. Additional assessments will include at the 
minimum: daily measurement of vital signs (temperature, pulse, and blood pressure); daily 
history directed physical examinations; the collection and grading (for consistency) of each 
stool; and weighing of all loose or watery stools. Stool specimens or rectal swabs (if unable to 
produce a stool) will also be evaluated for grossly visible blood and tested for occult blood using 
hemoccult. Hemoccult testing may be limited to two samples per day if positive. Additionally, 
one sample per day will be cultured for the presence of the challenge strain. All subjects will 
receive antibiotic therapy 5 days after challenge inoculation (unless meeting criteria for early 
treatment). 
Subjects will be eligible for discharge from the inpatient unit on day 8 if they have started their 
antibiotic treatment on day 5 and passed 2 consecutive stools culture-negative for S. sonnei at 
least six hours apart. In the event stool cultures are still growing S. sonnei; subjects will remain 
in the inpatient unit until their stool is culture-negative. 
Following an evaluation and history directed physical examination; subjects will be discharged, 
with follow-up on study days 14±2 28±2 and 56±4 to provide additional stool, saliva (cohorts 2-5 
only) and blood specimens, according to the Schedule of Events in Appendix A, for safety 
checks or immunology monitoring. Subjects will be given an ice-pack and cooler for collection of 
a stool sample at home and instructed to bring the sample with them to the follow-up visit. At 
approximately study day 180, the subjects will be contacted by phone for supplemental 
surveillance to track the occurrence of any medically significant new chronic illnesses, 
pregnancy, or serious health event. 

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Any subject who wishes to withdraw early from the inpatient portion of the study will be treated 
with antimicrobials and should have two negative stool cultures before they are discharged. 
These subjects will be requested to adhere to the same follow-up schedule as all other subjects. 
Dose escalation will occur in a stepwise fashion and will be dependent upon the assessment of 
safety parameters and by meeting criteria for advancement to the next dose. A Safety 
Monitoring Committee (SMC) established as an advisory committee to the study sponsors for 
this study, along with the research monitor, will review inpatient safety data and SAEs through 
day 14 from each dose level prior to making a recommendation for advancement to the next 
dose level. The SMC would recommend one of the following alternatives: advancing to the next 
dose level, halting, additional subjects at the current dose, or decreasing the current dose. The 
next inpatient group will not be admitted until a minimum of 4 weeks have elapsed from the time 
of the preceding group’s challenge to ensure adequate time for safety monitoring. Subjects 
participating in one dose level will not be permitted to participate in a subsequent dose level. 
The inpatient research unit where this study will be conducted is part of CCHMC and is well 
equipped and suited for clinical research studies on adult subjects. Only clinical research 
studies are performed on this unit which is capable of accommodating up to 24 inpatients at a 
time. Rooms can be utilized as private or semi-private rooms (depending on the needs of 
particular studies). Each room has its own private bathroom. The inpatient unit has controlled 
access. Our research clinic, located on the same floor as the inpatient unit, will be used for the 
outpatient portion of this study. 
To minimize bias on study outcomes, an independent outcome adjudication committee, 
comprised of up to 3 individuals, independent of the study sponsors and investigative team, will 
review all study endpoints (diarrhea, fever, etc.) after study completion (defined as after day 180 
of the final cohort). The role of the committee will be to (1) review and confirm all primary 
endpoint cases; (2) review all protocol-specified entry criteria, adherence, and compliance 
issues to ascertain classification in the per-protocol and other study populations; and (3) provide 
guidance regarding secondary and other endpoint classifications to include agreement on 
objective criteria for classification of endpoints. Specific duties and responsibilities will be 
outlined by charter prior to the start of the study. The members of the adjudication committee 
may also serve as members of the SMC. 
 
4.1 Study Outcome Measures 
 
4.1.1 Primary Outcome Measures 
Shigellosis is defined as shedding of S. sonnei in the stool accompanied by moderate-severe 
diarrhea and/or dysentery along with moderate fever or one or more severe intestinal 
symptoms. All stools will be collected, weighed and graded to assess for diarrhea. Only Grade 
3, 4 and 5 stools will be considered in the criteria for diarrhea. Grade 3, 4 and 5 stools within 
any 24 hour period will be added to determine the highest severity grade for the episode. When 
there are no loose stools for 48 hours, the episode will be considered resolved. The end date 

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for the episode will be the date and time of the last Grade 3, 4, or 5 stool. The clinical disease 
endpoint is shigellosis and the primary clinical endpoints are shown below: 
o Diarrhea- 
 
Moderate- 4-5 loose or watery Grade 3-5 stools or 400-800 gram/Grade 3-5 
stools per 24 hours 
 
Severe- 6 or more loose or watery Grade 3-5 stools or >800 gram/Grade 3-5 
stools per 24 hours or requires medical intervention 
o Dysentery- a Grade 3, 4, or 5 stool with gross blood on at least 2 occasions and 
reportable constitutional symptoms. 
o Moderate Fever- Oral temperature of ≥38.5°C 
o Symptoms - one or more severe intestinal symptoms 
 Occurrence of abnormal clinical laboratory values within 11 days post challenge.
The severity of these outcomes will be graded based on the definitions outlined in the AE 
Section 9.2.1. 
 
4.1.2 
Secondary Outcome Measures 
1. Immunogenicity 
a. Systemic Immunogenicity: 
i. Serum IgA, IgM and IgG response to Shigella antigens: S. sonnei 
LPS, S. sonnei Invaplex, IpaB, IpaC and IpaD 
b. Mucosal immunogenicity: 
i. IgA and IgG Antibody Secreting Cells (ASC) and Antibody in 
Lymphocyte Supernatant (ALS) response to Shigella antigens: S. 
sonnei LPS, S. sonnei Invaplex, IpaB, IpaC, IpaD; Fecal IgA response 
to Shigella antigens: S. sonnei LPS, S. sonnei Invaplex, IpaB, IpaC 
and IpaD; and Salivary IgA response to Shigella antigens. 
c. Cell Mediated Immunogenicity: Lymphocyte proliferation after ex vivo 
stimulation of PBMCs with Shigella-related antigens (IpaB, IpaC, IpaD, 
Invaplex, LPS, and appropriate controls). The phenotype of the immune 
response will be characterized as Th1 and Th2 based on the cytokines 
secreted during the proliferation assay using a multiplex assay format. 
Cytokines to be measured include, but not limited to; IL-2, IL-4, IL-5, IL-10, IL- 
12, IL-17, IL-18 and IFN- for Th1/Th2. These investigational assays are 
required to better understand the immune response to Shigella infections. 
d. B Memory Cell assays: B memory cells (BM) are long lived plasma cells that 
provide an anamnestic or recall response in the host upon reexposure to the 
same antigen. The response is usually of greater magnitude, is faster and 

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better quality and has been correlative of protection in live Shigella vaccine 
studies [25, 26]. In this study IgG and IgA BM cell responses will be measured 
as Enzyme-Linked ImmunoSpot (ELISPOT) assays with PBMCs to S. sonnei 
LPS, S. sonnei Invaplex, IpaB, IpaC and IpaD. PBMCs will be expanded in 
vitro with appropriate mitogens prior to measuring BM responses to specific 
Shigella antigens. 
e. Expanded Immunology and Systems Biology: Specific samples will be 
collected as part of this study to support future evaluations in systems 
biology. Cells and serum samples will be collected for use in a variety of 
omics-based work such as transcriptomic and cytokine analysis. 
2. Fecal shedding of Shigella – Daily during the inpatient stay, a sample of stool will 
be screened for the presence of the S. sonnei strain. Refer to the Manual of 
Procedures (MOP). If there is no stool sample, collect a rectal swab and streak 
on HEA as described in the MOP. 
 
4.1.3 
Exploratory Outcome 
4.1.3.1 
Stool Polymerase Chain Reaction (PCR) to assess quantitative stool 
burden of S. sonnei 
 
5. STUDY ENROLLMENT AND WITHDRAWAL 
 
5.1 Subject Inclusion Criteria 
To be eligible to participate, each subject must fulfill all of the following criteria: 
1. Male or non-pregnant female between 18 and 49 years of age (inclusive). 
2. General good health defined as (a) no significant medical illness, (b) no clinically 
significant physical examination findings and (c) no screening laboratory values 
significantly outside the normal limits of the testing laboratory within 45 days of 
challenge. 
3. Demonstrate comprehension of the protocol procedures and knowledge of study by 
passing a written examination (pass grade ≥70%) on day -1. 
4. Willing to sign an informed consent form (ICF). 
5. Willingness to participate for an inpatient stay lasting up to 11 days and an outpatient 
follow-up lasting 6 months from challenge. 
6. Willing to not smoke during the inpatient stay. 
7. Available for all planned follow-up visits. 
8. Negative serum pregnancy test at screening and negative urine pregnancy test on the 
day of admission to the inpatient phase for female subjects of childbearing potential. 

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Females of childbearing potential must agree to use an effective method of birth control 
(birth control pills, injection hormonal contraceptive, implant hormonal contraceptive, 
hormonal patch, IUD, sterilization by hysterectomy or tubal ligation, spermicidal products 
and barrier methods such as cervical sponge, diaphragm, or condom) within two months 
before challenge through day 180. Abstinence is acceptable. A woman is eligible if she 
is monogamous with a vasectomized partner. 
9. Willing to not donate blood for up to 6 months after completion of the inpatient phase of 
the study. 
10. Willing to refrain from participation in another investigational vaccine or drug trial at least 
until after completion of the 6 month follow-up safety call. 
 
5.2 Subject Exclusion Criteria 
Subjects who meet any of the following criteria at baseline will be excluded from participation: 
1. Presence of a significant medical condition (e.g. psychiatric conditions, alcohol or illicit drug 
abuse/dependency, or gastrointestinal disease, such as peptic ulcer, symptoms or 
evidence of active gastritis or gastroesophageal reflux disease, inflammatory bowel 
disease), or other laboratory abnormalities which in the opinion of the investigator 
precludes participation in the study. 
2. Immunosuppressive illness or IgA deficiency 
3. Positive serology results for HIV, HBsAg, HCV, or RPR (syphilis) antibodies. 
4. Evidence of inflammatory arthritis on exam and/or HLA-B27 positive. 
5. Family history of inflammatory arthritis. 
6. Significant abnormalities in screening lab hematology or serum chemistry, as determined 
by PI. 
7. Allergy to fluoroquinolones or trimethoprim-sulfamethoxazole 
8. Fewer than 3 stools per week or more than 3 stools per day as the usual frequency. 
9. History of diarrhea in the 2 weeks prior to planned inpatient phase 
10. Use of antibiotics during the 7 days before receiving the challenge inoculum dosing 
11. Use of prescription and/or OTC medications that contain Imodium, acetaminophen, aspirin, 
ibuprofen, and other non-steroidal anti-inflammatory drugs, during the 48 hours prior to 
investigational product administration 
12. Travel within two years prior to dosing to countries where Shigella infection is endemic. 
13. Use of any medication known to affect the immune function [e.g., oral steroids, parenteral 
steroids, or high-dose inhaled steroids (>800 μg/day of beclomethasone dipropionate or 
equivalent and others): nasal and topical steroids are allowed] within 30 days preceding 
receipt of the challenge inoculum or planned use during the active study period. 

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14. Serologic evidence of Shigella sonnei (titer > 1:2500) 
15. A positive urine test for opiates 
16. A chronic disease (such as hypertension, hyperlipidemia or anxiety/depression) for which 
doses of prescription medications are not stable for at least the past 3 months. 
17. Have immunocompromised household contacts. 
18. A clinically significant abnormality on physical examination, including a systolic blood 
pressure >140 mm Hg or diastolic blood pressure >90 mm Hg, or a resting pulse >100 
beats/min or <55 beats/min (<50 beats/min for conditioned athletes). 
19. Pregnant, nursing, or plan to become pregnant within 6 months of receipt of the study 
product. 
20. In the 4 weeks following challenge, subject will be living with or having daily contact with 
elderly persons aged 70 years or more, diapered individuals, persons with disabilities, 
children <2 years old, or a woman known to be pregnant or nursing, or anyone with 
diminished immunity. This includes contact at home, school, day-care, nursing homes, or 
similar places. 
21. Work in a health care setting, day care center, or as a food handler in the 4 weeks following 
the challenge with S. sonnei. 
22. Use of any investigational drug or any investigational vaccine within 60 days preceding 
challenge, or planned use during the 6 months after receipt of the study agent. 
23. Have received a licensed, live vaccine within 28 days or a licensed inactivated vaccine 
within 14 days of receiving the challenge inoculum. 
24. Inability to comply with inpatient rules and regulations. 
25. Has any other condition that, in the opinion of the Investigator, would jeopardize the safety 
or rights of a participant or would render the subject unable to comply with the protocol. 
26. Received blood or blood products within the past six months. 
 
6. TREATMENT ASSIGNMENT PROCEDURES 
 
6.1 Withdrawal 
 
6.1.1 
Reasons for Withdrawal 
A subject may be withdrawn from study participation if any clinical AE, laboratory abnormality, 
medical condition or situation occurs such that continued participation in the study would not be 
in the best interest of the subject. The US Food and Drug Administration (FDA) also may elect 
to discontinue the study for safety reasons. The study may also be terminated at the direction of 
the study sponsors. 

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Subjects are free to withdraw from participation in this study at any time upon request, for any 
reason, specified or unspecified and without penalty or loss of medical benefits to which the 
subject is otherwise entitled. If voluntary withdrawal occurs, the subject will be asked to 
complete a final visit for safety evaluation and will receive appropriate care under medical 
supervision if symptoms of any AE related to participation in the study are continuing. The 
subject will be followed until the AE is resolved or until the subject’s condition becomes stable. 
Subjects may be withdrawn from the study for any of the following reasons: 
 Withdrawal of consent, deviation from the protocol, incidental illness, or an AE occurs such 
that continued participation in the study would not be in the best interest of the subject.
 
The reason for withdrawal should be documented in the subjects’ records, including the date 
of withdrawal/final assessment.
 
6.1.2 
Handling of Withdrawals 
If a subject decides to leave the inpatient facility after receipt of the challenge strain but before 
planned discharge; they will be strongly encouraged to at the minimum complete the antibiotic 
treatment before withdrawing from the study. Subjects who choose to withdraw from the 
inpatient portion of the study will be encouraged to complete all outpatient visits according to the 
same follow-up schedule as all other challenged subjects. 
 
6.2 Termination of Study 
During the clinical trial, if any subject develops an SAE determined to be related to study 
participation; the study will be reviewed by the SMC, along with the research monitor to 
determine whether the study will proceed or be discontinued. The FDA also may elect to 
discontinue the study for safety reasons. The study may also be terminated at the direction of 
the study sponsors. 
 
7. INVESTIGATIONAL PRODUCT 
 
7.1 Study Products Description 
 
S. sonnei 53G was initially isolated from a child with diarrhea in Tokyo in 1954. The seed was 
maintained at the Center for Vaccine Development, University of Maryland. A master cell bank 
(MCB) (BPR-327-00, Lot 0593) was manufactured by the Pilot Bioproduction Facility (PBF), 
Walter Reed Army Institute of Research (WRAIR), Silver Spring, MD, U.S. The lyophilized 53G 
strain of S. sonnei was manufactured under current Good Manufacturing Practice (cGMP) 
conditions at the WRAIR PBF. Testing completed on S. sonnei 53G are listed in Table 1. 

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7.1.1 
Acquisition 
For S. sonnei 53G; MCB seeds and Production Cell Bank (PCB Lot 0599) seeds were available 
for use and stored at the WRAIR Pilot Bioproduction Facility as 1 mL glycerol cultures. A single 
vial of PCB was used to carry out a 30-liter fermentation, and the contents were lyophilized after 
the cultures were suspended in a cryopreservative. The lyophilized vials are stored at -80ºC ± 
10ºC at the PBF. 

