A Phase 1 Randomized, Double-Blinded, Placebo-controlled, Dose-Escalation Study to Assess the Safety, Tolerability and Immunogenicity of Live Attenuated, Oral Shigella WRSS1 Vaccine in Bangladeshi Toddlers (12 to 24 Months Old)
Phase: PHASE1
Conditions: Diarrhea
Sponsor: PATH

Study Protocol: PR#16023 Version 5.0 
WRSS1 (VAC 049) 
 
10 July 2017 
1 
 
 
 
 
 
RRC APPLICATION FORM 
 
RESEARCH PROTOCOL  
Number: PR-16023 
Version No. 5.00 
Version date: 10 July 2017 
IND Number: 15335 
Sponsor Number: VAC 049 
 
FOR OFFICE USE ONLY 
RRC Approval: 
 Yes 
 No 
Date:12-APR-2016 
ERC Approval: 
 Yes 
 No 
Date:31-JUL-2016 
AEEC Approval: 
 Yes 
 No 
Date:      
External IRB Approval  
 Yes 
 No      Date:03 MAY 2016 
Name of External IRB: ___WIRB____________________________________ 
Protocol Title:* (maximum 250 characters including space)  
A Phase 1 Randomized, Double-Blinded, Placebo-controlled, Dose-Escalation Study to Assess the Safety, 
Tolerability and Immunogenicity of Live Attenuated, Oral Shigella WRSS1 Vaccine in Bangladeshi Toddlers (12 
to 24 months old) 
Short Title: (maximum 100 characters including space) Shigella WRSS1 Vaccine trial in Bangladesh 
 
Key Words:* WRSS1, Shigella sonnei Vaccine, Toddlers, Bangladesh, Safety, Immunogenicity 
Name of the Research Division Hosting the Protocol:* 
  Health Systems and Population Studies Division (HSPSD) 
  Nutrition and Clinical Services  Division (NCSD) 
  Infectious Diseases  Division (IDD) 
 
   Maternal and Child Health Division (MCHD) 
   Laboratory Sciences and Services Division (LSSD) 
   Other (specify) 
Has the Protocol been Derived from an Activity:*   
 No            
 Yes (please provide following information): 
Activity No.  :       
Activity Title:       
PI:       
Grant No.:        
Budget Code:       
 
Start Date:        
End Date:       
icddr,b Strategic Priority/ Initiative (SP 2015-8):* (check all 
that apply)  
   Reducing maternal and neonatal mortality 
   Controlling enteric and respiratory infections 
   Preventing and treating maternal and childhood malnutrition 
   Detecting and controlling emerging and re-emerging 
infections 
 
 
   Achieving universal health coverage 
   Examining the health consequences of climate 
change 
   Preventing and treating non-communicable 
diseases 
Research Phase (4 Ds):* (check all that apply) 
   Discovery 
   Development 
 
   Delivery 
   Evaluation of Delivery 
Anticipated Impact of Research:* (check all that apply) 
   Knowledge Production 
   Capacity Building 
 
   Informing Policy 
   Health and Health Sector Benefits 
   Economic Benefits 
Which of the Sustainable Development Goal This Protocol 
Relates to?:*                              
(Please 
visit: 
http://www.icddrb.net.bd/jahia/Jahia/....... 
for 
selecting SDG Code(s) 
  Yes (please select SDGs) 
  No 
Does this Protocol Use the Gender Framework:*                         
(Please visit: http://www.icddrb.net.bd/jahia/Jahia/pid/684  for 
Gender Alanysis Tool with instructions) 
  Yes (please complete Gender Analysis Tool) 
  No 
If ‘no’ is the response, its reason(s) in brief:  
Will this Research Specifically Benefit the Disadvantaged (economically, socially and/or 
otherwise): 
 Yes 
  No      
Does this Protocol use Behaviour Change Communication:     
  Yes 
  No     

Study Protocol: PR#16023 Version 5.0 
WRSS1 (VAC 049) 
 
10 July 2017 
2 
 
Principal Investigator (Should be icddr,b staff):*    Sex   
  Female    
  Male 
Dr. Rubhana Raqib  
 
(Position, phone no, extension no, cell, and email address ):  
Senior Scientist, Tel: 880-2-9827001-10 Ext:2404, Off: 880-2-9886734, 
Mobile:880-2-01713-040942; email:  rubhana@icddrb.orgDo you have ethics 
certification?  
 No    
 Yes (please attach in your CV below) 
Do you have RBM training certification?   
 No 
 Yes (please attach the 
certificate with CV below) 
Primary Scientific Division of the PI 
Infectious Diseases Division  
(IDD) 
 
Co-Principal Investigator(s) Internal:  Sex    
  Female    
  Male 
Dr. K Zaman 
 
(Position, phone no, extension no, cell, and email address ):  
Senior 
Scientist, 
Tel: 
880-2-9827001-10 
Ext:3806, 
Mobile:880-2-
01713047100; email:  kzaman@icddrb.org  
 
Signature or written consent of Co-PI: __________________ 
(electronic signature or email or any sort of written consent) 
[if more than one, please copy and paste this row for additional Co-PIs]  
 
Do you have ethics certification?   
 No    
 Yes (please attach in your CV 
below) 
Do you have RBM training certification?   
 No 
 Yes (please attach the certificate 
with CV below) 
Primary 
Scientific 
Division/ 
Programme  of the Co-PI 
Infectious Diseases Division  
(IDD) 
 
 
___________________________ 
Approval of the Respective Senior 
Director/ Programme Head 
 
 
 
(Signature) 
Co-Principal Investigator(s) - External: Sex    
 Female     
  Male 
      
Address (provide full official address, including land phone no(s), extension no. (if any), cell phone number, and email 
address).  
      
 
Signature or written consent of Co-PI: __________________ 
(electronic signature or email or any sort of written consent) 
[if more than one, please copy and paste this row for additional Co-PIs] 
Co-Investigator(s) - Internal:  Sex    
  Female    
  Male 
Dr. Nur Haque Alam 
 
(Position, phone no, extension no, cell, and email address ):  
Senior 
Scientist, 
Tel: 
880-2-9827001-10 
Ext:2346, 
Mobile:880-2-
01713093851; email:  nhalam@icddrb.org  
 
Signature or written consent of Co-I: __________________ 
(electronic signature or email or any sort of written consent) 
[if more than one, please copy and paste this row for additional Co-Is 
 
Do you have ethics certification?      
 No    
 Yes (please attach in your CV 
below) 
Do you have RBM training certification?   
 No 
 Yes (please attach the certificate
with CV below) 
Primary Scientific Division of the 
Co-I 
Nutrition and Clinical Services 
Division 
 
 
___________________________ 
Approval of the Respective Senior 
Director/ Programme Head 
 
 
 
(Signature) 

Study Protocol: PR#16023 Version 5.0 
WRSS1 (VAC 049) 
 
10 July 2017 
3 
 
Co-Investigator(s) - Internal:  Sex    
  Female    
  Male 
Dr. Firdausi Qadri      
 
(Position, phone no, extension no, cell, and email address ):  
Senior 
Scientist, 
Tel: 
880-2-9827001-10 
Ext: 
2413, 
Mobile:880-
01711595367; email: fqadri@icddrb.org  
 
Signature or written consent of Co-I: __________________ 
(electronic signature or email or any sort of written consent) 
[if more than one, please copy and paste this row for additional Co-Is 
Do you have ethics certification?      
 No    
 Yes (please attach in your CV 
below) 
Do you have RBM training certification?   
 No 
 Yes (please attach the certificate
with CV below) 
Primary Scientific Division of the 
Co-I 
Infectious Diseases Division  
(IDD) 
 
 
___________________________ 
Approval of the Respective Senior 
Director/ Programme Head 
 
 
 
(Signature) 
Co-Investigator(s) - Internal:  Sex    
  Female    
  Male 
Dr. Md. Jobayer Chisti      
  
(Position, phone no, extension no, cell, and email address ):  
Scientist, Tel: 880-2-9827001-10 Ext: 2317, Mobile: 880-2-01749292703; 
email: chisti@icddrb.org 
 
 
Signature or written consent of Co-I: __________________ 
(electronic signature or email or any sort of written consent) 
[if more than one, please copy and paste this row for additional Co-Is 
Do you have ethics certification?      
 No    
 Yes (please attach in your CV 
below) 
Do you have RBM training certification?   
 No 
 Yes (please attach the certificate
with CV below) 
Primary Scientific Division of the 
Co-I 
Nutrition and Clinical Services 
Division 
 
 
___________________________ 
Approval of the Respective Senior 
Director/ Programme Head 
 
 
 
(Signature) 
Co-Investigator(s) - Internal:  Sex    
  Female    
  Male 
Dr. Dilruba Ahmed       
  
(Position, phone no, extension no, cell, and email address ):  
Head, Clinical Microbiology, Phone: +880-2-9827001-10; Ext:  2446 
Mobile: +880-01713093858; email: dahmed@icddrb.org 
 
 
Signature or written consent of Co-I: __________________ 
(electronic signature or email or any sort of written consent) 
[if more than one, please copy and paste this row for additional Co-Is 
Do you have ethics certification?      
 No    
 Yes (please attach in your CV 
below) 
Do you have RBM training certification?   
 No 
 Yes (please attach the certificate
with CV below) 
Primary Scientific Division of the 
Co-I 
Laboratory Sciences and Services 
Division 
 
 
___________________________ 
Approval of the Respective Senior 
Director/ Programme Head 
 
 
 
(Signature) 
Laboratory Investigator(s) – External:  Sex  
  Female     
  Male 
Address (provide full official address, including land phone no(s), extension no. (if any), cell phone number, and email 
address):  
Dr. Malabi Venkatesan 
Bacterial Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, MD 20910, tel nos. 301-319-
9764 
(w), 
301-605-4869 
(blackberry), 
email: 
Malabi.m.venkatesan.civ@mail.mil,  
Malabi.venkatesan@us.army.mil 
 