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Table 1. Tests Completed on S. sonnei 53G 
Test 
Result 
Final Container Lot Evaluation 
Pass; White cake with no visible particulates 
Viability and stability 
Pass; > 4×10^8 cfu/vial and > 50% Form I cfu (WRAIR 
Pilot Bioproduction Facility and WRAIR Dept. of Live 
Shigella Vaccines) 
Biochemical identification 
Pass; Shigella sonnei (WRAIR Multidrug-resistant 
organism Repository and Surveillance Network) 
Antibiotic Sensitivity 
Pass; Sensitive to expected antibiotics, including 
ciprofloxacin, TMP-SMX, and tetracycline (WRAIR 
Multidrug-resistant organism Repository and Surveillance 
Network) 
pH 
Report as tested; pH 6.5 (WRAIR Pilot Bioproduction 
Facility) 
Moisture Content 
Pass; < 3% (BioReliance) 
Invasion assay 
Pass; Invasive in HeLa cells (WRAIR Dept. of Live 
Shigella Vaccines) 
Plaque assay 
Pass; Plaque-positive (WRAIR Dept. of Live Shigella 
Vaccines) 
Virulence assay 
Pass; Virulent, Sereny-positive in the guinea pig 
keratoconjunctivitis model (WRAIR Dept of Live Shigella 
Vaccines) 
Presence of lytic bacteriophage 
Pass; Negative (PATH Vaccine Solutions and Charles 
River Laboratories) 
Microbial Limits Test 
Pass; Meets USP <61> and <62> criteria of acceptance 
for the Microbiological Examination of Non-Sterile 
Products for Microbial Enumeration Test for Yeast and 
Mold and Tests for Specified Microorganisms: Bile- 
tolerant Gram-negative bacteria, Escherichia coli, 
Salmonella spp., Pseudomonas aeruginosa, 
Staphylococcus aureus, Clostridia spp., and Candida 
albicans (BioScreen Testing Services) 

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Product Storage, Stability, and Expiration 
Vials containing the challenge strain will be stored in the Investigational Pharmacy in a -80ºC ± 
10ºC freezer that is under a temperature-monitoring program. On the day of challenge; 
lyophilized frozen vials of Shigella sonnei 53G strain (Lot 1794) will be prepared by an 
Investigational Pharmacist. 
The viability and stability of Shigella sonnei 53G has been measured by removing vials from 
storage and doing colony counts as well as evaluating the percentage of Form I phenotype by 
colony immunoblots with monoclonal antibodies to either IpaB or IpaC. These studies were 
carried out at the PBF and the Department of Live Shigella Vaccines. Viability of Lot 1794 and 
stability testing is scheduled to occur annually. The data for Shigella sonnei 53G are given in 
Table 2. 
 
 
Table 2. Viability and Stability Testing of Shigella sonnei 53G 
 
Date 
Viability (cfu/vial) 
Stability (% Form I cfu) 
2013 Feb 25 
3.9×109 
> 80% 
2013 Jun 3 
3.2×109 
> 80% 
2013 Aug 26 
2.8×109 
> 80% 
2013 Nov 25 
5.2×109 
> 80% 
2014 Feb 26 
1.9×109 
> 80% 
2014 Aug 13 
3.6×109 
> 80% 
2015 Mar 04 
4.8×109 
> 80% 
 
Microbiological materials 
As detailed in the Manual of Procedures (MOP), WRAIR will ship specialized microbiological 
materials to CCHMC for training and clinical trial use. 
 
7.1.2 
Preparation/Handling 
Lyophilized, frozen vials of S. sonnei 53G will be maintained in Investigational Pharmacy and 
stored at -80ºC ± 10ºC until the day of challenge. On the day of challenge, the requisite number 
of vials will be removed from the freezer and placed on ice and allowed to thaw for 30 minutes. 
Then 2 mL of cold sterile water for injection (SWI) will be added to each vial. To ensure 
complete rehydration of the challenge strain along with homogeneous mixing of each vial’s 
contents; the vials will remain on ice for another 15 minutes with intermittent swirling of the 
suspension. All vials then will be combined and diluted in cold, sterile normal saline 0.9% to 

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arrive at the desired concentration. The diluted challenge material will be kept on ice until 
administration. At the time of reconstitution, an aliquot will be removed for testing by colony 
count to be able to document the actual dose administered to the subjects. Plating and colony 
count determination will be performed as described in the MOP regarding culturing of stool 
samples for Shigella. Time of reconstitution will be noted. The maximum hold time is 2 hours on 
ice between the reconstitution and administration of the challenge product to the subjects. 
One mL of the challenge solution at the appropriate concentration will be added to 30 mL of 
sterile normal saline 0.9%. Two grams of sodium bicarbonate (NaHCO3) in 120 mL of SWI will 
be placed in a second cup. 
 
7.1.3 
Administration 
Subjects will fast for 90 minutes prior to receiving the challenge inoculum and 90 minutes after 
challenge. At the indicated time, the subjects will be gathered together for dose distribution. 
Subjects will drink the 120 mL of sodium bicarbonate to neutralize gastric acidity and then drink 
the challenge suspension within 5 minutes of drinking the sodium bicarbonate. Care will be 
taken to ensure that a minimum amount of time, not to exceed 2 hours, is spent between the 
reconstitution of the challenge inoculum and the oral administration of the inoculum to the 
subjects. 
Shigella sonnei 53G is classified as a BSL-2 category organism. Personnel will take appropriate 
precautions for working with BSL-2 organisms and wear proper protective equipment such as 
lab coats, gloves and, if necessary, masks to shield themselves and others from spills and 
inadvertent mishaps. Spills will be treated according to SOP (Standard Operating Procedure). 
All unused reconstituted inoculum and materials containing the bacteria will be disposed of 
properly in biohazard bags. All exposed materials and all disposable materials will be discarded 
in the biohazard waste for pick up and decontamination as per CCHMC policy. 
 
7.1.4 
Accountability/Final Disposition for the Investigational Products: 
Study personnel will maintain inventory of Shigella sonnei 53G inoculum according to the SOP 
“Investigational Product Accountability” of the CCHMC Investigational Drug Service. 
The FDA requires accounting for the disposition of all investigational products. The investigator 
is responsible for ensuring that a current record of product disposition is maintained and 
dispensed only at an official study site by authorized personnel as required by applicable 
regulations and guidelines. Records of product disposition as required by federal law consist of 
the date received, date administered, quantity administered, and the subject to whom the drug 
was administered. 
The pharmacist will be responsible for maintaining accurate records of the shipment and 
dispensing of the study product, and all efforts will be made to protect the integrity of the test 
article. The pharmacy records must be available for inspection by the sponsor’s representative 
and is subject to inspection by a regulatory agency (e.g., FDA) at any time. 

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At the termination of the study, all unused product must be returned to the study sponsor or 
designee (or destroyed if not feasible to return). A written explanation will be required for any 
product not returned to the study sponsor, stating the reason it was not returned. Study vials 
and other contaminated materials will be disposed of using appropriate biohazard precautions. 
Any test article that pharmacy personnel are instructed to dispose of will be destroyed per site 
destruction policies. 
 
7.1.5 
Ancillary Supplies Description 
Oral administration of an antibiotic will be started for all subjects on study day 5 unless they 
meet the requirements for early treatment (see Section 8.1.4). This treatment is to ensure 
clearance of the challenge organism from their bodies prior to discharge. The first line treatment 
is ciprofloxacin 500 mg BID x 3 days; the alternate treatment is trimethoprim-sulfamethoxazole 
(Bactrim) 160/800 mg BID x 5 days. Ciprofloxacin is a broad-spectrum antibacterial agent 
belonging to the fluoroquinolone family of antibiotics. Bactrim is a sulfonamide antibiotic 
combination of trimethoprim and sulfamethoxazole used in the treatment of a variety of bacterial 
infections. Possible AEs to ciprofloxacin and trimethoprim-sulfamethoxazole are described in 
Section 2.3.1. 
 
8. STUDY SCHEDULE 
 
A Study Schedule of Events is provided in Appendix A. 
 
8.1 Clinical 
 
8.1.1 
Screening (day -45 to -2) (may occur in 2-3 visits) 
 
Presentation of complete description of study to subjects 
 
Review inclusion/exclusion criteria 
 
Sign ICF 
 
Medical interview 
 
Medical history 
 
History of all current medications as well as those taken within the past 28 days 
 
Vital signs (temperature, pulse, and blood pressure) will be taken and recorded 
 
Complete physical examination 
 
Collect lab samples as described in Section 8.2.1 
Subjects, who meet all eligibility criteria and sign the study consent form, will be eligible for 
enrollment in the study. A low creatinine value, a low ALT value and/or a low AST value are/is 
acceptable for study inclusion. No lower limits of lab values for creatinine, ALT, and AST are 

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considered to be clinically significant. Acceptable laboratory values are set forth in Appendix B. 
Subjects will be informed of their screening test results by phone. Those with results that 
exclude them from eligibility will be given the test results by phone unless the results are 
sensitive in nature. Subjects who have a positive HIV, Hep B or C test will be asked to return to 
the clinic to receive their results. Excluded subjects will be advised to seek further evaluation 
from their primary care provider. Subjects that are found positive for HLA-B27 will be counseled 
on their risk for ankylosing spondylitis or related diseases and referred to the primary care 
provider for further questions. 
To ensure comprehension of the study, all subjects will have to pass a written examination 
before they are eligible for inclusion (minimum passing grade is 70%). Participants will be given 
a sample test at the time of screening. On the day of admission to the inpatient unit, a written 
test will be administered which the participants will have 2 chances to pass successfully. If 
subjects are unable to score 70% or greater on the test after the second attempt, they will be 
excluded from the study. 
 
8.1.2 
Admission day (study day –1) 
After admission and prior to receipt of the challenge inoculum: 
 
A complete physical examination will be performed by a study investigator 
 
Inclusion and exclusion criteria will be reviewed to reassess ongoing eligibility 
 
Comprehension test will be administered 
 
Medical interview 
 
Medical history will be reviewed 
 
Concomitant medications will be reviewed 
 
Vital signs (temperature, pulse and blood pressure) will be taken and 
recorded 
 
A urine pregnancy test (HCG) will be performed for all female subjects 
 
Stool will be collected and sent to the lab as described in Appendix A and 
Section 8.2.2.6 for baseline stool sample for fecal IgA. Challenge is not 
dependent on results but efforts will be made to collect 2 samples = between 
day -5 and day 0 prior to challenge 
 
Blood will be collected for PBMC, serology, cytokines and systems biology 
 
Saliva will be collected (cohorts 2-5 only) for salivary IgA between day -1 and 
day 0 any time prior to challenge 
 
8.1.3 
Challenge day (study day 0) 
Challenge administration 

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Study investigator will conduct a medical interview and physical examination and a review of 
inclusion/exclusion criteria, concomitant medications, and medical history. Subjects will fast for 
90 minutes prior to receiving the challenge inoculum and 90 minutes after receipt. Prior to 
administration of the challenge product; subjects will drink approximately 120 mL solution of 
bicarbonate buffer to neutralize the stomach acidity. Within 5 minutes, subjects will drink 
approximately 30 mL of the S. sonnei suspension. Challenge preparation is described in Section 
7.1.2. If any subject vomits following ingestion of the study agent, they will not be re-dosed. A 
note will be made in the study record regarding the apparent amount of the vomitus (small, 
moderate or large). The subject still will continue in the study and undergo all study related 
procedures. 
Post Challenge 
Subjects will be closely monitored for any symptoms/events occurring within the first 90 minutes 
post challenge. The following will be conducted and recorded after 90 minutes: 
 
Physical assessment: Vital signs (temperature, pulse, and blood pressure) will be 
checked and history targeted physical exam will be conducted. 
 
Adverse Events: Symptoms will be assessed for adverse events and concomitant 
medications will be reviewed. 
 
8.1.4 
Inpatient stay (study days 0-8). 
Daily clinical assessment: Each study day will be considered midnight to 23:59. At least daily, 
a study investigator will assess every subject and administer a targeted questionnaire to capture 
symptoms/signs of illness (including diarrhea, abdominal pain/abdominal cramps, gas, anorexia, 
nausea, headache, myalgia, malaise, arthralgia, fever, vomiting). Additionally, based on 
symptomatology; a history-directed physical examination will be performed. Study staff will take 
vital signs, (temperature, pulse, and blood pressure) and review the subject stool log with 
investigator. Additional medically indicated clinical investigations will be performed based on the 
study physicians’ judgment. Illness labs may be collected on any participant experiencing Grade 
3 or higher illness symptoms and may include chemistry (sodium, potassium, BUN, creatinine 
total bilirubin, ALT, AST) and hematology (WBC, hemoglobin, neutrophil and lymphocyte count, 
platelets) evaluations. A urine dipstick may be performed to capture urine specific gravity  
results in real time. 
A study physician will be on-call 24 hours/day. Adverse events, medical history and concomitant 
medications will be reviewed daily and entered into the source documents. 
Subjects will be asked to collect all stools and bring them to the study staff. Qualified medical 
personnel will evaluate the stool for consistency and the presence/absence of visible blood 
and/or mucus. Stools that are loose or watery (grades 3-5) will be weighed, and if there is visible 
blood, a hemoccult test will be performed to confirm presence of occult blood. Hemoccult testing 
may be limited to two samples per day if positive. Study staff will record the information in the 
subject’s source document. If a subject is unable to produce a stool by midnight, a rectal swab 
will be obtained. 

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Saliva will be collected at several time points during the inpatient stay, according to the 
Schedule of Events in Appendix A. 
To assess diarrhea grade, study personnel will use the following definitions for grading of stool 
samples: 
 
Grade 1 (Normal stool)– Firm, tootsie roll consistency 
 
Grade 2 – Soft, pudding consistency, not firm but holds some shape 
 
Grade 3 (Loose stool)– Takes the shape of the container, thick gravy consistency/brown 
watery, opaque watery, chocolate milk consistency 
 
Grade 4 (Watery stool)- Opaque colored, watery consistency 
 
Grade 5- “rice water”, soapy water consistency 
Assessment and Management of Diarrhea, Vomiting, Dysentery, and Dehydration 
Only Grade 3, 4 and 5 stools will be considered in the criteria for diarrhea. Grade 3, 4 and 5 
stools within any 24 hour period will be added to determine the highest severity grade for the 
episode. When there are no loose stools for 48 hours, the episode will be considered resolved. 
The end date for the episode will be the date and time of the last Grade 3, 4, or 5 stool. 
For subjects who experience diarrhea, all loose or watery stools will be weighed, graded (for 
consistency), and evaluated by qualified medical personnel for grossly visible blood. Prior to 
study start-up, study personnel will receive training and written instructions on how to weigh and 
evaluate stools for consistency and the presence of blood. If visible blood is noted on a grade 3, 
4 or 5 stool, a hemoccult test will be performed to confirm presence of occult blood. Testing may 
be limited to two samples per day if hemoccult positive. 
The assessment of any subject who experiences diarrhea (any Grade 3 – 5 stool) will include a 
targeted physical assessment and vital signs (temperature, heart rate, blood pressure). Blood 
pressure will be monitored for diastolic hypotension until diarrhea has resolved (see Table 4). 
Subjects developing Grade 2 adverse event for diarrhea symptoms also will have urine specific 
gravity and weight assessed daily until Grade 2 adverse event symptoms have resolved. If any 
Grade 3 adverse event for diarrhea symptoms occur vital signs will be obtained every 4 hours + 
1 hour in addition to the procedures listed above. Participants experiencing Grade 3 symptoms 
or higher illness symptoms may have illness labs collected. 
Oral rehydration will be initiated and recorded for subjects with a Grade 2 or higher adverse 
event of diarrhea, vomiting, dysentery, or dehydration. The hydration of subjects developing 
diarrhea will be maintained with oral electrolyte solutions (i.e. Pedialyte, Gatorade) by offering at 
least 1.5 mL for each gram of diarrheal stool lost, as tolerated. Subjects who develop vomiting 
will be offered at least 1.0 mL for each gram of emesis lost, as tolerated. Subjects will be 
evaluated by a physician who will consider intravenous hydration, for subjects who: 
 
cannot tolerate oral fluids, 
 
experience fluids loss that exceeds their ability to drink replacement fluids, 

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 
have >1000 mL deficit in intake in a 24 hour period, 
 
experience weight loss of more than 5%, 
 
have urine specific gravity >1.030 for 12 hours, 
 
have syncope or near-syncope, 
 
Grade 3 tachycardia, or 
 
Grade 3 hypotension. 
If the subject is unable to tolerate fluids by mouth and intravenous fluids are deemed necessary 
by the assessing physician, a 0.9% sterile saline solution 1-liter bolus will be administered 
intravenously. This bolus will be repeated as needed to resolve Grade 2 and above symptoms 
at the discretion of assessing physician. Fluid maintenance will be continued using 0.45% NaCl, 
5% dextrose, and 20 mEq KCL per liter at a rate of 100mL/hr until subject is able to resume oral 
rehydration. If the subject develops grade 4 hypotension (see Table 4) despite the aggressive 
fluid management, the subject will be transferred from the inpatient unit to a hospital setting. 
Study day 5 
All subjects will be treated (unless meeting criteria for early antibiotic treatment) with a 3-day 
course of ciprofloxacin 500 mg bid in order to eradicate S. sonnei from the intestinal tract of 
subjects. Trimethoprim (160)-sulfamethoxazole (800) bid x 5 days will be used as alternate for 
those subjects who cannot tolerate ciprofloxacin. 
 
 
Criteria for early antibiotic treatment (before study day 5): 
 
Diarrhea (any severity stool Grade 3 or higher) and two or more of the following 
symptoms: severe abdominal pain/cramps, severe nausea, severe headache, severe 
myalgia, severe arthralgia, gross blood in ≥2 stools, moderate fever (38.5ºC), or any 
vomiting. 
 
Subjects who experience unexpectedly severe events such as symptomatic hypotension 
(disproportionate to volume loss), renal dysfunction, or altered mental state (e.g. 
somnolence) at the discretion of the investigators. 
 
A study physician determines early treatment is warranted for other reasons. 
 
Subjects receiving early antibiotic treatment will remain on the unit until the planned day 
of discharge (day 8) 
 
8.1.5 
Study day 8 (Planned discharge) 
Subjects will be eligible for discharge from the inpatient unit on day 8 after they have met all of 
the following criteria: 
1. Antibiotic treatment has been initiated on day 5 (or earlier if protocol criteria are met). 

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2. Two stool samples (collected at least six hours apart) are negative for S. sonnei. 
3. All symptoms are resolved or resolving. 
4. The subject is able to maintain normal hydration status. 
 
Subjects who still have S. sonnei isolated from their stool on the planned day of discharge will 
remain on the unit until they are no longer shedding and all criteria are met. Subjects who meet 
all discharge criteria and were started on a 5 day course of Trimethoprim instead of the 3-day 
course of Cipro will receive instructions to take the remaining doses at home as prescribed. 
Subjects still shedding 48 hours after initiation of ciprofloxacin will be switched to Trimethoprim 
(1 tablet twice a day for five days). 
Following a history-directed physical exam performed by a study investigator and review of 
concomitant medications, medical history, and a symptom questionnaire to evaluate for 
reactogenicity, subjects will be discharged with instructions to return for follow-up visits on study 
days 14±2, 28±2d and 56±4d to provide additional specimens for safety checks or immunology 
monitoring, described in Section 8.2.2. Subjects will be given an ice-pack and insulated bag for 
collection of stools at home and subsequent transport of specimens to study site on days 14, 
and 28, 
Discharge Instructions 
Subjects will be provided with verbal and written instructions, a memory aid, and digital 
thermometer to take home and record symptoms and body temperature daily starting at 
discharge and for the next 5 days. Adverse events that occur during this time will be recorded 
on and collected from the Memory Aid. Subjects will be instructed to take any remaining doses 
of study antibiotic per the written schedule provided. Medications (including study antibiotic) 
taken during this period will also be recorded on and collected from the Memory Aid. 
Subjects will be asked to notify the study staff promptly if they develop fever, vomiting, or 
diarrhea during the 5 days after discharge and may be asked to return to the study site for 
evaluation. 
 
8.1.6 
Early Discharge 
If a subject decides to withdraw consent and leave the unit early, he/she will be counseled about 
the risk of transmission of Shigella to close contacts. Prior to release of the subject, a 1-gram 
dose of ciprofloxacin will be given to the subject and directly observed that the antibiotic is taken 
on the unit. This dose of ciprofloxacin has been shown to be 100% effective in eliminating 
infection and symptoms for treatment of shigellosis not caused by S. dysenteriae type 1. The 
subject will be sent home with four additional 500 mg tablets of ciprofloxacin to complete the 3- 
day course of therapy at home. He/she will be asked to read and sign the form contained in 
Appendix C. 
Efforts will be made to encourage participants to complete the inpatient stay. Participants who 
have been released from the inpatient unit before study day 8 will be asked to maintain the 
study schedule, including return visits for day 10 stool submission and the inpatient 

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immunogenicity blood draws that they missed to be collected, with the plan to be within 1 day of 
the scheduled collection. Study staff will attempt to contact subjects by phone to evaluate their 
well-being. 
 
8.1.7 
Study day 14 (+2 day) and 28 (±2 days) 
Outpatient visit: On study day 14, subjects will bring a stool specimen for fecal IgA processing, 
culture, PCR, microbiome. If no stool is available, 2 rectal swabs will be obtained for culture and 
PCR (fecal IgA only can be done on bulk stool). On study day 28, subjects will bring a stool 
specimen for microbiome. Subjects will bring their stool specimen within 2- 4 hours of collection 
(or within 8 hours for stool stored in an insulated bag with an ice pack). On both study days 
saliva will be collected (cohorts 2-5 only), and blood will be collected for serum IgA, IgG and IgM 
assays. On day 14, blood will be collected for safety labs and ALS. ALS samples will be 
processed within 2 hours from the time of collection. A review of the Memory Aid will be 
performed to evaluate any reactogenic symptoms since discharge from the inpatient unit 
(including diarrhea, abdominal pain/abdominal cramps, gas, anorexia, nausea, headache, 
myalgia, malaise, arthralgia, fever, and vomiting). Adverse events, medical history and 
concomitant medications will be reviewed. Subjects will be given an ice pack and insulated bag 
on study days 14 for collection of stools at home and subsequent transport of specimens to 
study site on study days 28. 
On day 28, a blood sample for serology (IgA, IgG, and IgM), and memory B-cell assay will be 
collected from all subjects and a urine pregnancy test (HCG) will be performed for all female 
subjects. 
Adverse events, medical history, pregnancy, and concomitant medications will be reviewed. 
History directed physical examinations will be performed as needed prior to discharge from the 
clinic. 
 
8.1.8 
Study day 56 (± 4 days) 
Outpatient visit: The final outpatient study visit will be done on study day 56±4. Blood will be 
collected for serology (IgA, IgG and IgM) and memory B-cell assay. Saliva will be collected 
(cohorts 2-5 only). A medical interview and history-directed physical examination will also be 
performed if needed. 
Adverse events, medical history, pregnancy, and concomitant medications will be reviewed. 
Female subjects who report pregnancy between the time of challenge and the final six month 
phone call will be followed through delivery or until pregnancy outcome is known. 
 
 
 
8.1.9 
Study day 180 (± 14 days) 
Telephone interview: A telephone interview will occur to query subjects about any medically 
significant new chronic illnesses, pregnancy, or serious adverse event. As part of the interview, 

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the ROME III, a questionnaire designed to classify functional gastrointestinal disorders, such as 
irritable bowel syndrome, will be administered. If subjects report any abnormal symptoms, 
including new onset of joint pains and arthritis symptoms, then an extra follow up visit may be 
arranged. 
If subjects report any research related injuries or SAEs, they will be followed periodically until 
the events are resolved or become stable. Follow-up process can be both by telephone and/or 
asking the subjects to visit the study site, which depends on their conveniences and/or the 
severity of the particular event. 
 
8.1.10 Unscheduled Visits 
If an unscheduled visit occurs, a member of the clinical study team (PI, Co-Investigator, Study 
Coordinator, or Clinical Nurse) will interview and evaluate the subject to determine the cause of 
the visit and provide care as needed and information documented on a supplemental visit form. 
Adverse events and concomitant medications will be reviewed. 
 
8.2 Laboratory Evaluations and Procedures 
 
8.2.1 
Laboratory Evaluations (blood volumes are estimates only) 
 
8.2.1.1 
Screening (study days -45 to -2) 
 
Approximately 48 mL blood will be collected: 
o Hematology 
 
CBC (WBC, Hgb, platelets, neutrophil and lymphocyte count): (3 mL) 
o Biochemistry 
 
Na+, K+, Creatinine (BUN will be obtained only if creatinine is above the 
normal range) Total bilirubin, aspartate aminotransferase/alanine 
aminotransferase (AST/ALT) (7.5 mL) 
 
HLA-B27 (10 ml) 
 
HIV, HCV, RPR (syphilis), and HBsAg: (10 mL) 
o Serum βHCG pregnancy test, all females (5mL) 
o Serology 
 
anti-S. sonnei LPS serum IgG titer: (5 mL SST) 
 
Systems biology work (3 x 2.5 mL PAXgene tubes) 
 
Urine: 
o Urine test for opiates (5 – 10mL urine in sterile screw top container) 
 
 
Stool: 

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o A total of 2 stool samples will be collected for baseline fecal IgA sample between day 
-5 and prior to receipt of the challenge inoculum on day 0 (challenge not contingent 
upon results). 
Acceptable Laboratory Values for Eligibility and Defining Normal Values 
Screening labs may be repeated once if outside the normal limits but considered not clinically 
significant by the site clinician. Refer to Appendix B for reference ranges. 
 
 
WBC and neutrophil counts below the laboratory normal will be allowable if they are in 
keeping with the levels seen in a condition that is prevalent in our population known as 
“benign ethnic neutropenia.” 
 
A low creatinine value, a low total bilirubin value, a low ALT value and/or a low AST 
value are/is acceptable for study inclusion. No lower limits of lab values for creatinine, 
total bilirubin, ALT, and AST are considered to be clinically significant. 
 
8.2.1.2 
Inpatient Baseline (study day -1) 
 
Approximately 112.5 mL blood will be collected: 
o Serology 
 
Antibody (IgG, IgA, IgM) serology assays: (10 mL SST) 
o Cell-based 
 
ASC: (50 mL in EDTA tubes) 
 
ALS: (10 mL EDTA tubes) 
 
Memory B-cells: (30 mL in EDTA tubes) 
 
Systems biology work (3 x 2.5 mL PAXgene tubes) 
 
Cytokines (5 mL SST tube) 
o βHCG pregnancy test (for all females) (>5 mL urine in sterile screen top container) 
will be performed 
 
Stool: One stool sample, for culture, PCR, fecal IgA testing, baseline microbiome and 
transcriptome testing will be collected prior to challenge inoculation. 
 
Saliva (cohorts 2-5 only): A baseline saliva sample will be collected prior to challenge 
inoculation. 
 
8.2.1.3 
Inpatient Study Days (study days 0 – 8) 
 
Stool- Every day, an aliquot of stool will be collected for culture, PCR, microbiome and 
transcriptomics as detailed in the Schedule of Events, Appendix A. Stool processed in 
modified buffered glycerol saline (BGS) will be used for quantitative culture and PCR. As 
scheduled by Appendix A, one Oak ridge tube for fecal IgA (days 0, 3, 7 and 14), 2.0 gm 
for microbiome and two 0.5 gm aliquots saved in RNAlater for transcriptomics will be 
processed and kept frozen at -80ºC ± 10ºC. If no stools are passed on a given day, 2 
rectal swabs will be collected for culture and PCR. 
 
Saliva samples will be collected (cohorts 2-5 only) on days 1, 3, 5, and 7 and assessed 
for antigen-specific antibody titers and total IgA concentration. 

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 
Blood will be collected on 
o Day 0 (4 hrs. and 12 hrs. post-inoculation) (approximately 25 mL blood) for: 
 
Systems Biology (3 x 2.5 mL PAXgene tubes) 
 
Cytokines (5 mL SST tube) 
o Day 1 (24 hours post-inoculation) (approximately 12.5 mL blood) for 
 
Systems Biology (3 x 2.5 mL PAXgene tubes) 
 
Cytokines (5 mL SST tube) 
o Day 3 (approximately 5 mL blood) for 
 
Cytokines (5 mL SST tube) 
o Day 5 (approximately 62.5 mL blood will be collected) for: 
 
ASC: (50mL in EDTA tubes) 
 
Systems biology (3 x 2.5 mL PAXgene tubes) 
 
Cytokines (5 mL SST tube) 
o Day 7 (approximately 70 mL blood will be collected) for: 
 
Serology 
 
Antibody (IgG, IgA, IgM) serology assays: (10 mL SST) 
 
Cellular-based: 
 
ASC: (50 mL in EDTA tubes) 
 
ALS: (10 mL in EDTA tubes) 
 
8.2.1.4 
Study day 14+2 day 
 Stool will be collected for fecal IgA, culture, microbiome, and PCR as described in Section 
8.2.2.
 Blood (approximately 30.5 mL) will be collected for:
o Serology 
 
Antibody (IgG, IgA, IgM) serology assays: (10 mL SST) 
o Hematology 
 
CBC (WBC, Hgb, platelets, and neutrophil and lymphocyte count: (3 mL) 
o Biochemistry 
 
Na+, K+, BUN/Cr (BUN will be obtained only if creatinine is above normal 
values in Appendix B), Total bilirubin, aspartate aminotransferase/alanine 
aminotransferase (AST/ALT) (7.5 mL) 
o Cellular-based 
 
ALS: (10 mL – EDTA tube) 
 Saliva will be collected and assessed for antigen-specific antibody titers and total IgA 
concentration

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8.2.1.5 
Study day 28±2d 
 Stool will be collected for microbiome as described in Section 8.2.2
 Blood (approximately 50.5 mL) will be collected:
o Serology 
 
Antibody (IgG, IgA, IgM) serology assays: (10 mL SST) 
o Hematology 
 
CBC (WBC, Hgb, platelets, and neutrophil and lymphocyte count: (3 mL 
EDTA tube) 
o Biochemistry Na+, K+, BUN/Cr (BUN will be obtained only if creatinine is above 
normal values in Appendix B), Total bilirubin , aspartate aminotransferase/alanine 
aminotransferase (AST/ALT) (7.5 mL) 
o Cellular-based 
 
Memory B-cell: (30 mL in EDTA tubes) 
 Saliva (cohorts 2-5 only) will be collected and assessed for antigen-specific antibody titers 
and total IgA concentration
 
8.2.1.6 
Study day 56±4d 
 
Blood (approximately 40 mL) will be collected: 
o Serology 
 
Antibody (IgG, IgA, IgM) serology assays: (10 mL SST) 
o Cellular-based 
 
Memory B-cell: (30 mL in EDTA tubes) 
 Saliva (cohorts 2-5 only) will be collected and assessed for antigen-specific antibody titers 
and total IgA concentration
 
8.2.2 
Laboratory Assays 
 
8.2.2.1 
Stool Culture: 
The Laboratory for Specialized Clinical Studies (LSCS) at CCHMC will assay for the presence  
of S. sonnei. The first procedure is qualitative and involves streaking swabs containing fecal 
material to Hektoen Enteric Agar (HEA) plates and will be done at LSCS. The second procedure 
is quantitative and involves plating serial dilutions of stool suspensions (in BGS) to HEA plates 
and will be done at WRAIR. All HEA plates will be incubated at 37±1o C overnight. For each 
subject from the qualitative cultures, up to two blue-green (non-lactose fermenting) colonies will 
be picked from swab-streaked HEA plate and tested by agglutination by S. sonnei polyvalent 
Group D antiserum at LSCS. Colonies that agglutinate will be recorded as positive for presence 
of S. sonnei. HEA plates used for the serial dilutions that contain blue-green colonies will be 
processed using a colony blot procedure specific for the detection of S. sonnei. The number of 
positive colonies for each dilution will be recorded and used to calculate the cfu per gram of 

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stool. These procedures will follow the MOP for culture, isolation and identification for Shigella 
sonnei from stool specimens. 
 