Signature or written consent of Co-I: __________________ 
(electronic signature or email or any sort of written consent) 
[if more than one, please copy and paste this row for additional Co-Is] 

Study Protocol: PR#16023 Version 5.0 
WRSS1 (VAC 049) 
 
10 July 2017 
4 
 
Co-Investigator(s) – External:  Sex  
  Female     
  Male 
Address (provide full official address, including land phone no(s), extension no. (if any), cell phone number, and email 
address):  
Dr. Abdullah Baqui, Professor,  Department of International Health, Johns Hopkins Bloomberg School of 
Public Health,  Johns Hopkins University, Room E-8138, 615 N Wolfe St., Baltimore, MD 21205, USA; Phone: 
410-955-3850; Email: abaqui@jhu.edu 
 
 
Signature or written consent of Co-I: __________________ 
(electronic signature or email or any sort of written consent) 
[if more than one, please copy and paste this row for additional Co-Is] 
 
Student Investigator(s) - Internal:  Sex  
  Female     
  Male 
      
(Position, phone no, extension no, cell, and email address ):  
 
      
 
Signature or written consent of Student Investitor: __________________ 
(electronic signature or email or any sort of written consent) 
 
Have ethics certificate?  
 No    
 Yes  (If Yes, please attach to your CV below) 
Students Affiliation      
 
 
___________________________ 
Approval of the Respective Senior 
Director/ Programme Head 
  
 
(Signature) 
Student Investigator(s) - External: Sex  
  Female     
  Male 
 
Address (provide full official address, including land phone no(s), extension no. (if any), cell phone number, and email 
address):  
      
 
Signature or written consent of Student Investitor: __________________ 
(electronic signature or email or any sort of written consent) 
 
Collaborating Institute(s): Please provide full official address 
 
Institution # 1 
 
 
Country 
USA 
Contact person 
Tushar Tewari, MD 
Department 
(including Division, Centre, Unit) 
Senior Medical Officer, PATH 
Institution 
(with official address) 
PATH Vaccine Solutions (PVS) 
A-9 Qutab Institutional Area  
USO Road 
New Delhi 110067, India 
Directorate 
(in case of GoB i.e. DGHS) 
      
Ministry (in case of GoB) 
      

Study Protocol: PR#16023 Version 5.0 
WRSS1 (VAC 049) 
 
10 July 2017 
5 
 
 
Institution # 2 
 
Country 
USA 
Contact person 
Dr Malabi M. Venkatesan 
Department 
(including Division, Centre, Unit) 
Bacterial Diseases Branch  
Institution 
(with official address) 
Walter Reed Army Institute of Research,  
Silver Spring, MD, United States 
Directorate 
(in case of GoB
i.e. DGHS)  
      
Ministry (in case of GoB) 
       
Institution # 3 
 
Country 
USA 
Contact person 
Professor Abdullah Baqui  
Department 
(including Division, Centre, Unit) 
Department of International Health 
Institution 
(with official address) 
Johns Hopkins Bloomberg School of Public Health,  Johns 
Hopkins University, Room E-8138, 615 N Wolfe St., Baltimore, 
MD 21205 
Directorate 
(in case of GoB
i.e. DGHS) 
      
Ministry (in case of GoB) 
       
Note: If less than or more than three collaborating institutions, please delete or insert blocks as needed. 
Contribution by the Members of the Scientific Team: 
 
 
Members’ Name 
Contribution 
Research 
idea/ 
concept 
Study 
design 
Protocol 
writing 
Respond 
to external 
reviewers’ 
comments 
Defending 
at IRB 
Developing 
data 
collection 
Tool(s) 
Data 
Collection 
Data analysis/ 
interpretation 
of results 
Manuscript  
writing 
Dr. Rubhana Raqib  
 
 
 
 
 
 
 
 
 
Dr. K Zaman 
 
 
 
 
 
 
 
 
 
Dr. A. Baqui 
 
 
 
 
 
 
 
 
 
Dr. Dilruba Ahmed   
 
 
 
 
 
 
 
 
 
Dr. Firdausi Qadri 
 
 
 
 
 
 
 
 
 
Dr Malabi Venkatesan 
 
 
 
 
 
 
 
 
 
Dr. Nur Haque Alam 
 
 
 
 
 
 
 
 
 
Dr Jobayer Chisti 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Study Protocol: PR#16023 Version 5.0 
WRSS1 (VAC 049) 
 
10 July 2017 
6 
 
Study Population: Sex, Age, Special Group and Ethnicity 
 
Research Subject: 
   Human             
   Animal  
 
   Microorganism 
   Other (specify): _____________ 
 
Sex: 
   Male             
   Female  
   Transgender 
 
Age: 
   0 – 4 years 
   5 – 10 years 
   11 – 17 years 
   18 – 64 years 
   65 + 
Special Group: 
   Pregnant Women 
   Fetuses 
   Prisoners 
   Destitutes  
   Service Providers 
   Cognitively Impaired 
   CSW 
   Expatriates 
   Immigrants 
   Refugee 
   Others (specify): ________________ 
 
Ethnicity: 
  No ethnic selection (Bangladeshi) 
  Bangalee 
  Tribal group 
   Other (specify): ________________ 
 
NOTE: It is icddr.b’s policy to include men, women, children and transgender in its research projects involving participation 
of humans, unless there is strong justification(s) for their exclusion.  
 
Consent Process: (Check all that apply) 
 
  Written 
  Oral 
  Audio 
  Video 
  None 
Language: 
   Bangla 
   English 
   Other (specify: _______________ 
 
Project/Study Site: (Check all that apply) 
  Chakaria  
  Bandarban 
  Dhaka Hospital  
  Kamalapur Field Site/HDSS  
  Mirpur (Dhaka) 
  Matlab DSS Area 
  Matlab non-DSS Area 
  Matlab Hospital 
  Mirzapur 
 
 
   Bianibazar (Sylhet) 
   Kanaighat (Sylhet) 
   Jakigonj (Sylhet) 
   Other community in Dhaka 
        Name: _________________________________          
   Other sites in Bangladesh 
        Name: ___ CTU in icddr,b ______________ 
   Multi-national Study 
        Name of the country______________________   
Project/Study Type: (Check all that apply) 
 
  Case Control Study 
  Clinical Trial (Hospital/Clinic/Field)* 
  Community-based Trial/Intervention 
  Cross Sectional Survey 
  Family Follow-up Study 
  Longitudinal Study (cohort or follow-up) 
  Meta-analysis 
  Programme Evaluation 
 
   Programme (Umbrella Project) 
   Prophylactic Trial 
   Record Review 
   Secondary Data Analysis 
        Protocol No. of  Data Source: _____________ 
   Surveillance/Monitoring 
   Systematic Review 
   Other (specify): ____Vaccine Trial________ 
 
 *Note: International Committee of Medical Journal Editors (ICMJE) defines Clinical Trial as “Any research project that 
prospectively assigns human participants to intervention and comparison groups to study the cause-and-effect 
relationship between a medical intervention and a health outcome”. 
PI of the RRC- and ERC-approved Clinical Trials should provide necessary information to IRB Secretariat (Research 
Administration) for registration and uploading into relevant websites (usually at the https://register.clinicaltrials.gov/). 
They should also provide relevant information to the IRB Secretariat in the event of amendment/modification after 
their approval by RRC and ERC.           

Study Protocol: PR#16023 Version 5.0 
WRSS1 (VAC 049) 
 
10 July 2017 
7 
 
Biological Specimen: 
 
a)  Will the biological specimen be stored for future use?          
 Yes            
 No            
 Not applicable 
b)  If the response is ‘yes’, how long the specimens will be 
preserved?     
____N/A______  years 
c) What types of tests will be carried out with the preserved 
specimens? 
N/A  
d) Will the consent be obtained from the study participants for use 
of the preserved specimen for other initiative(s) unrelated to this 
study, without their re-consent? 
 Yes            
 No            
 Not applicable 
e) Will the specimens be shipped to other country/ countries? 
If yes, name of institution(s) and country/ countries?   
 Yes            
 No            
 Not applicable 
f) 
If shipped to another country, will the surplus/unused specimen 
be returned to icddr,b?   
If the response is ‘no’, then the surplus/unused specimen must 
be destroyed. 
 Yes            
 No            
 Not applicable 
g) Who will be the custodian of the specimen at icddr,b?  
PI of the study 
h) Who will be the custodian of the specimen when shipped 
outside Bangladesh?    
N/A 
i) 
Who will be the owner(s) of the specimens?        
icddr,b 
j) 
Has a MoU been signed with regards to collection, storage, use 
and ownership of specimen?                                    
If the response is ‘yes’, please attach a copy of the MoU..   
       If the response is ‘no’, appropriate justification should be 
provided for not signing a MoU. 
 Yes            
 No            
 Not applicable 
An MoU is not required as specimens will not 
be shipped outside icddr,b. 
Proposed Sample Size: 
 
Sub-group (Name of subgroup e.g. Men, Women) and Number 
 
Name 
Number 
(1) Healthy Male and Female Children (12-24 mo)   
64 
Total sample size 
64 
                                                                                                                                                  
Determination of Risk: Does the Research Involve (Check all that apply) 
  Human exposure to radioactive agents? 
  Foetal tissue or abortus? 
  Investigational new device? 
        Specify: __________________ 
  Existing data available from Co-investigator? 
 
 
  Human exposure to infectious agents? 
  Investigational new drug? 
  Existing data available via public archives/sources? 
  Pathological or diagnostic clinical specimen only? 
  Observation of public behaviour? 
    
  New treatment regime? 