8.2.2.2 
Microbiology and Molecular Biology: 
2-3 grams of frozen stool in BGS (or a rectal swab, if no bulk stool is collected) will be used for 
quantitative culture on HEA. Non-lactose fermenting colonies will be tested for the expression of 
IpaB using a colony immunoblot assay and the presence of the IpaH locus by PCR as described 
in the MOP. Whole genome sequencing will be performed on selected colonies as described in 
the MOP. 
 
8.2.2.3 
Antibody secreting cells (ASC) and Antibody in Lymphocyte 
Supernatant (ALS): 
The EDTA tubes will be maintained at room temperature and processing (including 
centrifugation) will begin within 2 hours of collection. Peripheral blood mononuclear cells 
(PBMC) will be cryopreserved and to minimize variability due to testing on different days, the 
cells will be assayed in a batch when the complete series of blood collections from a cohort is 
available. An ELISPOT will be used to enumerate S. sonnei LPS. IpaB, IpaC, IpaD and S. 
sonnei Invaplex specific IgA and IgG ASCs per 106 PBMC. This procedure will follow the MOP 
for “Detection of Shigella antigen(s) specific Antibody Secreting Cells (ASC) in Blood of Human 
and/or Monkey by ELISPOT”. The ALS assay will measure the secretion of Shigella-specific 
antibodies from peripheral blood lymphocytes (PBLs) and will follow the SOP for Purification of 
α4β7+ lymphocytes from frozen peripheral blood mononuclear cells via magnetic separation. 
 
8.2.2.4 
Memory B-cell Assay: 
The detection of antigen-specific memory B cells induced after challenge with S. sonnei 53G will 
be evaluated in frozen PBMCs collected on day -1 (or any time before challenge on day 0), 28 
and 56 by assaying mitogen-expanded PBMCs by ELISPOT for antigen-specific IgA-secreting 
ASCs using established protocols and the SOP for expansion of human PBMCs to measure 
Shigella-specific IgG and IgA memory B cells by ELISPOT. 
 
8.2.2.5 
Serum Immunoglobulins: 
Blood (10 mL) will be collected in a tiger top SST on days -1, 7, 14, 28 and 56 for IgA, IgM and 
IgG assay by ELISA [27-29]. The serum will be separated and stored frozen for determination of 
specific antibody responses against S. sonnei LPS and Invaplex protein-antigens. This 
procedure will follow the SOP “Enzyme linked immunosorbent assay (ELISA) to Shigella 
antigen-specific serum IgG, IgA and IgM antibodies from human serum samples.” Serum 
collected for serology may also be used for future SBA testing according to the SOP 
“Bactericidal assay against Shigella sonnei.” 
 
8.2.2.6 
Fecal Secretory IgA: 
IgA (total and S. sonnei specific) will be determined in fecal extracts using standard ELISA on 
days -1, 0 (back-up), 3, 7, and 14. The stool sample (4-5gm) will be placed in a 30 mL Oakridge 
tube and frozen at -75ºC ± 10ºC until ready for the assay. IgA will be recovered from stool 
samples using a soybean trypsin inhibitor-EDTA procedure. These procedures will follow the 

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SOP for enzyme linked immunosorbant assay (ELISA) to measure Shigella antigen-specific 
IgG, IgA and IgM antibodies from fecal extract samples, the SOP for enzyme linked 
immunosorbant assay (ELISA) to measure the total antibody concentration in fecal extract 
samples, and the SOP for processing of human fecal samples to extract fecal IgA. 
 
8.2.2.7 
Salivary IgA: 
Saliva collection will follow the schedule as described in Appendix A. Collection of saliva 
samples will be performed utilizing synthetic oral swabs (Oracol Swab; Malvern Medical). 
Salivary samples will be collected from all subjects in cohorts 2-5. The subject will gently rub the 
sponge portion of the Oracol Swab along their gum line for approximately one minute. This 
procedure has been used in many research studies without untoward risks to subjects [30-32]. 
There is a minimal risk of choking from or ingesting the swab as it is attached to a handle for 
ease of use. Some individuals might experience temporary dry mouth. Subjects will be 
instructed not to eat or drink anything, including chewing gum, for approximately 10 minutes 
prior to saliva sample collection. Subjects will be instructed to avoid drinking alcohol or using 
mouthwash for approximately 24 hours and to avoid caffeinated beverages for approximately 12 
hours prior to collecting the sample. Saliva samples will be assessed for antigen-specific 
antibody titers from pre- and post-challenge after adjusting for total IgA. Saliva will be collected 
and processed according to the MOP. 
 
8.2.2.8 
Systems Biology/Microbiomics/Transcriptomics: 
Blood will be collected in PAXgene tubes during the pre-screen and on days -1, 0 (4 hrs. and 12 
hrs. post-inoculation), 1, and 5. Up to two stool samples for microbiomics and transcriptomics 
will be collected on days detailed in Appendix A. These samples will be processed and stored 
as described in the MOP. Analysis of these samples is considered exploratory and will be 
performed at a future date. 
 
8.2.2.9 
Cytokines: 
Blood samples will be collected in SST tubes on days -1, 0 (4 hours and 12 hours post- 
inoculation), 1 (24 hours post-inoculation), 3, and 5. Samples will be frozen at -80°C± 10ºC and 
stored for future use. 
 
8.3 Concomitant Medications/Treatments: 
Any concomitant medication deemed necessary for the welfare of the subject during the 
inpatient/challenge phase of the study may be given at the discretion of the Investigator. 
However, it is the responsibility of the Investigator to ensure that details regarding the 
medication are recorded in full in the subject’s source documents and electronic Case Report 
Forms (eCRFs), including any changes in medication that have occurred during the study. 
All medications taken by the subject during 28 days prior to the challenge period will be 
recorded. All medications taken by a subject during the study (from challenge through Day 56 
post challenge) will be recorded. Any change in dose, dosing schedule, or specific medication 
administered during the study must also be recorded. 

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In order to avoid the masking of a potential elevated body temperature, prescription and OTC 
medications that contain acetaminophen, aspirin, ibuprofen, and other non-steroidal anti- 
inflammatory drugs will not be allowed during the 48 hours prior to challenge administration. 
Following investigational challenge administration, the above medications will be allowed if 
medically required and reported on the eCRF. Medications taken during the outpatient post- 
challenge phase will be reported by the study subjects. 
 
8.4 Specimen Preparation, Handling, and Shipping 
 
8.4.1 
Instructions for Specimen Preparation, Handling, and Storage: 
Approximately 456.5 mL of blood (including screening and illness labs) will be collected from 
each subject over the study. Blood samples will be separated into lymphocyte fractions using a 
ficoll-hypaque density gradient technique and the lymphocyte fraction saved and frozen pending 
future testing. 
Saliva samples will be collected (cohorts 2-5 only) utilizing synthetic oral swabs for salivary IgA. 
Immediately after production, subject will place stool sample for fecal IgA into a sealed container 
with an ice pack, and brought to the clinic within 8 hours of production. All stool samples will be 
transported to the LSCS at CCHMC. 
All specimens will be labeled with subject code and recorded in specimen tracking log. Subjects 
will be asked to sign a separate consent form (Sample donation form) to allow the investigators 
to use their stored specimens in the future. Specimens may be used for research purposes 
related to shigellosis and other enteric pathogens. Use of these specimens in future studies will 
require approval by the appropriate Institutional Review Board (IRB). A subject’s refusal to grant 
consent for such use of their stored specimens will not affect his/her eligibility to participate in 
the current protocol. 
Stool specimens will be transported within CCHMC in certified double containers appropriately 
marked in accordance with laboratory specific operating procedures and approved Institutional 
Biosafety Committee protocols. Groups of specimens will be transported to the lab in a 
Styrofoam container with lid and lined with absorbent material. 
Laboratory staff that handle shipments have received certified training and certified dangerous 
good containers must be used. For the laboratory, the laboratory-specific operating procedures 
and specific Institutional Biosafety Committee approved procedures will be used. 
In the isolation facility, standard approved infection control procedures will be used for handling 
of biohazardous waste and disinfection of the inpatient unit. During stool collections, the 
remaining stool in the plastic stool collector will be disinfected with bleach for twenty minutes 
and then flushed into the hopper located in the stool processing area. The disinfected stool 
collector will then be discarded in hazardous waste. 

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8.4.2 
Study Product Shipment 
 
Live Shigella sonnei Product (53G) (lot #1794) 
Lyophilized vials of the challenge material (S. sonnei 53G, lot# 1794) will be shipped directly to 
CCHMC prior to the clinical trial. The following procedure is followed by WRAIR PBF for 
shipment of products, including lyophilized vials of live bacteria, which are stored at -80ºC ± 
10ºC: 
 
Place all frozen packs in a -80ºC ± 10ºC freezer for a minimum of 48 hours before 
shipment to ensure packs are frozen solid. 
 
Place dry ice or one layer of freezer packs in the bottom of the shipping container. 
 
For vials or tubes, place them in a biohazard container and place the biohazard 
container on top of dry ice or one layer freezer packs (centered, if possible). Ship with a 
temperature monitor and tape or otherwise secure the temperature monitor in the 
biohazard container or biohazard bag and activate temperature monitor. Seal the 
shipping container with shipping tape. 
 
Place a stamped copy of Form F-040-XX (and Form F-229-XX if requested by PIs and/or 
CRADA partners) and all paperwork going with the shipment in a plastic bag or envelope 
and attach it to the top of the shipper lid. Place the appropriate handling labels (e.g., 
“Freeze Upon Arrival”), on the outside of the shipping box. The QA ICS (Quality 
Assurance Inventory Control System) must complete the carrier-shipping document with 
the appropriate information, which must include overnight priority shipping status. 
 
Shigella antigens, negative and positive controls 
As detailed in the study MOP, WRAIR will ship specialized Shigella antigens and negative and 
positive controls directly to the LSCS at CCHMC for training and clinical trial use. 
All antigens and control sera will be supplied in 1.0-2.0 mL Nunc® cryovials. The number of 
vials sent and schedule of shipment will be specified in the MOP. 
 
Microbiological materials 
As detailed in the MOP, WRAIR will ship specialized microbiological materials to the LSCS at 
CCHMC for training and clinical trial use. 
 
9. SAFETY REPORTING AND SAFETY MONITORING 
 
Regulatory requirements including the Food and Drug Administration (FDA) regulations, 
International Conference on Harmonisation (ICH) Guidelines for Good Clinical Practice (GCP), 
and European Union (EU) Clinical Trials Directive set forth safety monitoring and reporting 
responsibilities of study sponsors and investigators to ensure the safety and protection of 
human subjects participating in clinical trials. 

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It is the policy of CCHMC that all research involving biohazardous materials will be conducted in 
a safe manner and in compliance with the NIH Guidelines for Research Involving Recombinant 
or Synthetic Nucleic Acid Molecules (NIH Guidelines), with the CDC’s publication Biosafety in 
Microbiological and Biomedical Laboratories, and with all federal, state, local and institutional 
regulations in order to protect employees, patients, visitors and the greater community and 
environment at large. The CCHMC IBC shall review all Recombinant DNA Research under its 
jurisdiction in accordance with these guidelines. CCHMC IBC approval will be received prior to 
final IRB approval and initiation of this trial. 
 
9.1 Responsibilities 
Investigators participating in this clinical trial are responsible for and will: 
 
Evaluate subject safety including assessment of reactogenic symptoms and AEs for 
seriousness, severity, and causality 
 
Immediately (within 24 hours of site awareness) notify the study sponsors of SAEs 
 
Provide detailed written reports, including necessary documentation requested by 
the study sponsors or IRB/independent ethics committee (IEC), promptly following 
immediate initial reports 
 
Inform the IRB/IEC of AEs as required by applicable regulatory requirements 
 
9.2 Definitions 
The investigator is responsible for documentation of AEs according to the detailed guidelines 
set out below. Subjects will be instructed to contact the investigator immediately should she/he 
manifest any signs or symptoms of reactogenicity they perceive as serious during the study 
period as well as immediate reporting (within 24 hours of site awareness) for any new onset 
symptoms irrespective of severity from Day 0 through Day 56. Approximately six months after 
the challenge, the subjects will be contacted by phone to document any intervening medically 
significant new chronic illnesses or serious health events. 
 
9.2.1 
Adverse Event (AE) 
Adverse Event: ICH guideline E6 defines an AE as any untoward medical occurrence in a 
patient or clinical investigation subject administered a pharmaceutical product regardless of its 
causal relationship to the study treatment. An AE can therefore be any unfavorable and 
unintended sign (including an abnormal laboratory finding), symptom, or disease temporally 
associated with the use of medicinal (investigational) product. The occurrence of an AE may 
come to the attention of study personnel during study visits and interviews or by a challenge 
recipient presenting for medical care. 
All AEs, solicited or unsolicited, must be graded for severity and relationship to study product 
(see below). 

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Solicited Adverse Event 
Solicited Adverse Events – reactogenicity events (serious or non-serious) beginning the day of 
administration of study agent until five days after discharge (recorded per memory aid) from the 
inpatient unit and include diarrhea, abdominal pain/abdominal cramps, gas, anorexia, nausea, 
headache, myalgia, malaise, arthralgia, fever, and vomiting. Any unresolved events will be 
followed to resolution or stabilization. 
Unsolicited Adverse Event 
Unsolicited Adverse Events- non-reactogenicity events (serious and/or non-serious) occurring 
from the time of study enrollment to conclusion of study. Any unresolved events will be followed 
to resolution or stabilization. 
FDA defines an AE as any untoward medical occurrence associated with the use of a drug in 
humans, whether or not considered drug related. 
Adverse events characterized as intermittent require documentation of onset and duration of 
each episode. 
 
Table 4 Assessment of Solicited Adverse Events 
Adverse Event 
Severity 
Parameter 
Fever 
1 
100.4°F - 101.1°F (38.0-38.4°C) 
2 
101.2°F - 102.0°F (38.5-38.9°C) 
3 
102.1°F - 104°F (39.0°C - 40°C) 
4 
> 104°F (>40°C) 
Hypertension 
(systolic) 
1 
141-150 mm Hg 
2 
151-160 mm Hg 
3 
>161mm Hg 
4 
ER visit of hospitalization for malignant hypertension 
Hypotension 
(systolic) 
1 
85-89 mm Hg 
2 
80-84 mm Hg 
3 
<80 mm Hg 
4 
ER visit or hospitalization for hypotensive shock 
Hypotension 
(diastolic) (with 
symptoms of 
dehydration) 
1 
10 mm Hg decrease upon standing within 3 minutes of sitting or lying 
supine 
2 
20 mm Hg decrease upon standing within 3 minutes of sitting or lying 
supine 
3 
>30 mm Hg decrease upon standing within 3 minutes of sitting or lying 
supine 

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Table 4 Assessment of Solicited Adverse Events 
Adverse Event 
Severity 
Parameter 
4 
ER visit or hospitalization for hypotensive shock 
Bradycardia 
1 
50-54 bpm* 
2 
45-49 bpm 
3 
<45 bpm 
4 
ER visit or hospitalization for arrhythmia 
Tachycardia 
1 
101-115 bpm 
2 
116-130 bpm 
3 
>130 bpm 
4 
ER visit or hospitalization for arrhythmia 
Nausea 
1 
mild or transient; maintains reasonable intake 
2 
moderate discomfort; intake decreased significantly; some activity limited 
3 
no significant intake and requires medical intervention 
4 
hospitalization required 
Abdominal 
Cramping 
1 
No interference with daily activities 
2 
Some interference with daily activities not requiring medical intervention 
3 
Prevents daily activities and requires medical intervention 
4 
ER visit or hospitalization 
Vomiting 
1 
1 episode within a 24-hour period 
2 
2 episodes within a 24-hour period 
3 
>2 episodes within a 24-hour period and requires medical intervention 
4 
ER visit or hospitalization for hypotensive shock 
Diarrhea** 
1 
2-3 Grade 3-5 stools (loose or watery) or <400 g/Grade 3-5 (loose or 
watery) stools per 24 hours 
2 
4-5 Grade 3-5 stools (loose or watery) or 400-800 g/ (loose or watery) 
Grade 3-5 stools per 24 hours 
3 
6 or more Grade 3-5 stools(loose or watery) or >800 g/Grade 3-5 (loose  
or watery) stools per 24 hours and requires medical intervention 
4 
ER visit or hospitalization for hypotensive shock 
Subjects should be at rest for all vital sign measurements. Oral temperature; no recent hot or cold beverages. 
*Not considered Grade 1 bradycardia in conditioned athletes. 
**The end of a diarrheal episode occurs when a volunteer does not pass any Grade 3-5 stool within 24 hours. 

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9.2.2 
Serious Adverse Event (SAE) 
An adverse event or suspected adverse reaction is considered “serious” if, in the view of either 
the investigator or study sponsors, it results in any of the following outcomes: 
 
Death; 
 
A life-threatening event*; 
 
Inpatient hospitalization or prolongation of existing hospitalization; 
 
A persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions; or 
 
A congenital anomaly/birth defect. 
 
Important medical events that may not result in death, be life threatening, or require 
hospitalizations may be considered serious when, based upon appropriate medical 
judgment, they may jeopardize the patient or subject and may require medical or 
surgical intervention to prevent one of the outcomes listed in this definition. Examples of 
such medical events include allergic bronchospasm requiring intensive treatment in an 
emergency room or at home, blood dyscrasias or convulsions that do not result in 
inpatient hospitalization, or the development of drug dependency or drug abuse. 
* Life-threatening adverse events. An adverse event is considered “life-threatening” if, in the 
view of either the investigator or study sponsors, its occurrence places the patient or 
subject at immediate risk of death. It does not include an adverse event, had it occurred in 
a more severe form, might have caused death. 
All SAEs must be documented and followed until the event either resolves, subsides, stabilizes, 
disappears or is otherwise explained, or the subject is lost to follow-up, but not longer than 6 
months after the last receipt of test article. Any follow-up activities, with additional or changed 
information completed on the SAE form, have to be reported within 7 calendar days of receipt of 
the new information. Clinically significant laboratory abnormalities will be followed up until they 
have returned to normal or until stable. Reports relative to the subsequent course of an AE 
noted for any subject must be submitted to the study sponsors. The outcome of SAEs should be 
assessed in the same manner as all AEs. 
Unexpected 
An unexpected AE is an adverse reaction, the nature or severity of which is not consistent with 
the applicable product information (e.g., investigator’s brochure [IB] for an unapproved 
investigational medicinal product). Unexpected refers to an experience that has not been 
previously observed. This includes events that are more serious than expected or occur more 
frequently than expected. 
Expected 
Any adverse experience, the nature, severity or frequency of which is consistent with the current 
IB; or with the risk information described in the investigational plan or protocol or consent form. 

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Procedures to be followed in the Event of Abnormal Laboratory Test Values or Abnormal 
Clinical Findings: 
Laboratory test abnormalities will be analyzed based on the grading scale provided in the 
protocol. Adverse events and SAEs should be documented and reported appropriately. 
Adverse events will be followed until resolved or considered stable. Lab values (that are Grade 
1 severity) will be followed to resolution or stabilization. Any lab values that are Grade 2 or 
greater severity will be followed at least until they are Grade 1 severity. AEs may be followed 
further at the discretion of the site principal investigator or appropriate sub-investigator. 
Expedited Safety Report 
An expedited safety report is documentation in appropriate form and format summarizing an 
SAE that meets expedited safety reporting criteria, submitted within the required reporting time 
frame of applicable regulatory authorities and/or IRBs/IECs of participating countries. 
 
9.3 Safety Reporting Procedures 
 
9.3.1 
Reporting Interval 
All reactogenicity will be captured and recorded Day 0 through 5 days after discharge from the 
inpatient unit. All non-serious AEs will be collected Day 0 through Day 56. All SAEs and New 
Onset Chronic Medical Conditions will be documented from the first administration of study 
product (Study Day 0) through Study Day 180. 
All SAEs will be followed until resolution even if this extends beyond the study-reporting period. 
Resolution of an AE is defined as the return to pretreatment status or stabilization of the 
condition with the expectation that it will remain chronic. 
At any time after completion of the study, if the investigator becomes aware of an SAE that is 
suspected to be related to study product, the investigator will report the event to the study 
sponsors, see Section 9.3.2. 
 
9.3.2 
Notification of the Study Sponsors of Serious Adverse Events 
 
All SAEs must be reported immediately (within 24 hours of site awareness) by the investigator, 
whether or not regarded as possibly attributable to the test articles or antibiotic. Any unexpected 
fatal or life threatening experience after inoculation with the challenge strain will be reported to 
the FDA, within 7 calendar days. SAE reports will be provided to the study sponsors, Research 
Monitor, SMC and the IRBs; contact information provided below. The investigator must report 
SAEs immediately, within 24 hours of site awareness, by telephone, fax or e-mail (if 
appropriate) as described in the protocol. This initial notification may include minimal, but 
sufficient information to permit identification of the reporter, the subject, the test articles, SAEs, 
and date of onset. The investigator should not wait for additional information to fully document 
the event before submitting notification. An acknowledgement letter from the sponsor will 

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confirm the first notification. The report is then to be followed by submission of a completed SAE 
Report Form provided by the IND-Sponsor as soon as possible, but not more than 3 working 
days past the initial report, detailing relevant aspects of the SAE in question. All investigator 
actions and event outcomes must also be reported. 
 
SAE Report Forms are to be used for documentation of these various aspects regarding the 
event. Hospital records and autopsy reports should be obtained if applicable. 
 
An Independent Research Monitor has been assigned for this study. The independent Research 
Monitor may discuss the protocol with the investigators, interview subjects, and consult with 
others outside the study about the research and shall have the authority to stop the protocol, 
remove subjects from the protocol, and take any necessary steps to protect the safety and well- 
being of subjects until the IRB can assess the Monitor’s Report. The study investigators will 
consult with the Research Monitor on issues related to subject enrollment and continued 
participation as needed. The Research Monitor is required to review all unanticipated problems 
involving risk to subjects, SAEs, and all subject deaths associated with the protocol, and provide 
an unbiased written report of the event. Reports for unanticipated problems involving risks to 
subjects or others must be promptly forwarded to the USAMRMC ORP HRPO, in addition to the 
IRB. At a minimum, the Research Monitor should 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 should indicate concurrence or non-concurrence with the details of the report provided 
by the investigator. Reports for events determined by either the investigator or Research 
Monitor to be related or unrelated to participation and reports of events resulting in death should 
be promptly forwarded to the IRBs. 
 
Sponsor Notification 
For this project, sponsors include funding agencies (PATH and DoD) and the IND-Sponsor 
(PATH). 
 
The investigator must report all SAEs immediately, within 24 hours of site awareness, to site 
sponsors whether or not the event is considered related to the study product, and provide to the 
study sponsors the following information via email (preferred) or telephone: 
 
 
Protocol Investigational New Drug (IND) number, investigational product, investigator 
name and contact number 
 
 
Subject identification number 
 
 
Serious adverse event, onset date, date of investigational product administration, 
severity, relationship, and subject’s current status 
 
AND Email (preferred) or fax the following documents to the Study Sponsors’ Regulatory Affairs 
office: 
 
 
Cover sheet 

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 
Adverse Event Case Report Form 
 
 
Supplemental 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 
 
The investigator will assess all SAEs as being either related or unrelated to the 
administered product. 
 
When submitting SAE reports via email, the subject line of each email notification may read as 
follows: 
 
SAFETY REPORT – IND #  
, Protocol Log #  
, Subject ID#  
, Event term 
 
In order to comply with regulations mandating study sponsors notification of specified SAEs to 
the FDA within 7 calendar days of site awareness, investigators must submit additional 
information as soon as it is available. The study sponsors will report related, unexpected SAEs 
to the FDA as specified in 21 CFR. 
 
The investigator must report these additional immediately reportable events (within 24 hours of 
site awareness) to the study sponsors: 
 
 
Any withdrawal of consent during the study 
 
 
Pregnancy or intent to become pregnant 
 
 
A protocol deviation that jeopardizes the safety of a subject or scientific integrity of the 
study 
 
IRB Notification 
 
Unanticipated problems involving risk to subjects or others, SAEs related to participation in the 
study, and all subject deaths will be promptly reported (within 7 calendar days of site 
awareness) by phone, email, or fax to CCHMC IRB. A complete written report will follow the 
initial notification. 
 
Investigators are required to forward safety information provided by the study sponsors 
to the IRB. All SAEs will be reported to the CCHMC IRB according to the appropriate 
guidelines. 
 
Investigators are required to report adverse events that fit the following criteria within 7 
calendar days of the time the investigator becomes aware of them (see below for contact 
information): 

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Event is UNANTICIPATED. An unanticipated event is any adverse experience where the 
nature, severity or frequency is not identified in the investigator brochure or described in the 
protocol. Events which are already cited in the investigator brochure or protocol are not 
unanticipated and do not have to be reported to the CCHMC IRB 
 
Event is POSSIBLY RELATED to the study design, procedures, or drug/device. If the AE is 
clearly not related to the study drug, device, procedures, or washout process, it would not 
represent a risk to other subjects in the research and, therefore, does not have to be 
immediately reported to the CCHMC IRB, but would be reported according to CCHMC IRB 
guidelines. 
 
CCHMC IRB Contact Information: 
 
Cincinnati Children’s Hospital Medical Center (CCHMC) IRB: 
3333 Burnet Ave. MLC 7040 
Cincinnati, OH 45229 
Phone: 513-636-2754 
Fax: 513-639-1321 
Email: orcra@cchmc.org 
 
Other Notifications 
 
 
Independent Research Monitor 
 
 
SMC 
 
 
FDA (by IND-sponsor) per CBER reporting requirements 
 
9.3.3 
Regulatory Reporting for Studies Conducted Under IND 
Following notification from the investigator, the IND Sponsor will report any suspected adverse 
reaction that is both serious and unexpected. The IND Sponsor will report an adverse event as 
a suspected adverse reaction only if there is evidence to suggest a causal relationship between 
the drug and the adverse event. The IND Sponsor will notify the FDA and all participating 
investigators (i.e., all investigators to whom the study sponsors are providing drug under its 
INDs or under any investigator’s IND) in an IND safety report of potential serious risks from 
clinical trials or any other source, as soon as possible, but in no case later than 15 calendar 
days after the sponsor determines that the information qualifies for reporting as specified in 21 
CFR Part 312.32. The IND Sponsor will also notify FDA of any unexpected fatal or life- 
threatening suspected adverse reaction as soon as possible but in no case later than 7 calendar 
days after the sponsor’s initial receipt of the information. Relevant follow-up information to an 
IND safety report will be submitted as soon as the information is available. Upon request from 
FDA, the IND Sponsor will submit to FDA any additional data or information that the agency 
deems necessary, as soon as possible, but in no case later than 15 calendar days after 
receiving the request. 

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All serious events designated as “not related” to study products will be reported to the FDA at 
least annually in a summary format. 
 
9.3.4 
Reporting of Pregnancy 
Subjects will be screened by pregnancy test before participation in this study and instructed to 
use effective contraception. Sexually active females will have to use birth control (birth control 
pills, injection hormonal contraceptive, implant hormonal contraceptive, hormonal patch, IUD, 
sterilization for effective contraceptive methods, spermicidal products and barrier methods are 
considered acceptable) within two months of challenge and during the entire study. 
Although not AEs, pregnancies are reportable events captured through Study Day 180. 
Pregnancy outcome will be reported (e.g., any premature terminations, elective or therapeutic, 
and any spontaneous abortions or stillbirths, as well as the health status of the mother and child 
including date of delivery and infant’s gender and weight). In general, pregnancies should be 
followed until birth or outcome is known (See Section 9.3.4), although the exact length of follow- 
up will be determined by each individual product profile and protocol and pending the subject’s 
permission. 
 
9.4 Investigator’s Assessment of Adverse Events 
The determination of seriousness, severity, and causality will be made by an on-site investigator 
defined as a study clinician licensed to make medical diagnoses and listed on the FDA 1572 
(site principal investigator or sub-investigator) who is qualified (licensed) to diagnose AE 
information, provide a medical evaluation of AEs, and classify AEs based upon medical 
judgment. This includes but is not limited to physicians, physician assistants, and nurse 
practitioners. 
 
9.4.1 
Assessment of Seriousness 
Event seriousness will be determined according to the protocol definition of an SAE (Section 
9.2.2). 
 
9.4.2 
Assessment of Severity 
Adverse events with objective parameters will be graded as listed in Table 4 Section 9.2.1. 
Events with subjective parameters will be graded as: 
 Mild (Grade 1): No interference with daily activities.
 Moderate (Grade 2): Some interference with daily activities not requiring medical 
intervention.
 Severe (Grade 3): Prevents daily activities and requires medical intervention.
 Life threatening (Grade 4): ER visit or hospitalization.

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A physician will be called to evaluate any subject for intravenous hydration who cannot tolerate 
oral fluids, experiences fluid loss that exceeds their ability to drink replacement fluids, has 
>1000 mL deficit in intake in a 24 hour period, experiences weight loss of more than 5%, has a 
urine specific gravity >1.030 for 12 hours, has syncope or near-syncope, Grade 3 tachycardia, 
or Grade 3 hypotension. 
Changes in the severity of an AE should be documented to allow an assessment of the duration 
of the event at each level of intensity to be performed. Adverse events characterized as 
intermittent require documentation of onset and duration of each episode. 
The severity of AEs not specifically defined above will be graded based on the Guidance for 
Industry: Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in 
Preventive Vaccine Clinical Trials dated September 2007 and the NIH comments on the draft 
guidance (Docket No. 2005D-0155, August 2005). These guidance documents will be utilized to 
grade the severity of clinically significant laboratory abnormalities with final determination made 
by the Principal Investigator on a per subject basis. 
 
9.4.3 
Assessment of Association 
The clinician’s assessment of an AE's relationship to the study product is part of the 
documentation process, but it is not a factor in determining what is or is not reported in the 
study. If there is any doubt as to whether a clinical observation is an AE, the event should be 
reported. All AEs will be assessed and documented by clinicians for relationship to study 
product. The association assessment categories that will be used for this study are: 
 
Related – There is a reasonable possibility that the study product caused the adverse event. 
Reasonable possibility means that there is evidence to suggest a causal relationship 
between the study product and the adverse event.
 
Not Related – There is not a reasonable possibility that the administration of the study 
product caused the event.
The investigator must provide an assessment of association or relationship of AEs to the study 
product based on: 
 Temporal relationship of the event to the administration of study product
 Whether an alternative etiology has been identified
 Biological plausibility
 Existing therapy and/or concomitant medications
 
9.4.4 
Assessment of Reactogenicity 
Reactogenicity events are AEs that are common and known to occur for Shigella and will be 
collected and graded using a grading scale based on functional assessment or magnitude of 
reaction. The following reactogenicity events will be solicited beginning the day of administration 

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of study agent until five days after discharge from the inpatient unit: diarrhea, abdominal 
pain/abdominal cramps, gas, anorexia, nausea, headache, myalgia, malaise, arthralgia, fever, 
and vomiting. The grading scales/criteria for these solicited symptoms are listed in Table 4 in 
Section 9.2.1. 
 
9.5 Safety Monitoring 
 
9.5.1 
Safety Monitoring Committee 
A Safety Monitoring Committee (SMC) will be established and will be comprised of one 
Independent Safety Monitor (ISM) and a committee of outside members with expertise in 
infectious diarrheal diseases and/or clinical trials. The SMC will advise the study sponsors 
regarding trial safety. The SMC will operate under the rules of a charter that will be written at the 
organizational meeting of the SMC. At a minimum, the SMC should comment on the safety 
event and the outcomes of the event or problem. In the case of an SAE or death, the SMC will 
need to comment on the relationship of the event to participation in the study. The SMC also 
should indicate whether it concurs with the details of the report provided by the study 
investigator. Reports for events determined by either the investigator, SMC or Research Monitor 
to be possibly or definitely related to participation and reports of events resulting in death should 
be promptly forwarded to the IRB. The SMC and Research Monitor will review available safety 
data for each cohort prior to making a recommendation on whether or not to proceed to the next 
cohort. The SMC would recommend one of the following alternatives: advancing to the next 
dose level, halting, additional subjects at the current dose, or decreasing the current dose. Ad 
hoc meetings may be required if a halting rule occurs. 
 