Study Protocol: PR#16023 Version 5.0 
WRSS1 (VAC 049) 
 
10 July 2017 
8 
 
 
Will the information be recorded in such a manner that study participants can be identified from the 
information directly or through identifiers linked to the study participants? 
Yes 
 
No 
 
Does the research deal with sensitive aspects of the study participants’ sexual behaviour, alcohol use or 
illegal conduct such as drug use? 
Yes 
 
No 
 
 
  Could information on study participants, if available to people outside of the research team: 
 
a) Place them at risk of criminal or civil liability? 
Yes 
 
No 
 
b) Damage their financial standing, reputation or employability, or social rejection, or lead to 
stigma, divorce etc.? 
Yes 
 
No 
 
 
Do you consider this research: (check one) 
 
   Greater than minimal risk 
 No more than minimal risk 
   Only part of the diagnostic test 
 
Note: Minimal Risk: The probability and the magnitude of the anticipated harm or discomfort to participants is not greater than those ordinarily 
encountered in daily life or during the performance of routine physical, psychological examinations or tests, e.g. the risk of drawing a small amount of 
blood from a healthy individual for research purposes is no greater than when the same is performed for routine management of patients. 
Risk Group of Infectious Agent and Use of Recombinant DNA 
 
a) Will specimens containing infectious agent be collected? 
 Yes            
 No            
 Not applicable 
b) Will the study involve amplification by culture of infectious 
agents? 
 Yes            
 No            
 Not applicable 
c) If response to questions (a) and/or (b) is ‘yes’, to which Risk 
Group (RG) does the agent(s) belong? (Please visit 
http://www.icddrb.net.bd/jahia/Jahia/pid/684 to review list of 
microorganism by Risk Group) 
 RG1           
 RG2         
 RG3           
 RG4 
d) Does the study involve experiments with recombinant DNA? 
 Yes            
 No            
 Not applicable 
Does the study involve any biohazards materials/agents or microorganisms of risk group 2, 3, or 4 (GR2, GR-3 or 
GR4)? 
 
 
 Yes            
 No            
 
[If the response is ‘yes’] I, (print name of the PI) affirm that we will use the standard icddr,b laboratory 
procedures for biosafety of the hazardous materials/agents or microorganisms in the conduction of the study.  
 
 
_________________________________                                                                                        _____________ 
Signature of  the Principal Investigator                                                                                                  Date 

Study Protocol: PR#16023 Version 5.0 
WRSS1 (VAC 049) 
 
10 July 2017 
9 
 
Dissemination Plan: [please explicitly describe the plans for dissemination, including how the research findings would be 
shared with stakeholders, identifying them if known, and the mechanism to be used; anticipated type of publication (working 
papers, internal (institutional) publication, international publications, international conferences/seminars/workshops/ 
agencies. [Check all that are applicable] 
 
Dissemination type 
Response 
Description (if the response is a yes) 
Seminar 
for 
icddr,b 
scientists/ staff 
 No 
 Yes 
Will be shared with icddr,b scientists and staff through Centres’ 
Scientific forum  
Internal publication 
 No 
 Yes 
 
Working paper 
 No 
 Yes 
 
Sharing with GoB (e.g. 
DGHS/ 
Ministry, 
others) 
 No 
 Yes 
Will be shared with government officials in meetings/workshops 
Sharing with national 
NGOs 
 No 
 Yes 
 
Presentation at national 
workshop/ seminar 
 No 
 Yes 
Data will be shared in meetings in form of presentation 
Presentation 
at 
international workshop/ 
conference 
 No 
 Yes 
In the event of a presentation at an international workshop, icddr,b, 
WRAIR and PVS will collaborate accordingly on all presentation 
materials including PowerPoint slides and/or submitted abstract 
Peer-reviewed 
publication 
 No 
 Yes 
Potential publications will be written collaboratively between 
PVS, WRAIR and icddr,b; PVS requires draft manuscripts be 
reviewed and approved internally prior to journal submission.  
Sharing 
with 
international agencies 
 No 
 Yes 
 
Sharing with donors 
 No 
 Yes 
icddr,b and PVS will disseminate generated data as appropriate to 
donors, including the BMGF 
Policy brief 
 No 
 Yes 
 
Other 
 
 
 
Other 
 
 
 
` 
Funding: 
Is the protocol fully funded? 
 Yes 
 No 
If the answer is yes, please provide 
sponsor(s)’s name 
1. PATH Vaccine Solutions (PVS) 
       455 Massachusetts Avenue NW, Suite 1000  
Washington, DC 20001  
2. 
Is the protocol partially funded? 
 Yes 
 No 
If the answer is yes, please provide 
sponsor(s)’s name 
1. 
2. 
 
If fund has not been identified: 
Is the proposal being submitted for funding? 
 Yes 
 No 
If yes, name of the funding agency 
1. 
2. 

Study Protocol: PR#16023 Version 5.0 
WRSS1 (VAC 049) 
 
10 July 2017 
10 
 
 
Conflict of interest: 
Do any of the participating investigators and/or member(s) of their immediate families have an equity relationship (e.g. 
stockholder) with the sponsor of the project or manufacturer and/or owner of the test product or device to be studied or 
serve as a consultant to any of the above? 
 No 
 Yes (please submit a written statement of disclosure to the Executive Director, icddr,b) 
Proposed Budget: 
Dates of Proposed Period of Support              
 
            Cost Required for the Budget Period ($)  
 
(Day, Month, Year - DD/MM/YY)                      
    
 Beginning Date : January 2017 
  
 End Date           : 1 year from start 
 
 
 
 
 
 
Years 
Direct Cost 
Indirect 
Cost 
Total Cost 
Year-1 
750,135 
109,970 
860,105 
Year-2 
      
      
0 
Year-3 
      
      
0 
Year-4 
      
      
0 
Year-5 
      
      
0 
Total 
750,135 
109,970 
860,105 
 
Certification by the Principal Investigator: 
 
I certify that the statements herein are true, complete and accurate to the best of my knowledge. I am aware that any false, 
fictitious, or fraudulent statements or claims may subject me to criminal, civil, or administrative penalties. I agree to accept 
the responsibility for the scientific conduct of the project and to provide the required progress reports including updating 
protocol information in the NAVISION if a grant is awarded as a result of this application. 
 
I also certify that I have read icddr,b Data Policies and understand the PIs’ responsibilities related to archival and sharing of 
research data, and will remain fully compliant to the Policies. (Note: The Data Policies can be found here: 
http://www.icddrb.org/who-we-are/data-policies) 
 
 
___________                                                                                                                   ____________ 
Signature of PI                                                                                                                                         Date              
                                       
 
Approval of the Project by the Division Director of the Applicant: 
 
 
The above-mentioned project has been discussed and reviewed at the Division level. 
 
 
 
                         
                 ___________ 
      
Name of the Division Director 
Signature 
                Date of Approval 

Study Protocol: PR#16023 Version 5.0 
WRSS1 (VAC 049) 
 
10 July 2017 
11 
 
TABLE OF CONTENTS 
 
 
RRC APPLICATION FORM .................................................................................................................. 1 
PROJECT SUMMARY ........................................................................................................................ 15 
1.0 STUDY HYPOTHESIS .................................................................................................................. 19 
1.1 Primary Study Hypothesis ......................................................................................................... 19 
1.2 Secondary Study Hypotheses..................................................................................................... 19 
2.0 OBJECTIVES ................................................................................................................................ 19 
2.1 Primary Study Objective ........................................................................................................... 19 
2.2 Secondary Study Objectives ...................................................................................................... 19 
2.3 Exploratory Objectives ............................................................................................................. 19 
3.0 BACKGROUND OF THE PROJECT INCLUDING PRELIMINARY OBSERVATIONS AND 
SCIENTIFIC RATIONALE .................................................................................................................. 20 
3.1 Clinical Significance and Pathogenicity of Shigella1 ................................................................... 20 
3.2 WRSS1 as a prototype live attenuated S. sonnei vaccine for descending age study ......................... 23 
3.3 Development of a ΔvirG S. sonnei Vaccine strain WRSS1 .......................................................... 23 
3.4 Previous Vaccine studies ........................................................................................................... 24 
3.4.1 Preclinical studies with Shigella sonnei live attenuated vaccine (WRSS1) .................................... 24 
3.4.2 Previous human use of WRSS1 ...................................................................................................... 24 
3.5 Rationale for Current Study and Planned Future Development ..................................................... 30 
3.5.1 A Rationale for exploratory objectives ........................................................................................... 31 
3.6 Study Site ................................................................................................................................ 32 
4.0 RESEARCH DESIGN AND METHODS ......................................................................................... 32 
5.0 STUDY POPULATION .................................................................................................................. 33 
5.1 Eligibility Criteria..................................................................................................................... 33 
6.0 RECRUITMENT, SCREENING, RANDOMIZATION, AND MASKING PROCEDURES ................ 34 
6.1 Recruitment ............................................................................................................................. 34 
6.2 Screening ................................................................................................................................. 35 
6.3 Randomization ......................................................................................................................... 35 
6.4 Masking procedures .................................................................................................................. 35 
7.0 STUDY PROCEDURES/EVALUATIONS ...................................................................................... 36 
7.1 Clinical Definitions .................................................................................................................. 36 
7.1.1 Grading system for diarrhea ........................................................................................................... 36 
7.1.2 Inpatient Phase (All cohorts – Day 0 to Day 1) .............................................................................. 36 
7.1.3 Outpatient Phase ............................................................................................................................. 37 
7.2 Concomitant Medications/Treatments ........................................................................................ 37 
7.2.1 Antimicrobials ................................................................................................................................ 37 
7.2.2 Antipyretics ..................................................................................................................................... 37 
7.3 Laboratory Evaluations ............................................................................................................. 37 
7.3.1 Clinical Laboratory Evaluations ..................................................................................................... 37 
7.3.2 Research Laboratory Evaluations ................................................................................................... 37 
8.0 STUDY SCHEDULE ...................................................................................................................... 38 
8.1 Screening Visit (Days -30 to -9) ................................................................................................ 38 
8.2 Day of First Vaccination (V0) ................................................................................................... 39 
8.3 Inpatient Period (V1) ................................................................................................................ 40 
8.4 Discharge (V1) ......................................................................................................................... 40 