9.5.2 
Research Monitor 
The Research Monitor will function as an independent safety advocate for subjects per DoD 
Instruction 3216.02. An independent research monitor is required to review all unanticipated 
problems involving risk to subjects or others, SAEs, and all subject deaths associated with the 
protocol and provide an unbiased written report of the event. At a minimum, the research 
monitor should comment on the outcomes of the event or problem and, in the case of an SAE or 
death, comment on the relationship to participation in the study. The research monitor should 
also indicate whether he or she concurs with the details of the report provided by the study 
investigator. Reports for events determined by either the investigator or research monitor to be 
possibly or definitely related to participation and reports of events resulting in death should be 
promptly forwarded to the IRB. The Research Monitor will review available safety data for each 
cohort prior to making a recommendation on whether or not to proceed to the next cohort. The 
Research Monitor would recommend one of the following alternatives: advancing to the next 
dose level, halting, additional subjects at the current dose, or decreasing the current dose. Ad 
hoc meetings may be required if a halting rule occurs. 
An interim safety report will compile findings from the preceding group with the PI’s 
interpretation and plan to go forward. The SMC will review aggregate safety data for increased 

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rate of occurrence of serious suspected adverse reactions. An interim safety report for each 
dose level will be prepared and reviewed by the SMC and the Research Monitor. The SMC will 
convene a meeting or a conference call and provide comments and recommendations before 
the next higher dose level can proceed. The SMC report and decision will be reviewed and 
approved by the study sponsors. The SMC report and decision will then be sent to the principal 
investigator for submission to the IRB. The decision to advance to the next highest dose level 
will be based solely on the safety profile at the tested dose level. 
Criteria for advancing to the next level will include: 
 A minimum of 4 weeks has elapsed from the time of the preceding challenge to ensure 
adequate time for safety monitoring.
Shigellosis is defined as shedding of S. sonnei in the stool accompanied by moderate-severe 
diarrhea (and/or dysentery) along with moderate fever, or one or more severe intestinal 
symptoms. 
The SMC reviewing the data to determine the approximate percentage of subjects who met 
definition of shigellosis and the primary clinical endpoints as shown below: 
o Diarrhea- 
 
Moderate- 4-5 loose or watery stools (Grades 3-5) or 400-800 g/Grade 3-5 
stools per 24 hours 
 
Severe- 6 or more loose or watery stools (Grades 3-5) or >800 g/Grades 3-5 
per 24 hours or requires medical intervention 
o Dysentery- a Grade 3, 4, or 5 stool with gross blood on at least 2 occasions and 
reportable constitutional symptoms. 
o Moderate Fever- Oral temperature of ≥38.5°C 
o Symptoms - one or more severe intestinal symptoms 
 If the attack rate (AR) (the percentage of subjects who met clinical endpoints) is less than 
60%, the next cohort will receive a higher inoculum, based on deliberations between the 
investigative team and the SMC.
 ARs at or above 60% may result in verification of the attack rate at the same inoculum dose 
or a decrease in the inoculum to as low as 100 cfu based on deliberations between the 
investigative team and the SMC.
 
9.5.3 
Halting Criteria/Rules: 
The study will be halted for SMC review/recommendation if: 
 
Any individual experiences an SAE related to the investigational products 
 
The same unanticipated severe Grade 3 or greater AEs, such as in safety laboratory 
data, occurs in two or more subjects. (This does not include two or more subjects 

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experiencing severe (Grade 3) AEs such as diarrhea, abdominal cramps, fever, or other 
symptoms included in the clinical definition of shigellosis) determined to be related to the 
investigational products. 
The study sponsors retain the authority to suspend additional enrollment and study 
interventions/administration of study product for the entire study, as applicable. 
 
10. CLINICAL MANAGEMENT OF EVENTS 
 
10.1 Adverse Event Management 
 
Procedures to be followed in the Event of Abnormal Laboratory Test Values or Abnormal 
Clinical Findings: 
Laboratory parameters that are relevant to safety, study outcome measures, and/or clinical 
outcome will be collected. Abnormal laboratory values based on hematology and clinical 
chemistry (white blood cell count, hemoglobin, neutrophil and lymphocyte count, platelets, 
SGOT/AST, SGPT/ALT, total bilirubin, BUN [BUN will be obtained only if creatinine is above 
normal range], creatinine, sodium, and potassium) after challenge will be considered an AE in 
accordance with the table of laboratory toxicity grading scale (Appendix B) and should be 
recorded on the appropriate page of the Case Report Form (CRF). 
Adverse events will be followed until resolved or considered stable. Any safety lab values that 
are Grade 1 severity will be followed to resolution or stabilization. Any safety lab values that are 
Grade 2 or greater severity will be followed until they are less than Grade 2 severity or followed 
further at the discretion of the site principal investigator or appropriate sub-investigator. 
Additional clinical laboratory evaluations may be performed at other times as required to follow 
up a serious or SAE or as deemed necessary by the investigator. Non-clinically significant 
abnormal laboratory values that do not meet adverse event definition(s) will not be recorded as 
AEs. 
 
Type and Duration of Follow-up of Subjects after Adverse Events 
Any research related injuries and AEs would be followed periodically until the events are 
resolved or become stable. Follow-up process can be both by telephone and/or asking the 
subjects to visit the study site, which depends on their conveniences and/or the severity of the 
particular event. 
Investigators should follow-up AEs until the final study visit. This may include repeat safety 
laboratory analysis. For follow-up of SAEs, see Section 9.2.2. 
 
10.1.1 Non-Administration of Study Product in an Individual Subject 
An individual may be removed from further study participation or not administered the study 
product if: 

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 
The subject experiences an SAE unrelated to the investigational product (event will be 
discussed with the SMC and Research Monitor so as to determine if the event precludes 
further participation and dosing). 
 
The investigator deems that stopping the investigational product administration is in the 
best interest of the subject. 
 
The subject does not wish to continue with the study. 
 
The subject is lost to follow-up. 
Withdrawal from the trial due to these last two points, if not AE related, does not need to be 
reported other than as part of routine annual reporting to the FDA and IRBs. 
 
10.1.2 Pregnancy 
Female subjects who are found to be pregnant within 180 days after receiving the challenge 
dose will be followed according to the protocol visit schedule and will also be followed through 
delivery or until pregnancy outcome is known. 
 
11. CLINICAL MONITORING 
 
11.1 Site Monitoring Plan 
Site monitoring is conducted to ensure that the human subject protection, study procedures, 
laboratory, study intervention administration, and data collection processes are of high quality 
and meet the study sponsors’, ICH E6 and, when appropriate, regulatory guidelines. 
Site monitoring for the study will be contracted by the study sponsors. Monitoring visits by a 
designated clinical monitor will be scheduled to take place at the initiation of the study, during 
the study at appropriate intervals and after the last subject has completed the study and all 
laboratory results are available. During the site visits, the clinical monitor will review informed 
consent statements, study-specific MOP, and required regulatory documents. The monitor will 
verify the completeness and accuracy of the CRFs by comparing them with the source 
documents. During some of the monitoring visits the actual implementation of study procedures 
will be observed. A separate monitoring plan document will be developed to describe who will 
conduct the monitoring, at what frequency monitoring will be done, and what level of detail the 
monitoring will be conducted. The monitoring plan will include the number of subject charts to be 
reviewed, which/what proportion of data fields and what will be monitored, who will be 
responsible for conducting the monitoring visits, and who will be responsible for ensuring that 
monitoring findings are addressed. 
 
The Investigational Pharmacist (or designated individual) will be responsible for accountability of 
the challenge product and will document receipt, use, return, or destruction of any unused 
challenge product. Accountability documentation will be reviewed by a monitor during site 
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12. STATISTICAL CONSIDERATIONS 
 
12.1 
Study Hypotheses 
 
Hypotheses for primary objectives 
1. A human challenge model of Shigella sonnei infection using a lyophilized formulation 
of the challenge strain can be established. 
2. We hypothesize an increasing rate of shigellosis with increasing doses of S. sonnei 
53G and that at least 1 dose level will induce shigellosis in ≥60% of subjects. 
 
Hypotheses for secondary objectives 
1. S. sonnei 53G will induce a measurable serum or fecal immune response in at least 
80% of subjects. 
2. Fecal shedding of S. sonnei 53G will be detectable by PCR or culture on one or more 
occasions in at least 80% of subjects. 
 
12.2 
Sample Size Considerations 
 
The aim to select a S. sonnei strain 53G dose with a ≥ 60% attack rate can be accomplished in 
10 subjects with a two-sided exact 95% confidence interval of 26 – 88%. Increasing the number 
of subjects by 20 for the selected inoculum/fasting regimen (total of 25 subjects at a given 
inoculum dose) will provide greater confidence (39 - 79%) that the target 60% attack rate will be 
achieved in future applications of the challenge model. 
 
Follow-up studies evaluating tolerable immunogenic doses will be required with larger numbers 
of subjects in order to better define the safety profile. All subjects who receive S. sonnei 53G will 
be included in the safety analyses. Safety data, including AEs, physical exam assessments, and 
laboratory tests will be listed by subject. Adverse events will be coded with the number and 
proportion of subjects reporting a given event summarized by group. Immune response data will 
be summarized using similar methodology. 
 
12.2.1 Planned Interim Analyses 
There are no planned formal statistical interim analyses. The SMC, along with the Research 
Monitor will review the data prior to each dose escalation; however, this may not involve any 
hypothesis testing and will not be considered in estimating the precision of any estimates made 
at the conclusion of the study. 

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12.2.2 Analysis Plan 
This is a Phase 1 study and as such the analyses will focus on using descriptive techniques to 
estimate rates, means and their respective confidence intervals. No imputation is planned for 
missing data. 
Rates of local and systemic solicited and unsolicited AEs will be tabulated by level of severity. All 
rates will be determined with two-sided exact 95% confidence intervals. 
During each day of the inpatient period, subjects will be monitored for loose stools (not meeting the 
diarrhea definition), diarrhea, dysentery, nausea, vomiting, abdominal cramps, fever, headache, 
abdominal tenderness, abdominal distention or otherwise abnormal abdominal exam. 
Descriptive statistics and graphical summaries will be presented. 
The planned statistical evaluation is based on the proportion of subjects meeting prospectively 
defined clinical, microbiological and immunological endpoints. The attack rate will be calculated for 
all study groups, using the standard definition of: (# with endpoint / # receiving inoculum) x 100%. 
Summary tables will also be created to detail quantitative and temporal features of the illness such 
as diarrhea stool frequency and volume, maximum temperature observed, and time to illness and 
infection. Continuous variables will be analyzed using nonparametric statistics. 
Safety: The frequency of symptoms by severity will be calculated, as will frequency  and 
duration of fecal shedding as determined by culture and PCR (exploratory aim). Changes in 
clinical laboratory values will be considered significant if they are beyond the accepted normal 
values. Tables will be prepared to list each commonly observed adverse event, the number of 
subjects who experienced an event at least once, and the rate of subjects with adverse event(s). 
Adverse events will be divided into defined severity grades (mild, moderate, severe and life- 
threatening) based on the maximum observed severity for each subject. 
Immunology: Basic summary statistics as described above will be computed for all  
immunology endpoints at time points where such data is available. 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 two-sided exact 95% confidence intervals. Geometric mean titers will 
also be determined and presented with their two-sided exact 95% confidence intervals. All 
statistical tests will be interpreted in a two-tailed fashion using an alpha = 0.05. 
 
13. 
DATA HANDLING/RECORD KEEPING/SOURCE DOCUMENTS 
 
The EMMES Corporation will serve as the Statistical and Data Coordinating Center for this 
study, and will be responsible for data management, quality review, analysis, and reporting of 
the study data. 
The site principal investigator is responsible to ensure the accuracy, completeness, legibility, 
and timeliness of the data reported. 

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Data collection forms will be derived from the eCRFs to record and maintain data for each 
subject enrolled in the study. All data collection forms should be completed in a neat, legible 
manner to ensure accurate interpretation of data. Black or blue ink is required to ensure clarity 
of reproduced copies. When making a change or correction, cross out the original entry with a 
single line and initial and date the change. Do not erase, overwrite, or use correction fluid or 
tape on the original. 
Data reported in the eCRF derived from data collection forms should be consistent with the data 
collection forms or the discrepancies should be explained. 
EMMES will provide guidance to investigators on making corrections to the data collection 
forms and eCRFs. 
The site will maintain appropriate medical and research records for this trial, in compliance with 
ICH E6 (R1), Section 4.9, and regulatory and institutional requirements for the protection of 
confidentiality of subjects. Authorized representatives of the study sponsors, their designees, 
and appropriate regulatory agencies will be permitted to examine (and when required by 
applicable law, to copy) clinical records which include, but are not limited to, hospital records, 
clinical and office charts, laboratory notes, memoranda, evaluation checklists, pharmacy 
dispensing records, recorded data from automated instruments, copies or transcriptions certified 
after verification as being accurate and complete, microfiches, photographic negatives, 
microfilm or magnetic media, x-rays, and subject files and records kept at the pharmacy, at the 
laboratories, and medico-technical departments involved in the clinical trial for the purposes of 
quality assurance reviews, audits, and evaluation of the study safety and progress. 
 
13.1 
Data Capture Methods 
Clinical data (including AEs, concomitant medications, and reactogenicity data), clinical 
assessments, (including reported symptoms by the subject, assessment of stool, vital signs, and 
physical examinations), and clinical laboratory data (including safety labs) will be entered into a 
computerized data entry system. The data system includes password protection and internal 
quality checks to identify data that appear inconsistent, incomplete, or inaccurate. Clinical data 
will be entered directly from the source documents. 
 
13.1.1 
Types of Data 
 
Data for this study will include safety, laboratory (immunologic, microbiologic, and PCR), and 
outcome measures (e.g., reactogenicity, immunogenicity, microbiology). Interim reports will 
include the rate of accrual, including demographics (e.g. age, race, and ethnicity), as well as 
reports of all SAEs. Data will be locked when all data queries have been completed. Final 
analysis will await completion of the study by all subjects and the locking of the database. 

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13.2 
Study Records Retention 
Study documents will be retained for a minimum of 2 years after the last approval of a marketing 
application and until there are no pending or contemplated marketing applications, or when at 
least 2 years have elapsed since the formal discontinuation of clinical development of an 
investigational product. These documents will be retained for a longer period, however, if 
required by local regulations. No record will be destroyed without the written consent of the 
study sponsors. 
CCHMC will maintain appropriate medical and research records for this trial, in compliance with 
ICH E6, Section 4.9, regulatory and institutional requirements for the protection of confidentiality 
of subjects. CCHMC will permit authorized representatives of the study sponsors and regulatory 
agencies to examine (and when required by applicable law, copy) clinical records for the 
purposes of clinical site monitoring, quality assurance reviews, audits, and evaluation of the 
study safety and progress. 
It is the policy of the US Army Medical Research and Materiel Command (USAMRMC) that data 
sheets are to be completed for all subjects participating in research (Form 60-R, Volunteer 
Registry Data Sheet). The data sheets will be entered into this Command’s Volunteer Registry 
Database. The information to be entered into this confidential data base includes the subject’s 
name, address, and Social Security Number; study title; and dates of participation. The intent of 
this data base is twofold: first, to readily answer questions concerning an individual’s 
participation in research sponsored by USAMRMC; and second, to ensure that USAMRMC can 
exercise its obligation to ensure research subjects are adequately warned (duty to warn) of risks 
and to provide new information as it becomes available. The information will be stored at 
USAMRMC for a minimum of 75 years. The Volunteer Registry Database is a separate entity 
and is not linked to the study database. The Form 60-R will be mailed to USAMRMC for 
storage. 
 
13.3 
Source Documents 
The site will maintain appropriate medical and research records for this trial, in compliance with 
ICH E6 (R1) GCP, Section 4.9, and regulatory and institutional requirements for the protection 
of confidentiality of study participants. The site will permit authorized representatives of the 
study sponsors and regulatory agencies to examine clinical records for the purpose of quality 
assurance reviews, audits, and evaluation of the study safety and progress. 
Study documentation will be coded and stored in locked cabinets in work areas that are 
accessible by badge only. Electronic study data will be coded and stored on computers that are 
accessible only by password and will be maintained in work areas that are accessible by badge 
only. 
Source data are all information, original records of clinical findings, observations, or other 
activities in a clinical trial necessary for the reconstruction and evaluation of the trial. Source 
documentation include, but are not limited to, hospital records, clinical and office charts, 
laboratory notes, memoranda, documentation of phone collection of study participants’ memory 

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aid information, pharmacy dispensing records, recorded data from automated instruments, 
copies or transcriptions certified after verification as being accurate and complete, microfiches, 
X-rays and study subject files and records kept at the pharmacy. 
 
13.4 
Protocol Deviations 
A protocol deviation is any noncompliance with the clinical trial protocol, GCP, or Manual of 
Procedures requirements. The noncompliance may be either on the part of the subject, the 
investigator, or the study site staff. As a result of deviations, corrective actions are to be 
developed by the site and implemented promptly. Deviations from the protocol will be reported 
promptly to any Institutional Review Boards responsible for the study. 
These practices are consistent with GCP: 
21 
CFR 50 and 56 and, as applicable, ICH6, 
4.5 Compliance with Protocol, Sections 4.5.1, 4.5.2, and 4.5.3, 
5.1 Quality Assurance and Quality Control, Section 5.1.1, and 5.2.0 
Noncompliance, Sections 5.2.1, and 5.2.2. 
It is the responsibility of the site to use continuous vigilance to identify and report deviations 
within 5 working days of identification of the protocol deviation, or within 5 working days of the 
scheduled protocol-required activity. All deviations must be promptly reported to the study 
sponsors. 
All deviations from the protocol must be addressed in study subject source documents. A 
completed copy of Deviation Form must be maintained in the Regulatory File as well as in the 
subject’s source documents. Protocol deviations must be sent to the local IRB/IEC per their 
guidelines. The site PI/study personnel is responsible for knowing and adhering to their IRB 
requirements. 
 
14. QUALITY CONTROL AND QUALITY ASSURANCE 
 
The site is responsible for conducting routine quality assurance (QA) and quality control (QC) 
activities to internally monitor study progress and protocol compliance. The Principal 
Investigator will provide direct access to all trial-related sites, source data/documents, and 
reports for the purpose of monitoring and auditing by the study sponsors, and inspection by 
local and regulatory authorities. The Principal Investigator will ensure all study personnel are 
appropriately trained and applicable documentations are maintained on site. 
Clinical monitors will verify that the clinical trial is conducted and data are generated, 
documented (recorded), and reported in compliance with the protocol, GCP, and the applicable 
regulatory requirements. Clinical monitoring reports will be submitted to the study sponsors. 

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15. ETHICS/PROTECTION OF HUMAN SUBJECTS 
 
15.1 
Ethical Standard 
The investigator will conduct the study in full conformity with the principles set forth in The 
Belmont Report: Ethical Principles and Guidelines for the Protection of Human Subjects of 
Research of the US National Commission for the Protection of Human Subjects of Biomedical 
and Behavioral Research (April 18, 1979) and codified in 45 CFR Part 46 and 21 CFR 50 and 
56 and the ICH E6; 62 Federal Regulations 25691 (1997). 
 
15.2 
Institutional Review Board 
CCHMC will provide for the review and approval of this protocol and the associated ICFs by 
their IRB located at CCHMC, 3333 Burnet Avenue, MLC 5020, Cincinnati, Ohio, 45229-3039, 
under the institution’s US Federal wide Assurance. Any amendments to the protocol or consent 
materials must also be approved before they are placed into use. Only those IRB members who 
are independent of the investigators and the study sponsors should provide an opinion on 
study-related matters. No deviations from or changes to the protocol will be initiated without 
prior approval of an appropriate amendment. 
 
16. INFORMED CONSENT PROCESS 
 
The informed consent process will be conducted on the first screening day in a private room at 
CCHMC. The consent will explain that subjects may withdraw consent at any time throughout 
the course of the trial. Extensive explanation and discussion of risks and possible benefits of 
this investigation will be provided to the subjects in understandable language. Study 
investigators or appropriately qualified study personnel who have been delegated authority by 
the study investigator will explain the purpose and the details of the protocol to the participant. 
An illiterate subject will not be enrolled for subject recruitment. Participants are required to read 
the consent form, fully discuss their concerns and ask questions until their complete satisfaction. 
They will be allowed to take their time before making a decision or take the form home for 
consideration and consultation with their family. Participants and clinical staff must sign and 
date the consent form. The original of the signed consent form will be filed in the participant’s 
source document, and a copy will be provided to the participant. 
The consent permission forms will describe in detail the study interventions/products/procedures 
and risks/benefits associated with participation in the study. The rights and welfare of the 
subjects will be protected by emphasizing that their access to and the quality of medical care 
will not be adversely affected if they decline to participate in this study. 
To ensure comprehension of the study, on the day of admission to the inpatient unit, a written 
test will be administered which the participants will have 2 chances to pass successfully. If 
subjects are unable to score 70% or greater on the test, they will be excluded from the study. 

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16.1 
Subject Confidentiality 
Each subject will be assigned a unique study identifier. All data collection sheets will identify the 
subject by a unique identifier, and the date. Names will not be used on any samples or in any 
publication of this study. All efforts will be made to protect the privacy of subjects. 
Subject confidentiality is strictly held in trust by the participating investigators, their staff, and the 
study sponsors and their agents. This confidentiality includes documentation, investigation data, 
subject’s clinical information, and all other information generated during participation in the 
study. 
This information and data will not be used by the site principal investigator or other study 
personnel for any purpose other than conducting the study. These restrictions do not apply to: 
(1) information which becomes publicly available through no fault of the site principal 
investigator or other study personnel; (2) information which is necessary to disclose in 
confidence to an IRB solely for the evaluation of the study; (3) information which it is necessary 
to disclose in order to provide appropriate medical care to a study subject; or (4) study results 
which may be published. 
The study monitor or other authorized representatives of the study sponsors or governmental 
regulatory agencies may inspect all documents and records required to be maintained by the 
investigator, including but not limited to, medical records (office, clinic, or hospital) and 
pharmacy records for the subjects in this study. The clinical study site will permit access to such 
records. 
No information concerning the study or the data generated from the study will be released to 
any unauthorized third party without prior written approval of the study sponsors and the 
subject. 
 
16.2 
Study Discontinuation 
Each subject has the right to withdraw from the trial at any time for any reason without affecting 
their right to treatment by the investigator. The investigator also has the right to withdraw the 
subjects in the event of intercurrent illnesses, AEs, a subject’s failure to comply with study 
procedures, or if it is determined to be in the subject’s best interest. If a subject withdraws for 
safety reasons, an AE or SAE will be reported as described in Sections 6.1. 
If a subject withdraws for other reasons, the withdrawal will be reported as part of the annual 
review process. If a subject chooses to withdraw from the study after challenge but before the 
time of planned administration of antibiotics, they will be asked to inform either the PI for the trial 
or an Associate investigator of the study and antibiotic therapy will be initiated. They will be 
encouraged to remain on the Inpatient Unit until a three-day course of treatment can be 
completed. If they choose not to; the study staff will observe the first dose of antibiotic and 
instruct the subjects how to complete the antibiotic therapy. They will also be encouraged to 
complete all remaining outpatient visits for the trial as described in Section 6.1 and Appendix A. 

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16.3 
Future Use of Stored Specimens 
Subjects will be asked to provide consent to allow the investigators to use their stored 
specimens in the future. Specimens will be used for research purposes related to the 
development of improved vaccines for shigellosis and other enteric pathogens at CCHMC or 
other external collaborative institutions. If the investigators have access to identifiers, then prior 
to future use of the samples, appropriate IRB approval will be obtained. Samples may be shared 
in a de-identified manner with non-study investigators and the level of IRB review will be 
discussed with the appropriate IRB prior to initiation of the study. A subject’s refusal to grant 
consent for such use of their stored specimens will not affect his/her eligibility to participate in 
the current protocol. Specimens will be stored in the de-identified state in which they were 
originally labeled after collection and stored in a secure freezer at CCHMC for 20 years. If 
samples still remain at the end of the 20 years of storage, they will be destroyed. Only study 
investigators or designated persons by the principle investigators will have access to the stored 
specimens. 
16.4 
Research Related Injuries 
All study-related medical care will be provided to subjects without cost. Should a subject be 
injured as a direct result of participating in this research project, s/he will be provided medical 
care by the staff of a military-affiliated medical center at no cost to the subjects, for that injury. 
The subjects will not receive any injury compensation, only medical care. The subjects will not 
be compensated for care if s/he chooses to seek care from his/her own physician. 
If a subject is injured because of participation in this research and is a DoD healthcare 
beneficiary (e.g., active duty in the military, military spouse or dependent), the subject is entitled 
to medical care for that injury within the DoD healthcare system, as long as the subject remains 
a DoD healthcare beneficiary. This care includes, but is not limited to, free medical care at DoD 
hospitals or clinics. 
During the challenge phase, subjects who require medical treatment beyond what can be 
provided safely on the inpatient unit at CCHMC will be transferred to a local full-service hospital 
for care. In the event this occurs, the study sponsors agrees to reimburse the hospital for all 
reasonable expenses incurred by that hospital in providing medical treatment and/or 
hospitalization reasonably necessary to address the subject’s medical needs. The Cincinnati 
Children’s Hospital has no plan to provide compensation to subjects if they experience injury or 
other bad effects which are not the fault of the study doctors. Subjects will only be treated for 
injuries that are directly caused by the research study. In the event this occurs, the study 
sponsors agree to reimburse the Hospital for all reasonable expenses incurred by the Hospital 
in providing medical treatment and/or hospitalization reasonably necessary to address any 
injury to a Subject that, in the reasonable judgment of Hospital and study sponsors, occurs 
directly as a result of the administration of the IMPs or performance of study procedures in 
accordance with the Protocol, but only to the extent such expenses are not: 
 
the result of a foreseeable side effect as indicated in the Protocol 

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 
reimbursed by (or submitted for reimbursement to) the Subject’s insurance or any 
governmental program or other third-party payer providing medical or hospital 
coverage; provided, however, that this provision shall not obligate Hospital to submit 
such costs to the prospective Subject’s insurance or any governmental program or 
other third-party payer coverage 
 
attributable to a failure of Hospital, or any of the Investigator Personnel, including PI, 
to adhere to the terms of the Protocol, study Sponsors’ written instructions or 
Applicable Law 
 
attributable to the negligence, recklessness or willful misconduct or omission of 
Hospital or any of its Investigator Personnel, including PI 
 
attributable to a pre-existing abnormal medical condition or underlying disease of the 
Subject or treatment that would have been provided to the Subject in the ordinary 
course notwithstanding participation in the study, or 
 
attributable to the failure of the Subject to follow the reasonable instructions of 
Investigator Personnel or Subject’s physician. 
Transportation to and from military hospitals or clinics will not be provided. No reimbursement is 
available if the subject incurs medical expenses to treat research-related injuries from outside or 
private providers. No compensation is available for research-related injuries. The subject is not 
waiving any legal rights. The subject should contact the PI if the subject believes he or she has 
sustained a research-related injury. The subject should contact the PI for any questions. 
Requests for other benefits, such as compensation for lost time from work, are processed 
independently of this protocol. Military members retain the right to pursue military disability 
benefits, and Federal civilian employees retain the right to pursue relief through established 
workers compensation processes, but neither military disability benefits nor workers 
compensation benefits are guaranteed. The right of other parties to seek redress against the 
United States Government is limited to that set forth by existing agency regulations and the 
Federal Tort Claims Act. The subject should understand that this does not constitute a waiver or 
release of legal rights. This issue is addressed in the ICF and will be discussed with the subject 
by the investigator or designee before the subject signs the ICF to participate in the study. 
 
17. PUBLICATION POLICY 
 
Following completion of the study, the investigator is expected to publish the results of this 
research in a scientific journal. The International Committee of Medical Journal Editors (ICMJE) 
member journals have adopted a trials-registration policy as a condition for publication. This 
policy requires that all clinical trials be registered in a public trials registry such as 
ClinicalTrials.gov [23], which is sponsored by the National Library of Medicine. Other biomedical 
journals are considering adopting similar policies. It is the responsibility of the sponsor to 
register this trial in an acceptable registry. Any clinical trial starting enrollment after 01 July 2005 

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must be registered on or before patient enrollment. For trials that began enrollment prior to this 
date, the ICMJE member journals will require registration by 13 September 2005, before 
considering the results of the trial for publication. 
The ICMJE defines a clinical trial as any research project that prospectively assigns human 
subjects to intervention or comparison groups to study the cause-and-effect relationship 
between a medical intervention and a health outcome. Studies designed for other purposes, 
such as to study pharmacokinetics or major toxicity (e.g., Phase 1 trials), would be exempt from 
this policy. 

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18. LITERATURE REFERENCES 
 
1. 
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. 
2. 
Ashkenazi, S., Shigella infections in children: new insights. Semin Pediatr Infect Dis, 
2004. 15(4): p. 246-52. 
3. 
Lamberti, L.M., et al., Estimating diarrheal illness and deaths attributable to Shigellae 
and enterotoxigenic Escherichia coli among older children, adolescents, and adults in 
South Asia and Africa. PLoS Negl Trop Dis, 2014. 8(2): p. e2705. 
4. 
DuPont, H.L., et al., Inoculum size in shigellosis and implications for expected mode of 
transmission. J Infect Dis, 1989. 159(6): p. 1126-8. 
5. 
Shakoor, S., A.K. Zaidi, and R. Hasan, Tropical bacterial gastrointestinal infections. 
Infect Dis Clin North Am, 2012. 26(2): p. 437-53. 
6. 
Ericsson, C.D., Travelers' diarrhea. Epidemiology, prevention, and self-treatment. 
Infect.Dis.Clin.North Am., 1998. 12: p. 285-303. 
7. 
Kosek, M., P.P. Yori, and M.P. Olortegui, Shigellosis update: advancing antibiotic 
resistance, investment empowered vaccine development, and green bananas. Curr Opin 
Infect Dis, 2010. 23(5): p. 475-80. 
8. 
Niyogi, S.K., Increasing antimicrobial resistance--an emerging problem in the treatment 
of shigellosis. Clin Microbiol Infect, 2007. 13(12): p. 1141-3. 
9. 
Shiferaw, B., et al., Antimicrobial susceptibility patterns of Shigella isolates in Foodborne 
Diseases Active Surveillance Network (FoodNet) sites, 2000-2010. Clin Infect Dis, 2012. 
54 Suppl 5: p. S458-63. 
10. 
Islam, D., et al., Evaluation of an intragastric challenge model for Shigella dysenteriae 1 
in rhesus monkeys (Macaca mulatta) for the pre-clinical assessment of Shigella vaccine 
formulations. APMIS, 2014. 122(6): p. 463-75. 
11. 
Gregory, M., et al., Development of an Aotus nancymaae model for Shigella Vaccine 
immunogenicity and efficacy studies. Infect Immun, 2014. 82(5): p. 2027-36. 
12. 
Black, R.E., et al., Prevention of shigellosis by a Salmonella typhi-Shigella sonnei 
bivalent vaccine. J Infect.Dis., 1987. 155: p. 1260-1265. 
13. 
Herrington, D.A., et al., Studies in volunteers to evaluate candidate Shigella vaccines: 
further experience with a bivalent Salmonella typhi-Shigella sonnei vaccine and 
protection conferred by previous Shigella sonnei disease. Vaccine, 1990. 8(4): p. 353-7. 
14. 
Mackowiak, P.A., S.S. Wasserman, and M.M. Levine, An analysis of the quantitative 
relationship between oral temperature and severity of illness in experimental shigellosis 
[see comments]. J Infect.Dis., 1992. 166: p. 1181-1184. 
15. 
Munoz, C., et al., Characteristics of Shigella sonnei infection of volunteers: signs, 
symptoms, immune responses, changes in selected cytokines and acute-phase 
substances. Am J Trop Med Hyg, 1995. 53(1): p. 47-54. 
16. 
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. 
17. 
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. 