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8.5 Outpatient Visits ....................................................................................................................... 40 
8.5.1 Outpatient Vaccinations (V28 and V56) ......................................................................................... 40 
8.5.2 Outpatient Assessments (V7, V28, V35, V56, V63, V84) ............................................................. 41 
8.6 Early Termination..................................................................................................................... 42 
9.0 STUDY INTERVENTION/INVESTIGATIONAL PRODUCT.......................................................... 43 
9.1 WRSS1 .................................................................................................................................... 43 
9.1.1 Biological Name and Structure ....................................................................................................... 43 
9.1.2 Chemistry, Manufacturing, and Control ......................................................................................... 43 
9.1.3 Final container and composition ..................................................................................................... 44 
9.2 Preparation, Administration and Dosage of Study Investigational Product, and Buffer .................. 44 
9.2.1 WRSS1 ........................................................................................................................................... 44 
9.2.2 Placebo ............................................................................................................................................ 44 
9.2.3 Sodium Bicarbonate ........................................................................................................................ 44 
9.3 Accountability Procedures for the Investigational Product(s) ....................................................... 44 
9.4 Assessment of Participant Compliance with Investigational Products ........................................... 45 
10.0 ASSESSMENT OF SAFETY ........................................................................................................ 45 
10.1 Adverse Events ....................................................................................................................... 45 
10.1.1 Adverse Event Definitions ............................................................................................................ 45 
10.1.2 Reactogenicity .............................................................................................................................. 46 
10.1.3 Guidelines for Determining Causality of an Adverse Event ......................................................... 46 
10.1.4 Follow-up of Adverse Events and Assessment of Outcomes ....................................................... 46 
10.1.5 Treatment of Adverse Events ....................................................................................................... 46 
10.1.6 Recording Adverse Events ............................................................................................................ 47 
10.1.7 Reporting Serious Adverse Events ............................................................................................... 47 
10.1.8 Follow-up of Serious Adverse Events .......................................................................................... 48 
10.2 Safety Oversight - Data Safety Monitoring Board (DSMB) ....................................................... 48 
10.3 Stopping Rules ....................................................................................................................... 49 
10.4 Six Month Follow-up Safety Surveillance ................................................................................ 49 
11.0 MONITORING BY SPONSOR’S REPRESENTATIVE ................................................................. 49 
11.1 Study Monitoring ................................................................................................................... 50 
11.2 Independent Auditing .............................................................................................................. 50 
11.3 Regulatory Agency Auditing ................................................................................................... 50 
12.0 SAMPLE SIZE CALCULATION AND OUTCOME PRIMARY AND SECONDARY VARIABLES50 
12.1 Introduction............................................................................................................................ 50 
12.2 Sample Size ........................................................................................................................... 50 
12.3 Data Analysis ......................................................................................................................... 51 
12.3.1 General Analytic Plan Associated Assessment of Safety and Immunogenicity ........................... 51 
12.3.2 Final Analysis Plan ....................................................................................................................... 52 
13.0 DATA SAFETY MONITORING PLAN (DSMP) AND DATA MANAGEMENT ........................... 52 
13.1 Database Development ............................................................................................................ 52 
13.2 Data Validation ...................................................................................................................... 53 
13.2.1 Source Data Verification .............................................................................................................. 53 
13.2.2 Definition of Source Data ............................................................................................................. 53 
13.2.3 Definition of Source Document .................................................................................................... 53 
13.3 Database Locking Procedures .................................................................................................. 53 
13.4 Record and Specimen Archival ................................................................................................ 53 
13.5 Screen Failures ....................................................................................................................... 53 
13.6 Protocol Deviations ................................................................................................................ 53 
14.0 OBLIGATIONS AND ROLES OF THE SPONSOR, INVESTIGATOR AND STUDY PERSONNEL54 

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15.0 ETHICS/PROTECTION OF HUMAN PARTICIPANTS ................................................................ 54 
15.1 Clinical Development Plan ...................................................................................................... 54 
15.2 Ethical Conduct of the Study ................................................................................................... 54 
15.3 Institutional Review Board ...................................................................................................... 55 
15.4 Informed Consent Process ....................................................................................................... 55 
15.5 Risk/Benefit ........................................................................................................................... 55 
16.0 USE OF ANIMALS ...................................................................................................................... 55 
17.0 PUBLICATION POLICY .............................................................................................................. 55 
18.0 FACILITIES AVAILABLE ........................................................................................................... 55 
19.0 LITERATURE CITED .................................................................................................................. 57 
 
   
  Check here if appendix is included 
 
 

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TABLE OF FIGURES AND TABLES 
 
FIGURE 1. YEARLY OVERALL ISOLATION OF SHIGELLA BY SERO-GROUPS IN DHAKA 
HOSPITAL, 2008-11 ............................................................................................................................ 21 
FIGURE 2: YEARLY OVERALL ISOLATION OF SHIGELLA BY SERO-GROUPS IN MIRPUR 
TREATMENT CENTRE, 2009-11 ........................................................................................................ 21 
FIGURE 3: YEARLY ISOLATION OF SHIGELLA BY SERO-GROUPS AMONG CHILDREN < 5 
YEARS OLD IN MIRZAPUR, 2010-2011 ............................................................................................. 22 
TABLE 
1A. 
SHIGELLA 
ISOLATION 
AMONG 
DIFFERENT 
AGE 
GROUPS.............................................................................................................................................22  
TABLE 1B. ISOLATION OF S. SONNEI AMONG AGE GROUPS..............................................................22 
TABLE 2.  RATE OF CLINICALLY RELEVANT SYMPTOMS AND RATE OF ANY SYMPTOM 
LIKELY TO BE REPORTED DURING AN INPATIENT TRIAL .......................................................... 25 
TABLE 3.  RATE OF CLINICALLY RELEVANT SYMPTOMS AFTER VACCINATION PROTOCOL 
NO. A-11538*...................................................................................................................................... 25 
TABLE 4.  ADVERSE EVENTS (CLINICALLY RELATED SYMPTOMS) IN PHASE 1B TRIAL OF 1.5 
X 104 CFU WRSS1 IN THAI ADULTS. ................................................................................................ 26 
TABLE 5. NUMBER OF PARTICIPANTS RECEIVING THE LIVE, ATTENUATED S. SONNEI 
VACCINE OR PLACEBO BY WRSS1 DOSE ...................................................................................... 27 
TABLE 6.  NUMBER (PERCENT) SOLICITED REACTOGENICITY AMONG CHILDREN RECEIVING 
WRSS1 OR PLACEBO ........................................................................................................................ 28 
TABLE 7. IMMUNE RESPONSE TO SHIGELLA SONNEI LPS AND INVAPLEX ANTIGEN IN 
CHILDREN 5-9 YEARS OLD AFTER THREE DOSES (AT DAY 63) OF THE LIVE, ATTENUATED S. 
SONNEI VACCINE OR PLACEBO BY WRSS1 DOSE ........................................................................ 29 
TABLE 8.  SERUM IMMUNE RESPONSE TO LPS OR INVAPLEX AFTER 3 DOSES OF WRSS1  
VACCINE BY DOSE COHORT AMONG CHILDREN 5 TO 9 YEARS OLD, ICDDR,B (UNBLINDED).
............................................................................................................................................................ 29 
TABLE 9.  FOLD INCREASE IN TITER FROM PRE-VACCINATION (DAY 0) TO POST THIRD DOSE 
(DAY 63) FOR COHORT B4 (CHILDREN RECEIVING 106 CFU WRSS1 OR PLACEBO).   ................. 30 
TABLE 10. VACCINE DOSE AND SUBJECT ALLOCATION BY COHORT ....................................... 33 
TABLE 11: VACCINE AND PLACEBO VOLUMES ............................................................................ 43 
TABLE 12.  ADVERSE EVENTS TO BE SOLICITED DAILY DURING THE FIRST 72-HOURS POST-
VACCINATION .................................................................................................................................. 46 
 
 
 
 
 
 
 

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Project Summary 
[The summary, within a word limit of 300, should be stand alone and be fully understandable.] 
Principal Investigator: Rubhana Raqib, PhD 
Co-Principal Investigator (Internal): K. Zaman, MD 
 