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18. 
Taylor, D.N., et al., Rifaximin, a nonabsorbed oral antibiotic, prevents shigellosis after 
experimental challenge. Clin Infect Dis, 2006. 42(9): p. 1283-8. 
19. 
Bodhidatta, L., et al., Establishment of a Shigella sonnei human challenge model in 
Thailand. Vaccine, 2012. 30(49): p. 7040-5. 
20. 
Porter, C.K., et al., The Shigella human challenge model. Epidemiol Infect, 2013. 141(2): 
p. 223-32. 
21. 
Gaston, J.S., Shigella induced reactive arthritis. Ann Rheum Dis, 2005. 64(4): p. 517-8. 
22. 
Gaston, J.S., How much HLA-B27 expression is needed for spondyloarthritis? J 
Rheumatol, 2008. 35(5): p. 738-40. 
23. 
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. 
24. 
Neal, K.R., J. Hebden, and R. Spiller, Prevalence of gastrointestinal symptoms six 
months after bacterial gastroenteritis and risk factors for development of the irritable 
bowel syndrome: postal survey of patients. BMJ, 1997. 314(7083): p. 779-82. 
25. 
Simon, J.K., et al., Antigen-specific IgA B memory cell responses to Shigella antigens 
elicited in volunteers immunized with live attenuated Shigella flexneri 2a oral vaccine 
candidates. Clin Immunol, 2011. 139(2): p. 185-92. 
26. 
Wahid, R., et al., Shigella antigen-specific B memory cells are associated with 
decreased disease severity in subjects challenged with wild-type Shigella flexneri 2a. 
Clin Immunol, 2013. 148(1): p. 35-43. 
27. 
Oaks, E.V., T.L. Hale, and S.B. Formal, Serum immune response to Shigella protein 
antigens in rhesus monkeys and humans infected with Shigella spp. Infect Immun, 1986. 
53(1): p. 57-63. 
28. 
Oaks, E.V., W.D. Picking, and W.L. Picking, Antibody response of monkeys to invasion 
plasmid antigen D after infection with Shigella spp. Clin Diagn Lab Immunol, 1996. 3(2): 
p. 242-5. 
29. 
Turbyfill, K.R., et al., Identification of epitope and surface-exposed domains of Shigella 
flexneri invasion plasmid antigen D (IpaD). Infect Immun, 1998. 66(5): p. 1999-2006. 
30. 
Vyse, A.J., B.J. Cohen, and M.E. Ramsay, A comparison of oral fluid collection devices 
for use in the surveillance of virus diseases in children. Public Health, 2001. 115(3): p. 
201-7. 
31. 
Gammie, A., R. Morris, and A.P. Wyn-Jones, Antibodies in crevicular fluid: an 
epidemiological tool for investigation of waterborne disease. Epidemiol Infect, 2002. 
128(2): p. 245-9. 
32. 
Griffin, S.M., et al., Development of a multiplex microsphere immunoassay for the 
quantitation of salivary antibody responses to selected waterborne pathogens. J 
Immunol Methods, 2011. 364(1-2): p. 83-93. 

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19. 
APPENDICES 
 
19.1 
Appendix A: Schedule of Events – for each Cohort 
Study Event 
Screening 
Day -45 
Day 
-1 
Day 
0 
Day 
1 
Day 
2 
Day 
3 
Day 
4 
Day 
5 
Day 
6 
Day 
7 
Day 
8 
Day 
14 
Day 
28 
Day 
56 
Day 
180 
Visit Number 
00A 
00B 
01 
01A 
01B 
01C 
01D 
01E 
01F 
01G 
01H 
O2 
O3 
O4 
O5 
Visit Window 
-45 to -2 
N/A 
N/A 
N/A 
N/A 
N/A 
N/A 
N/A 
N/A 
N/A 
N/A 
+2 
±2 
± 4 
±14 
Facility 
Outpatient 
Inpatient 
Outpatient 
Inpatient Period 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Review of Inclusion /Exclusion 
Criteria 
X 
X 
X 
Sign Informed Consent 
X 
Complete Written Test1 
X 
Medical interview2 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Physical examination3 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Vital Signs 4 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Hematology5 
X 
X 
X 
Biochemistry6 
X 
X 
X 
Hepatitis B, C, HIV and 
syphilis7 
X 
Urine for Opiates 
X 
Serum Pregnancy Test8 
X 
Urine Pregnancy Test 
X 
X 
HLA B27 antigen9 
X 
Concomitant Medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Review Medical History 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Reactogenicity10 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Collection of AEs 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Collections of SAEs, significant 
new chronic illnesses, 
pregnancy 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Serology11 
X 
X 
X 
X 
X 
X 
Stool culture for Shigella 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 

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Study Event 
Screening 
Day -45 
Day 
-1 
Day 
0 
Day 
1 
Day 
2 
Day 
3 
Day 
4 
Day 
5 
Day 
6 
Day 
7 
Day 
8 
Day 
14 
Day 
28 
Day 
56 
Day 
180 
Visit Number 
00A 
00B 
01 
01A 
01B 
01C 
01D 
01E 
01F 
01G 
01H 
O2 
O3 
O4 
O5 
Visit Windows 
-45 to -2 
N/A 
N/A 
N/A 
N/A 
N/A 
N/A 
N/A 
N/A 
N/A 
N/A 
+2 
±2 
± 4 
±14 
Facility 
Outpatient 
Inpatient 
Outpatient 
Challenge 
X 
Stool grading12 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Stool for IgA13 
X 
X 
X 
X 
X 
Stool for PCR 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Stool for Microbiome 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Stool for transcriptomics 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Blood for Transcriptome 
(systems biology)14 
X 
X 
X 
X 
X 
Antibody secreting cells (ASC) 
15 
X 
X 
X 
Antibody lymphocyte 
supernatant (ALS)16 
X 
X 
X 
Memory B-Cell17 
X 
X 
X 
Cytokines18 
X 
X 
X 
X 
X 
Salivary IgA19 
X20 
X 
X 
X 
X 
X 
X 
X 
Start antibiotic therapy21 
X 
Planned discharge22 
X 
Study completion 
X 
Post-study safety assessment 
(via telephone follow-up) 
X 
Total Blood volume (mL)23 
48 
112.5 
25 
12.5 
5 
62.5 
70 
30.5 
50.5 
40 
1 Sample test is provided to participants at screening and administered on day -1 
2 The medical interview during screening is to assess eligibility. During the inpatient and outpatient period, the interview will be to update interim medical history, monitor safety, and 
confirm ongoing eligibility. 
3 A complete physical will be done for the initial screening visit and admission to the inpatient unit. All other physical exams will be history directed. 
4 Vital signs (temperature, pulse, and blood pressure) will be obtained at screening and at least daily during inpatient study (more frequently if indicated per Management and 
Assessment of Diarrhea, Vomiting, Dysentery, and Dehydration guidelines in section 8.1.4). 
5 Hematology labs (CBC) include WBC, hemoglobin, neutrophil and lymphocyte count, and platelets (3 mL) (and as indicated per Management and Assessment of Diarrhea, 
Vomiting, Dysentery, and Dehydration guidelines in section 8.1.4). 
6 Biochemistry will include sodium, potassium, creatinine, total bilirubin, ALT and AST and A BUN will be obtained only if Creatinine is > 1.5. (7.5 ml) (Also obtain as indicated per 
Management and Assessment of Diarrhea, Vomiting, Dysentery, and Dehydration in section 8.1.4). 
7 Hepatitis B, C, HIV and syphilis (10mL) 

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8 Serum pregnancy test (5 mL) 
9 HLA B27 antigen (10 ml) 
10 Reactogenicity will be recorded on the memory aid starting on day of discharge and for the next 5 days. 
11 Serology (anti-S.sonnei LPS serum IgG) (5mL screening), (10 mL Day -1 to day 56); serum collected for serology may also be used for SBA testing 
12 Fecal samples will be collected post-challenge through day of discharge for assessment of diarrhea and dysentery. A rectal swab will be obtained for culture and PCR if subject 
unable to provide a stool sample by midnight each day. 
13 Two stool samples will be collected between Day -5 and time of challenge on Day 0 for fecal IgA. Challenge is not contingent upon the results. 
14 Transcriptomics (systems biology) - 3 x 2.5 mL PAXgene tubes. Blood for transcriptomics and cytokines on Day 0 will be done at 4 hours and 12 hours post-inoculation and 24 hours 
post –inoculation on Day 1. 
15 ASC=(50 mL in EDTA tubes); cells collected for ASC may also be used for magnetic separation testing 
16 ALS=(10 mL EDTA tubes); cells collected for ALS may also be used for magnetic separation testing 
17 Memory B-cells=30mL in EDTA tubes 
18 Cytokines = 5 mL SST tubes (4 hours and 12 hours post inoculation), day 1 (24 hours post –inoculum), days 3, 5 
19 Saliva samples will be collected in cohorts 2-5 only. 
20 A baseline saliva sample will be collected between day -1 and any time before challenge on day 0. 
21Ciprofloxacin 500 mg BID x 3 days or (alternate) trimethoprim/sulfamethoxazole 160/800mg BID x 5 days. Subjects may be treated earlier if they meet the criteria for early antibiotic 
treatment. Subjects will complete antibiotic treatments at home if they meet discharge criteria before completing the treatment period. 
22Subjects will be discharged upon meeting the discharge criteria, which may be beyond day 8. Subjects remaining on the inpatient unit past day 8 will continue to have daily 
evaluations until meeting discharge criteria. 
23 Approximate total blood volume to be collected at each visit. 

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19.2 
Appendix B: Toxicity Grading 
During screening, subjects will have blood drawn to determine if any clinical laboratory 
abnormalities exist that would preclude study participation. The clinical toxicity grading scale 
below is based on the FDA Guidance for Industry dated September 2007. In the case where the 
institutional normal parameters of the laboratory used for this study vary from the FDA 
Guidance, the institutional normal parameters were inserted in the table and will serve as the 
standard against which eligibility and adverse event reporting will be based. 
 
Toxicity grading of safety screening laboratory chemistry and blood-count results 
Laboratory 
Normal Range 
Mild 
(Grade 
1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially 
Life 
Threatening 
* 
Sodium, low, mEq/L 
136-145 
130-135 
123-129 
116-122 
< 116 or 
abnormal 
sodium with 
mental status 
changes or 
seizures 
Sodium, high, mEq/L 
136-145 
146-150 
151-157 
158-165 
> 165 or 
abnormal 
sodium with 
mental status 
changes or 
seizures 
Potassium, high, 
mEq/L 
3.5-5.1 
5. 2-6.0 
6.1-6.5 
6.6-7.0 
> 7.0 or 
abnormal 
potassium 
with life- 
threatening 
arrhythmia 
Potassium, low, 
mEq/L 
3.5-5.1 
3.0-3.4 
2.5-2.9 
2.0-2.4 
< 2.0 or 
abnormal 
potassium 
with paresis, 
ileus or life- 
threatening 
arrhythmia 
Blood Urea Nitrogen 
(BUN) mg/dL 
7-20 
>1.0-2.5 x 
ULN** 
2.6-5.0 x 
ULN** 
5.1–10 x 
ULN** 
>10 x ULN** 
Creatinine 
Male - <1.17 
Female- <0.95 
>1.0–1.5 x 
ULN** 
>1.5–3.0 x 
ULN** 
>3.0–6.0 x 
ULN** 
> 6.0 x ULN** 
or dialysis 
required 
Liver Function Tests 
((ALT, AST),) 
increase by factor 
ALT – 1.1x<78 
AST – 1.1x<37 
>1.0-2.5 x 
ULN** 
>2.5-5.0 x 
ULN** 
>5.0-10 x 
ULN** 
> 10 x ULN** 

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Total bilirubin – 
when accompanied 
by any increase in 
Liver Function Test; 
increase by factor 
0.1-1.1 
>1.0-1.25 
x ULN** 
1.26-1.5 x 
ULN** 
1.51-1.75 x 
ULN** 
> 1.75 x 
ULN** 
Hgb (female), g/dL 
11.7-15.7 
11.0-11.6 
9.5 – 10.9 
8.0-9.4 
< 8.0 
Hgb (male), g/dL 
13.0-17.7 
12.5-13.2 
10.5-12.4 
8.5-10.4 
<8.5 
WBC, increase, 
cells, 
x 103u/L 
4.5-11 
>11.0 – 
≤15.0 
>15 - ≤ 20 
>20.0 – 
≤25.0 
>25.0 
WBC, decrease, 
cells, x 103u/L 
4.5-11 
2.5-<4.5 
1.5-<2.5 
1.0-<1.5 
<1.0 
Absolute Neutrophil 
Count, x 103u/L 
1.5-7.7 
1.5-<1.8 
1.0-<1.5 
0.5-<1.0 
<0.5 
Lymphocytes, x 
103u/L 
1.0-4.8 
0.75-<1.0 
0.5-<0.75 
0.25-<0.5 
<0.25 
Platelets, decrease, 
cells/mm3 
134-466 
125,000- 
<135,000 
100,000- 
124,000 
25,000- 
99,000 
< 25,000 
Urine protein 
Trace 
1+ 
2+ 
≥3+ 
Hospitalization 
or dialysis 
Urine glucose 
0 
1+ 
2+ 
≥3+, 
Hospitalization 
for 
hyperglycemia 
Urine blood(non- 
menstruating) 
0-1 
3-10 
11-50 
>50 and/or 
gross blood 
Hospitalization 
or packed red 
blood cells 
(PRBC) 
transfusion 
* 
The clinical signs or symptoms associated with laboratory abnormalities might result in characterization of the 
laboratory abnormalities as Potentially Life Threatening. For example, a low sodium value that falls within a grade 
3 parameter (116-122 mE/L) should be recorded as a life-threatening hyponatremia event if the subject had a new 
seizure associated with the low sodium value. 
** ULN is upper limit of normal in lab. 
In addition, please note that the toxicity grading scale outlined in the FDA Guidance for Industry 
dated September, 2007 will also be used as reference values for grading laboratory 
abnormalities encountered during the trial (see Appendix B). However AEs, guided by the 
Appendix B, will be graded for severity based on comparisons to subject baseline values, 
presence of clinical symptoms and an assessment by the principal investigator. 

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19.3 Appendix C: Information Form if a Subject Prematurely Withdraws from 
the Trial 
 
Safety and Infectivity of an Oral Challenge with S. sonnei 53G 
 
I,  
, am withdrawing consent from the Shigella 
challenge study. I have been told that I am leaving the inpatient unit before I have passed two 
stool specimens that are negative for Shigella. 
 
I have been advised by Dr. Frenck that the Shigella strain could still be living in my intestines 
and could be passed to my close contacts, who could develop symptoms of Shigella infection 
(diarrhea, blood in stool, fever). If one of my close contacts develops diarrhea, I have been told 
that he/she may come to the Gamble Program Outpatient Clinic for a clinical evaluation and 
testing of his/her stool specimen free of charge. 
 
I have been told that each time I use the bathroom I should wash my hands well with soap and 
water, and dry them completely. I have been advised that I should continue these precautions 
until my final stool specimens have been shown to be negative for Shigella. 
 
I have been told that I should complete my antibiotic treatment at home to eliminate the Shigella 
from my intestine. Even though I am leaving the unit early, I have been told that it is very 
important for me to come to my outpatient follow-up visits to the Gamble Program Outpatient 
Unit to have my stools checked for the Shigella and to provide samples, according to the 
schedule that I have been given. I will receive a signed copy of this document and a copy will 
remain on in my study file. 
 
 
Signature of Subject 
Date 
 
 
Printed Name of Subject 
 
 
Signature of Witness 
Date 
 
 
Printed Name of Witness 
 
 
Signature of Principal Investigator 
Date