Research Protocol Title: A Phase 1 Randomized, Double-Blinded, Placebo-controlled, Dose-Escalation Study 
to Assess the Safety, Tolerability and Immunogenicity of Live Attenuated, Oral Shigella WRSS1 Vaccine in 
Bangladeshi Toddlers (12 to 24 months old) 
Proposed start date: January 2017                                               Estimated end date: 12 months from start  
Background (brief): 
a. Burden: Shigellosis remains a major public health problem and a major cause of morbidity in children 
under 5 years old in impoverished Asian populations. In a large multicenter study of Shigella diarrhoea in 
six Asian countries, Shigella was isolated from 5% of all diarrhoea episodes detected. S. flexneri was the 
most frequently isolated species followed by S. sonnei and S. boydii. S. sonnei is a significant cause of 
diarrhoeal infection in Bangladesh and is gradually increasing in prevalence with economic improvements. 
In a recent analysis of diarrheal bacterial isolates obtained over 4 years (Jan 2005 to Dec 2008) from 
patients admitted at the International Centre for Diarrheal Diseases, Bangladesh (icddr,b) in Dhaka, 20% 
were Shigella spp [1]. Shigella isolation rates were second only to V. cholera, which accounted for ~40% 
of the isolates. Of the Shigella isolates, S. flexneri and S. sonnei made up 59% and 11.2%, respectively [1]. 
The Dhaka Hospital surveillance data shows that S. sonnei is 2nd to S. flexneri cases and have been steadily 
increasing over the years (from 4-9% in the period 1980-2008 to about 27% in 2011-2012). Among 
children under the age of 5, the prevalence also increased from 11% in 2008 to 40% in 2011. In the Mirpur 
Treatment Centre, among children <5, the rate of S. sonnei (53%) was much higher compared to S. flexneri 
(18%) in 2011. In the GEMS study conducted in Mirzapur by Dr ASG Faruque, S. sonnei isolation rate 
was as high (25%) as S. flexneri (21%) in children <5 year. More recently, studies on stool specimens from 
GEMS and MAL-ED studies using molecular assays showed increased burdens for Shigella spp then 
shown by conventional methods among under 5 year children. 
b. Knowledge gap: Testing the potential of live Shigella vaccines in populations at need is a critical step to 
understanding their safety, attenuation level, and ability to generate a protective immune response. 
c. Relevance: Shigella appears to be more ubiquitous in impoverished Asian populations than previously 
thought; various new serotypes and antibiotic-resistant strains have emerged. Nearly all shigellae are 
resistant to nalidixic acid and cotrimoxazole.  In recent studies at icddr,b, resistance to ciprofloxacin in 
Shigella isolates increased from 1% in 2005 to 34% in 2008. Intra-familial transmission of Shigella carries 
a high risk for diarrhoea and persistence in the community. Immunization with an efficacious Shigella 
vaccine will prevent shigellosis, substantially reduce overall diarrhoea burden in the community and block 
the spread of multidrug-resistant Shigella strains. Cross-protective Shigella vaccines will be needed to 
prevent shigellosis in Asia. 
WRSS1 Vaccine: A live attenuated S. sonnei WRSS1 vaccine was constructed from parent strain Moseley by 
making a 212-bp deletion in virG (also known as icsA). S. sonnei WRSS1, like its parent strain Moseley, is 
invasive in the HeLa cell invasion assay.  Unlike Moseley however, the vaccine is negative in the Sereny test 
and does not form plaques in tissue culture monolayers, indicating loss of ability to spread intracellularly and 
intercellularly.  S. sonnei WRSS1 develops predominantly form I colonies (>90%) and agglutinates with S. 
sonnei O-specific antiserum, indicating retention and expression of the virulence plasmid. The WRSS1 is at the 
most advanced stage of development of the Shigella vaccines.  In an earlier trial completed in 2010, WRSS1 
was tested in a single, low, oral dose in Thai adults and was shown to be safe, immunogenic, and a promising 
vaccine candidate when given to healthy adults in a range of 103 to 104 CFU. In a follow-up study (PR#12054) 
carried out in Bangladesh, the WRSS1 vaccine was evaluated in healthy adults (18-45 years) and children (5-9 
years). The new study in toddlers aims to confirm increased colonization rates and improved immunogenicity 
of WRSS1 by administering 3 doses at gradually increasing dose levels.  
The study at icddr,b will evaluate up to four dose levels of vaccine to assess the potential for enhancement of 
immune responses when booster doses are given at one month intervals after the initial priming dose.  
 

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Hypothesis:  
Primary Hypothesis: WRSS1 will be safe and tolerable in the toddlers.  
Secondary Hypotheses:  
 
WRSS1 will generate a positive (4-fold rise) serum/plasma immune response detectable in one or more 
assays in at least 50% of vaccinees. 
 
WRSS1 will be able to replicate in the intestine 
Objectives:  
Primary Objectives:  
1. To evaluate the safety and clinical tolerability of oral Shigella sonnei WRSS1 vaccine in Bangladeshi 
toddlers by monitoring the occurrence and severity of clinical signs and symptoms after administration 
of up to three vaccine doses. 
Secondary Objectives:  
1. To evaluate the immune response to WRSS1 antigens in blood and stool following ingestion of 
WRSS1 vaccine 
2. To assess the ability to replicate and duration of fecal shedding of WRSS1 following ingestion  
Methods: This is a single site, Phase 1, double-blind, randomized, placebo-controlled, dose-escalation study in 
toddlers (12-24 months old).  The study will enroll approximately 64 toddlers (12-24 months old) to receive 
three doses of 3x103, 3x104, 3x105 or 3x106 CFU WRSS1 vaccine or placebo. In each cohort, the first dose and 
immediate safety evaluation will be conducted at the Clinical Trials Unit (CTU), an inpatient facility located on 
the icddr,b campus, where the volunteers will be admitted for observation for 24 hours post-vaccination.  
Second and third vaccinations for the applicable cohorts will take place on an outpatient basis in the CTU. 
Before enrolling participants in subsequent cohorts to receive a higher vaccine dose, the safety data from the 
previous cohort(s) (through Study Day 7) will be evaluated and reviewed by the Internal Protocol Safety Team 
(IPST) comprised of the study physician, a medical monitor from the CRO (The Emmes Corporation), the 
principal investigator, and the Medical Monitor from PVS.  Evaluations of safety via clinic visits, physical 
exams, solicited symptoms and laboratory evaluations will be conducted.  Shedding of vaccine strain will be 
assessed by culture and PCR methods in collected stool specimens. Immunogenicity will be studied by 
determining S. sonnei LPS and invaplex antigen-specific antibody titers and cytokine responses in lymphocyte 
supernatant (ALS), and serum/plasma. Serum bactericidal response against Shigella as a functional indicator of 
protection will be also studied. Antigen specific IgA response and cytokine responses in stool will be measured.  
Outcome measures/variables:  
1. Safety and tolerability of the WRSS1 vaccine 
2. Immunogenicity of the WRSS1 vaccine 
3. Intestinal replication of the WRSS1 vaccine 
 
 
 
 

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KEY ROLES 
      
Study Sponsor 
PATH Vaccine Solutions (PVS) 
455 Massachusetts Ave, NW 
Suite 1000 
Washington, DC 20001 
Data Management/Study Monitoring 
The Emmes Corporation 
401 N. Washington St., Suite 700 
Rockville, MD  20850, USA 
Principal Investigator 
Rubhana Raqib, PhD 
Senior Scientist & Head 
Immunobiology, 
Nutrition 
and 
Toxicology 
Laboratory, 
Infectious Diseases Division 
icddr,b 
68 Shaheed Tajuddin Ahmed Sarani, 
Mohakhali, Dhaka-1212, Bangladesh 
Co- Principal Investigator/Study Physician 
K. Zaman, MD, PhD 
Infectious Diseases Division  
icddr,b 
68 Shaheed Tajuddin Ahmed Sarani, 
Mohakhali, Dhaka-1212, Bangladesh 
Co- Investigator 
Dr Dilruba Ahmed, MD, PhD 
Laboratory Sciences and Services Division 
68 Shaheed Tajuddin Ahmed Sarani, 
Mohakhali, Dhaka-1212, Bangladesh 
 
Co- Investigator 
Nur Haq Alam, MD, PhD 
Nutrition and Clinical Services Division, icddr,b 
68 Shaheed Tajuddin Ahmed Sarani, 
Mohakhali, Dhaka-1212, Bangladesh 
 
Co- Investigator 
Professor Abdullah Baqui 
Johns Hopkins University 
Department of International Health 
Co- Investigator 
Dr Md. Jobayer Chisti 
Nutrition and Clinical Services Division, icddr,b 
68 Shaheed Tajuddin Ahmed Sarani, 
Mohakhali, Dhaka-1212, Bangladesh 
Study Coordinator 
Protim Sarker, PhD 
Infectious Diseases Division 
icddr,b 
68 Shaheed Tajuddin Ahmed Sarani, 
Mohakhali, Dhaka-1212, Bangladesh 
Icddr,b Study Physician 
Dr. Arifuzzaman, MD 
Infectious Diseases Division 
icddr,b 
68 Shaheed Tajuddin Ahmed Sarani, 
Mohakhali, Dhaka-1212, Bangladesh 
Emmes Medical Monitor 
Robert Lindblad, MD 
The Emmes Corporation 
Phone: 301-251-1161 
Fax: 301-251-1355 
Email: rlindblad@emmes.com  
PVS Medical Monitor 
Tushar Tewari, MD 
PATH Vaccine Solutions (PVS) 
A-9 Qutab Institutional Area 
USO Road 
New Delhi 110067, India 
PVS Protocol Specialist 
Nicole Maier, MS 
PATH Vaccine Solutions 
455 Massachusetts Ave, NW, Suite 1000 
Washington, DC 20001 
PVS Scientific Lead 
Tom Wierzba, PhD 
PATH Vaccine Solutions (PVS) 
455 Massachusetts Avenue NW, Suite 1000  
Washington, DC 20001  
 
 
 

Study Protocol: PR#16023 Version 5.0 
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LIST OF ABBREVIATIONS 
AE 
Adverse Event 
CTU 
Clinical Trials Unit, icddr,b  
DDSS 
Diarrhoeal Disease Surveillance Systems   
DSMB 
Data Safety Monitoring Committee 
ERC 
icddr,b  Ethical Review Committee 
FDA 
US Food and Drug Administration 
GEMS 
Global Enteric Multicenter Study 
HEENT 
Head/Ears/Eyes/Nose/Throat 
icddr,b  
INT 
International Centre for Diarrhoeal Disease Research, Bangladesh 
Immunobiology, Nutrition and Toxicology 
IPST 
Internal Protocol Safety Team  
IRB 
Institutional Review Board 
ISF 
Investigational Study File 
LDC 
Less Developed Countries 
mL 
Milliliter 
MM 
Medical Monitor 
ORS 
Oral Rehydration solution 
OTC 
Over the Counter 
PATH 
Program for appropriate technology in health 
PI 
Principal Investigator 
PVS 
PATH Vaccine Solutions 
SAE 
Serious Adverse Event 
SOP 
Standard Operating Procedure 
WIRB 
Western Institutional Review Board 
WRAIR 
Walter Reed Army Institute of Research 

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DESCRIPTION OF THE RESEARCH PROJECT 
1.0 STUDY HYPOTHESIS 
In a hypothesis testing research proposal, briefly mention the hypothesis to be tested and provide the scientific basis of the hypothesis, critically 
examining the observations leading to the formulation of the hypothesis. 
Does this research proposal involve testing of hypothesis: 
 No       
 Yes (describe below)  
1.1 Primary Study Hypothesis 
 
Orally administered WRSS1 will be safe and tolerable in toddler recipients and will cause no major 
side effects in vaccinees. 
1.2 Secondary Study Hypotheses 
 
WRSS1 will generate a positive (4-fold rise in serum/plasma) immune response detectable in one or 
more assays in at least 50% of vaccinees.  
 
WRSS1 will be able to replicate in the intestine as determined by fecal shedding. 
2.0 OBJECTIVES 
Describe the specific objectives of the proposed study. State the specific parameters, gender aspects, biological functions, rates, and processes that will 
be assessed by specific methods. 
2.1 Primary Study Objective 
To evaluate the safety and clinical tolerance of WRSS1 in Bangladeshi toddlers (12-24 months old) by 
monitoring the occurrence and severity of clinical signs and symptoms after administration of three vaccine 
doses. 
Safety will be assessed by analyses of the following primary endpoints (events), where the unit of analysis in 
each case will be the proportion of participants with at least one event: 
 
any solicited reactogenicity 
 
any unsolicited AEs 
 
any SAEs 
 
unsolicited AEs and SAEs judged as having a reasonable possibility that the study product caused the 
event 
2.2 Secondary Study Objectives 
 
To examine the immunogenicity of WRSS1.  
 
To assess the ability to replicate and duration of fecal shedding of WRSS1 following ingestion.  
The secondary immunogenicity endpoints are chosen to further assess the mucosal and systemic 
immunogenicity.  This will be done by assessing: 
 
Frequency and magnitude of antigen specific serum/plasma IgA, IgG and IgM antibody titers to S. 
sonnei LPS, and S. sonnei Invaplex antigens after administration of three vaccine doses 
 
Frequency and magnitude of antigen-specific fecal IgA antibody titer to S. sonnei LPS and S. sonnei 
Invaplex antigens after administration of three vaccine doses 
 
Frequency and magnitude of antigen (LPS and Invaplex)-specific IgG, IgA, and IgM antibody titers 
in lymphocyte supernatent (ALS) to S. sonnei LPS and Invaplex antigens 
Finally, fecal shedding of vaccine will be measured by the frequency and duration of detectable fecal 
presence of WRSS1 by culture and PCR. 
2.3 Exploratory Objectives 
 
To evaluate serum bactericidal activity and cytokine profile against WRSS1 in toddlers before and 
after vaccination. 
 

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3.0 BACKGROUND OF THE PROJECT INCLUDING PRELIMINARY OBSERVATIONS AND 
SCIENTIFIC RATIONALE 
Provide scientific validity of the hypothesis based on background information of the proposed study and discuss previous works on the research 
topic, including information on sex, gender and diversity (ethnicity, SES) by citing specific references. Critically analyze available knowledge 
and discuss the questions and gaps in the knowledge that need to be filled to achieve the proposed aims. If there is no sufficient information on 
the subject, indicate the need to develop new knowledge.  
3.1 Clinical Significance and Pathogenicity of Shigella1 
Shigellosis is a highly infectious acute diarrheal disease that is transmitted through direct fecal-oral contact or 
through contaminated food or water. Infection with Shigella sp results in a wide range of clinical symptoms 
from mild watery diarrhea to severe diarrhea with dehydration, general colonic invasion and generalized 
toxic inflammation. More than one quarter of these patients progress to overt dysentery manifested as 
multiple liquid stools often containing frank blood, with systemic symptoms such as fever and/or abdominal 
pain [2]. In the nineteenth century and early twentieth century, outbreaks of dysentery were often 
accompanied by >20% mortality rates from dehydration, shock, and/or hemolytic uremic syndrome [3]. A 
review of scientific literature published between 1966 and 1997 projected an annual incidence of over 160-
million shigellosis cases worldwide with over 1 million deaths [4]. February 2009 estimates from the WHO 
estimate 90 million episodes of shigellosis cases worldwide and 108,000 deaths [5] although data from most 
of Africa and many parts of SE Asia were not available. Even though the fatality rate for shigellosis has 
decreased in recent decades, infections are still common in areas with substandard sanitation and continue to 
be significantly underestimated. The increasing spectrum of Shigella antimicrobial resistance mandates 
continued public health interventions and vaccine development efforts [2,6].  
Shigella infections are widespread in less developed countries (LDCs). Studies in Pakistan [7] and 
Mozambique [8] identified Shigella in 4.8% and 6.7%, respectively, of cases of diarrhea compared to 0.4% in 
healthy controls. Similar estimates have been obtained in studies in 6 Asian countries, where Shigella was 
isolated from 5% of all diarrhea cases occurring in children under 60 months [2]. Using the more sensitive 
real-time PCR with Shigella-specific ipaH (the gene encoding invasion plasmid antigen H that is nearly 
exclusively derived from Shigella spp) primers detected Shigella spp in 33% of culture-negative stool 
specimens [2]. Another study in Vietnam using ipaH based PCR method also suggested that about 36% of all 
cases of watery diarrhea in LDCs may be attributable to shigellosis [9], and a more recent similar study in 
China has produced estimates of about 58% [10]. However, large population based data in Bangladesh 
comparing PCR method with the stool culture method to detect presense of Shigella is not available. Recent 
studies using molecular methods (PCR-Luminex strategy, multiplex real-time PCR panels, and customised 
TaqMan array card) revealed much higher infection status of children <5 years of age with Shigella and other 
enteric pathogens [11-13]. Ongoing multicenter studies of diarrheal disease burden, clinical manifestations, 
and etiology in sub-Saharan Africa will give an improved picture of the current impact of shigellosis in the 
most impoverished LDCs.  
There are four major serogroups of Shigella, namely, S. dysenteriae, S. flexneri, S. boydii, and S. sonnei. 
There is only one serotype of S. sonnei, while each of the other serogroups contain multiple serotypes and 
subtypes, and at least 54 serotypes or subtypes are currently recognized, while new serotypes continue to be 
identified. All estimates cited in this study are based on information collected from different sources and 
different time points, which includes individual studies carried out by icddr,b and the Diarrhoeal Disease 
Surveillance Systems (DDSS) at the Dhaka and Matlab Hospitals. These estimates may vary from one study 
to  another. In addition, we have tried to gather Shigella prevalence data from Bangladesh, neighbouring 
countries and other regions of the world to emphasize on the importance of this study.  
In a large multicenter study of shigellosis in six Asian countries during 2000-2004, Shigella was isolated 
from 5% of all diarrhoea episodes detected, among which S. flexneri was the most frequently isolated in 5 
countries (68%) followed by S. boydii and S. sonnei. In Thailand, S. sonnei was most frequently detected 
(about 85 %) [2]. The study found that shigellosis occurs in these 6 Asian sites at a rate 100 times higher than 
in industrialized countries. Rates of shigellosis were higher in Bangladesh sites followed in declining order 
by China, Pakistan, Indonesia, Vietnam and Thailand sites. 
Over the last 35 years, S. flexneri has been the dominant species (61%) followed by S. boydii (17%), S. 
sonnei (10%), S. dysenteriae (8%), and Shigella-like organism (3%) in Bangladesh  except during 1993-95 
when S. dysenteriae was dominant .  

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Figure 1: Yearly overall isolation of Shigella by Sero-Groups in Dhaka Hospital, 2008-2011 
 
However, in the last decade, the prevalence of S. sonnei increased from 8% in 2000 to 27% in 2010 while the 
prevalence of S. dysenteriae drastically reduced to 3% in 2010. Interestingly, since 2004, S. dysenteriae type 
1 is gradually on the decline and was not found at all in Bangladesh. S. sonnei is now a significant cause of 
diarrhoeal infection in Bangladesh constituting about 27% of all Shigella cases and is second to S. flexneri 
(49%) infections [1,14]. 
According to the Dhaka Hospital surveillance report of 2011, prevalence of shigellosis is 3% in Dhaka; 
among the total Shigella  cases S. sonnei is 2nd to S. flexneri cases and is quite high (Figure 1). S. sonnei 
cases have been steadily increasing over the years (from 4-9% in the period 1980-2008 to about 27% in 
2011-2012) [15]. Among children under 5 years of age, the prevalence rate has also increased from 11% to 
40% in 2011. 
Data from study sites in Mirpur (situated about 10.9 km from icddr,b), Matlab (104 km from Dhaka) and 
Mirzapur (72 km from Dhaka) have been provided below. 
Figure 2: Yearly overall isolation of Shigella by Sero-Groups in Mirpur Treatment Centre, 200914 
 
In the Mirpur Treatment Centre, the percentage of isolation of S. sonnei has been steadily increasing and has 
superseded S. flexneri isolation in 2011 (Figure 2). Among children <5year, the isolation rate was substantially 
higher for S. sonnei (53%) compared to S. flexneri (18%) in 2011.  
The GEMS study conducted in Mirzapur by Dr ASG Faruque showed that the overall isolation of Shigella 
was 43%. In this study, mild diarrhea cases were excluded while moderate to severe diarrhea cases were 
enrolled. From 2008 to 2010, isolation of S. sonnei steadily increased from 19% in 2008 to 38.5% in 2010. It 
was second to S. flexneri cases which gradually declined from 72% to 57.7% from 2008 to 2010. Among 
children under 5 years of age, S. sonnei isolation rate was higher (25.2%) than S. flexneri (21.3%). However, 
even among moderate to severe diarrhea cases in the GEMS study, the isolation rate of S. sonnei was higher 
than S. flexneri in these children. 
Another Diarrheal Disease Burden Study (2010-2011) was recently completed by Dr ASG Faruque in 
Mirzapur, which covered all diarrheal cases (mild, moderate, severe) including watery diarrhea and dysentery 
among all age groups. This study (Figure 5) also showed that the isolation of S. sonnei was quite high (25%) 
and was 2nd to S. flexneri isolation rates which was 53.7% [16]. Similarly, among children <5 years old S. 
58
9
25
9
59
7
26
9
55
22
17
6
48
25
16
10
0
10
20
30
40
50
60
70
   S. flexneri
   S. sonnei
   S. boydii
   S. dysenteriae
% Isolation
2008
2009
2010
2011
55
10
24
10
53
16
26
5
29
33
31
7
0
10
20
30
40
50
60
   S. flexneri
   S. sonnei
   S. boydii
   S. dysenteriae
% Isolation
2009
2010
2011

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sonnei isolation was high (Figure 3) though interestingly S. boydii also showed a dramatic increase within 1 
year from 3% to 28%. 
Figure 3: Yearly isolation of Shigella by sero-groups among children < 5 yrs old, Mirzapur, 2010-2011 
 
The prevalence of shigellosis is 5% in Matlab according to the Matlab Hospital surveillance data of 2011. It 
shows that the highest isolate was S. flexneri (73% to 81%) which remained steady for 4 consecutive years 
(2008 to 2011) while S. sonnei was the 2nd with a lower rate at 7-8%. The prevalance among children <5 
years old was similar with slightly increased rates in children <5 (11-15%). 
There are no recent surveillance data from field sites. However, data from Dhaka and Matlab Hospitals of 
icddr,b (Jan. 2013 – Sep. 2015) (Table 1) show that the prevalence of Shigella infection in Dhaka Hospital is 
2.3% (total enrolled 3355) while in Matlab Hospital it is 8.6% (total enrolled 7116). Interestingly, the 
isolation of S. Sonnei among patients was highest in 12-24 month old children both in Dhaka and in Matlab. 
Table 1A. Shigella isolation among different age groups  
 
 
Dhaka Hospital 
 
Matlab Hospital 
Age group 
S. flex 
S. boydii 
S. sonnei 
S. dys 
 
S. flex 
S. boydii 
S. sonnei 
S. dys 
0-11 m 
9 (31.0) 
5 (17.2) 
14 (48.3) 
1 (3.4) 
 
18 (85.7) 
2 (9.5) 
1 (4.8) 
0 (0.0) 
12-24 m 
16 (51.6) 
1 (3.2) 
14 (45.2) 
0 (0.0) 
 
26 (52.0) 
3 (6.0) 
20 (40.0) 
1 (2.0) 
24 – 59 m 
15 (57.7) 
1 (3.8) 
10 (38.5) 
0 (0.0) 
 
51 (75.0) 
2 (2.9) 
15 (22.1) 
0 (0.0) 
5-9 yr 
6 (50.0) 
2 (16.7) 
4 (33.3) 
0 (0.0) 
 
9 (90.0) 
1 (10.0) 
0 (0.0) 
0 (0.0) 
> 9 yr 
45 (62.5) 
11 (15.3) 
12 (16.7) 
4 (5.6) 
 
90 (70.9) 
10 (7.9) 
22 (17.3) 
5 (3.9) 
Table 1B. Isolation of S. sonnei among age groups  
 
Dhaka Hospital 
Matlab Hospital 
Age group 
n  (%) 
n  (%) 
0-11 m 
14 (25.9) 
1 (1.7) 
12-24 m 
14 (25.9) 
20 (34.5) 
24 – 59 m 
10 (18.5) 
15 (25.9) 
5-9 yr 
4 (7.4) 
0 (0.0) 
> 9 yr 
12 (22.2) 
22 (37.9) 
The publication by Kathryn Holt in Nature Genetics [17] indicates that antibiotic treatment and better 
sanitation alone will not be sufficient for controlling shigellosis because antibiotics are driving the evolution 
of Shigella strains and are not a long-term solution for the elimination of this global health problem. These 
data highlight the socio-economic development of Bangladesh that may have an impact on the epidemiology 
of Shigella spp and should guide future vaccine development strategies. While the Holt paper describes the 
evolution of S. sonnei, the hypothesis should apply to all Shigella. The conclusion is that vaccine 
development will be vital for containing the disease. Since all S. sonnei have the identical O-antigen, 
protection with a single vaccine is achievable. In contrast the control of S. flexneri may require coverage of 2 
or more S. flexneri serotypes, such as 2a, 3a and 6.   
61
31
3
5
46
26
27
3
0
10
20
30
40
50
60
70
   S. flexneri
   S. sonnei
   S. boydii
   S. dysenteriae
% Isolation
2010
2011

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3.2 WRSS1 as a prototype live attenuated S. sonnei vaccine for descending age study 
Parents and public health authorities recognize dysentery as a threat to child health and development in 
LDCs. While shigellosis is common at any age in developing countries, it is in infants and young children 
where the consequences of infection are most severe. Early childhood diarrheas, such as shigellosis, are 
associated with substandard growth and physical fitness [18] and with impaired cognitive function[19] during 
the first decade of life. A vaccine against shigellosis would be in demand for childhood immunization 
programs in many LDCs [20]. 
Most experts agree that the optimal Shigella vaccine will provide protection against multiple Shigella 
serotypes. The likely candidates for a multivalent vaccine include S. dysenteriae type 1, S. sonnei and several 
S. flexneri serotypes. S. sonnei is considered a critical component because S. sonnei is the main serogroup 
found in industrialized countries [2,3]. S. sonnei, although only responsible for about 10% of episodes, 
remains an important cause of morbidity and mortality in less developed countries. Recent studies at icddr,b 
suggest a rapidly increasing trend for S. sonnei infections in Bangladesh (icddr,b Surveillance reports; 
unpublished data by Dr ASG Faruque; see section 3.1).   
To accelerate the development of safe, effective, and affordable vaccines against Shigella, PATH Vaccines 
Solutions (PVS) is working with private- and public-sector partners throughout the world. PVS is pursuing a 
wide range of promising vaccine approaches and related research, in an effort to make Shigella vaccines 
available to children who live in endemic countries as quickly as possible.  Live attenuated Shigella vaccines 
offer a promising option based on earlier field studies of streptomycin dependent strains and other vaccine 
candidates [21].  Since that time, advances in molecular biology techniques made it possible to create 
attenuated vaccines by the introduction of well-defined, stable deletions on the Shigella virulence plasmid.  
WRSS1, the ΔvirG live attenuated S. sonnei vaccine developed by scientists at WRAIR [22], has proven so 
far to be a promising prototype candidate based on its history of safety, immunogenicity and stability. 
WRSS1 is intended to be an initial component of a trivalent Shigella vaccine, with similarly attenuated S. 
flexneri 2a and S. flexneri 3a vaccine candidates to be added over the course of the next few years. Currently, 
PVS is partnering with WRAIR and the icddr,b to evaluate prototype WRSS1 in a descending age study in 
Bangladesh, and will serve as the study sponsor. 
We recently completed protocol PR# 12054 (VAC 008) at icddr,b. The adult cohort was completed first and 
then children 5-9 years old. In the adult cohort 30 adults received 1 or 3 doses of WRSS1 and 9 received 
matched placebo. In the childrens cohort 48 children received 1 or 3 doses of WRSS1 and 16 received 
matched placebo. WRSS1 was safe at dose levels ranging from 103 to 106 CFU in both adults and children. 
Most relevant, all of the symptoms in children were rated as mild. None of the children excreted the vaccine 
strain, however, S. sonnei specific immune responses were observed in most children in the two high dose 
groups (105 and 106 CFU) (see section 3.4.2.4). Because the vaccine is both safe and immunogenic, it is 
justified to test the vaccine in the target population for the vaccine i.e. children 12-24 months old.   
3.3 Development of a ΔvirG S. sonnei Vaccine strain WRSS1  
Live attenuated S. sonnei WRSS1 vaccine was constructed from parent strain Moseley by making a 212-bp 
deletion in virG (also known as icsA) [22]. The parent strain was isolated from a laboratory worker at 
WRAIR accidentally infected in 1975 with a laboratory S. sonnei strain. The person experienced a classic 
case of shigellosis with fever, severe intestinal cramps, diarrhea and finally dysentery. The worker was 
hospitalized for 3 days and recovered with IV fluids and ampicillin treatment. The isolated strain was 
lyophilized and stored in ampoules as S. sonnei Moseley strain in the laboratory of Dr Sam Formal. The 
Moseley strain was used as the parent strain primarily because it exhibited a stable Form 1 phenotype (>90% 
smooth colonies) when incubated on TSA plates overnight at 37C [22]. More recently genomic sequencing 
of WRSS1 has revealed a chromosomal deletion of length 82 kb that occurred spontaneously during the 
construction of the strain. This deletion does not affect bacterial invasion of cultured cells, intracellular 
replication or colonization and immunogenicity in a rhesus monkey model of infection [23,24]. At 104 CFU, 
WRSS1 was safe and highly immunogenic in US and Israeli volunteers (see below).  
S. sonnei WRSS1, like its parent strain Moseley, is invasive as indicated by the HeLa cell invasion assay. 
Unlike Moseley however, the vaccine is negative for the Sereny test and does not form plaques in tissue 
culture monolayers, indicating loss of ability to spread intracellularly and intercellularly.  S. sonnei WRSS1 

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develops predominantly form I colonies (>90%) and agglutinates with S. sonnei O-specific antiserum, 
indicating retention and expression of the virulence plasmid that carries the O-antigen encoding genes. 
A WRSS1 research seed from Hartman and Venkatesan [22] was manufactured as a lyophilized vaccine 
under current Good Manufacturing Practice (cGMP) at the WRAIR Pilot BioProduction Facility in Silver 
Spring, Maryland. Frozen Master Cell Bank seeds and Production cell Bank (PCB) seeds were manufactured 
in April 1997, and a frozen PCB seed was used for bulk vaccine production in June 1997. The bulk vaccine 
was grown in a 40-liter fermenter using Tryptone-Yeast Extract broth, and the resulting bacterial pellet was 
suspended in a cryopreservative composed of 7.5% Dextran T10, 2% sucrose, and 1.5% glycerol in 
Dulbecco’s phosphate buffered saline (PBS), for lyophilization. All bovine animal products used in WRSS1 
manufacture were derived from herds of the United States, Australia, or New Zealand. The final cGMP 
product (lot 0451) was lyophilized in 377 multi-dose vials and frozen at -80°C on 27 June 1997. Lot 0451 has 
consistently yielded 1-2 x 1010 CFU per vial when reconstituted with 5 mL deionized water. The cultured 
colonies are approximately 90% form l, indicating that the vaccine expresses a complete (smooth) LPS O-
polysaccharide and is invasive. Lot 0451 passed the General Safety Test (i.p. injection of guinea pigs and 
mice) and the product did not cause keratoconjunctivitis in the guinea pig cornea (Sereny test). The cGMP 
vaccine also elicited protection against keratoconjunctivitis after two ocular immunizations of guinea pigs 
[22]. This protection was equivalent to that induced by the WRSS1 research seed harvested from trypticase 
soy agar (TSA) plates.  
Reversion of the mutant vaccine to wild type: The bacterial vaccine strain WRSS1 was constructed from 
parent wild type strain Moseley by making a 212-bp deletion in virG, a plasmid coded virulence gene. A 212 
bp gene deletion in bacteria is usually considered irreversible since the only way that it can revert is to 
recombine with a wild-type virG gene carried on the virulence plasmid. The virulence plasmid in Shigella is 
non-conjugative [25,26], meaning it cannot transfer itself from one bacteria to another without the help of a 
conjugative plasmid or helper plasmid. So, natural reversion of the virG deletion in WRSS1 cannot happen.  
3.4 Previous Vaccine studies  
3.4.1 Preclinical studies with Shigella sonnei live attenuated vaccine (WRSS1) 
For efficacy studies in guinea pigs, ocular immunizations with 3 x 108 to 4 x 108 CFU of S. sonnei WRSS1 
were performed on days 0 and 14, followed by challenge with 4 x 108 CFU of virulent strain S. sonnei 53G 
on Day 42 [22].  The S. sonnei 53G strain was isolated in 1954 from a 5 year old patient hospitalized with 
diarrhea in Tokyo, Japan and maintained at the CVD, Univ of MD. A Master Cell bank (Lot# 0593) was 
manufactured from a seed stock from the CVD at the WRAIR Pilot Bioproduction Facility. Glycerol stocks 
of 53G made from lot# 0593 were used for these animal studies. Guinea pigs immunized with WRSS1 and 
challenged with 53G were rated over a 5 day period as to time of development and severity of disease. 
Percentage of protection was defined as follows:  full, percentage of eyes with a rating of 0; partial, 
percentage of eyes with a rating of 1.  In animals immunized with S. sonnei WRSS1 grown on solid medium 
overnight, 11/16 eyes showed no signs of disease (69% complete protection) while 5 eyes showed mild 
conjunctivitis (31% partial protection).  When reconstituted WRSS1 vaccine Lot # 0451 was used as the 
immunizing agent, 10/16 eyes did not develop disease (63% complete protection) while 4 eyes developed 
mild disease (25% partial protection).  No significant difference was found in the levels of protection 
conferred by the two formulations. Immunogenicity of the vaccine was measured in guinea pigs by 
determining levels of serum immunoglobulin G (IgG) and IgA specific for S. sonnei O antigen.  The 
geometric mean titers demonstrated that both formulations were immunogenic and produced comparable 
serum IgG and IgA titers against the O antigen [22]. 
3.4.2 Previous human use of WRSS1 
3.4.2.1 Phase 1 Inpatient Study (Protocol No. 98-035) 
Two double-blinded, placebo controlled trials were conducted with lot# 0451 under IND 8266 (Protocol No. 
98-035) in which 27 healthy adult participants, aged 18-45, received WRSS1 at the Center for Vaccine 
Development (CVD), University of Maryland, Baltimore, MD.  In the first trial, three vaccine inocula (3 X 
103, 3 X 104, and 3 X 105 CFU) were evaluated.  In the second trial (conducted under an amendment of 
Protocol 98-035) additional participants received 3 X 105 CFU or 3 X 106 CFU [27].   

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WRSS1 was well tolerated by the 27 participants who ingested doses ranging from approximately 3.8 X 103 
to 3.6 X 106 CFU in that the only presumptive reactions characterized as “severe” were headaches reported 
by two vaccinees. Four participants, (one in the 3-log group and three in the 5-log group) evidenced transient 
fever of 37.8 C (100.00F) or greater).  Although six participants (22%) developed objective criteria of illness 
(fever and diarrhea), these illnesses were not debilitating, were transient, and did not occur in a clear dose-
related fashion.  No volunteer had dysentery or fever (≥ 38.9°C (102F)) or clinically significant laboratory 
abnormalities. 
One adverse event that was considered probably related to vaccine administration involved arthralgia in one 
subject.  This consisted of a complaint of arthralgia of the elbows and fingers on Day 1 post vaccination.  The 
arthralgia was minor in severity and was accompanied by mild fever and loose stools.  This event resolved by 
24 hrs and did not require any treatment or action.   
Table 2.  Rate of Clinically Relevant Symptoms and Rate of Any Symptom Likely to be reported 
during an Inpatient Trial 
Inoculum 
(CFU) 
No. 
of 
particip
ants 
Loose 
stool 
Fever (°F) 
Head-
ache 
Cramps Clinical 
symptomsa 
Any 
symptom 
None 
7 
2 
0 
0 
0 
0 
2 (29%) 
3x 103 
7 
3 
1 (101.2) 
3 
2 
1 (14%) 
4 (57%) 
3 x 104 
4 
0 
0 
1 
0 
0 
1 (25%) 
3 x 105 
10 
2 
2 (100.7, 100.8) 
1 
2 
3 (30%) 
3 (30%) 
3 x 106 
6 
2 
0 
1 
1 
2 (33%) 
4 (66%) 
a Two or more loose stools totalling at least 200 ml in 48 hrs, a single loose stool totalling at least 300 ml, dysentery 
(gross blood in stool) or fever of at least 100°F. 
Excretion of WRSS1 by vaccinees in the phase 1 trials was indicated by a clear evidence of intestinal vaccine 
replication as demonstrated by recovery of 104 to 107 CFU of WRSS1 per gram of stool in 22 of 27 (82%) 
vaccinees.  Vaccine was still being excreted by 66% of vaccinees when antibiotic treatment began (study day 
7).  WRSS1 was not detected in the stools of placebo controls who shared living quarters and toilet facilities 
with vaccinees. A single dose of WRSS1 induced a vigorous immune response including IgA anti-LPS ASC 
responses at levels which have been correlated with protection against shigellosis in participant challenge 
studies. These promising results suggested that WRSS1 should undergo further clinical development.   
3.4.2.2 Phase 1 Outpatient Study (Protocol No. A-11538) 
A second phase 1 dose-escalating, open label trial “Outpatient Study of Safety, Immunogenicity and 
Transmissibility of a Live-Attenuated  ΔvirG(icsA) Shigella sonnei Vaccine (strain WRSS1), Given as a 
Single Dose of 103, 104 or 105 Colony Forming Units to Healthy Adult Israeli Volunteers” was conducted 
under IND 10254 in Israel (Protocol No. A-11538) [28]. Three groups of 15 participants were enrolled as 
outpatient vaccinees. Each group received one dose of approximately 103, 104 or 105 CFU of the live 
attenuated S. sonnei vaccine WRSS1 at Simbec-Tel-Aviv Sourasky Medical Center Clinical Research Center 
[24]. There was no control group.  
In the two lower doses tested (5 x 103 CFU and 2 x 104 CFU) the vaccine was well tolerated with the most 
common complaint being mild abdominal pain followed by mild nausea.  At the lower doses only one of 30 
volunteers reported moderate diarrhea while 5 volunteers had mild diarrheal symptoms.  Among the 
vaccinees receiving 105 CFU there was a tendency to develop more fever, headache and anorexia. 
Table 3.  Rate of Clinically Relevant Symptoms after Vaccination Protocol No. A-11538 
Symptom 
No. of volunteers/total no. (%) 
 
103 and 104 CFUa 
105 CFU 
Any soft stool 
17/30 (57) 
10/15 (67) 
Any liquid stool 
6/30 (20) 
4/15 (27) 
Moderate diarrhea (>two liquid stools/24 h) 
1/30 (3) 
4/15 (27)b 
Fever (≥100°F) 
0/30 (0) 
2/15 (13) 
Total with moderate or severe gastrointestinal signs or symptomsc 
5/30 (17) 
5/15 (33) 

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a More signs and symptoms were reported by volunteers who ingested the 103-CFU dose than by volunteers who 
ingested the 104-CFU dose, and the data 
for these two doses were combined for the safety analysis.  
b The frequency of diarrhea significantly increased with the 105-CFU dose compared to the 103- and 104-CFU doses 
(P=0.036). 
c Diarrhea, nausea, or abdominal pain. 
 
More than 70% of the vaccinees excreted the vaccine for a period ranging between 1 and 23 days.  However, 
there was no detection of transmission of the vaccine strain to close contacts.  About 80% of the vaccinees 
receiving any of the doses showed a specific ASC response to the vaccine strain and there were no significant 
differences in the rate of the specific ASC response among the three groups receiving either 103 or 104 or 105 
CFU.  It was concluded that WRSS1 is a promising vaccine candidate when given to healthy adults in a range 
of 103 to 104 CFU.  Further placebo-controlled studies are needed to establish a solid safety record and to 
evaluate further the immunologic potential of the vaccine. 
3.4.2.3 Preliminary report on Phase 1b-2b Inpatient Study (DMID Protocol 09-0021) [29] 
A phase 1 and phase 2b blinded, placebo-controlled inpatient trial entitled “Safety, Immunogenicity and 
Efficacy Studies of WRSS1, a Live Attenuated Shigella sonnei Vaccine Candidate in Healthy Thai Adults” 
was conducted under IND 10254 (DMID Protocol No. 09-0021).  The study was conducted at the Vaccine 
Trial Centre, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok 10400 
Thailand (Punnee Pitisuttithum, MD, P.I.).  Coordination and laboratory support was provided by the Armed 
Forces Research Institute of Medical Sciences (AFRIMS), 315/6 Rajvithi Road, Bangkok 10400 Thailand 
(Ladaporn Bodhidatta, MD, co-P.I.).  The study was part of a NIAID, NIH grant to AFRIMS (COL Carl J. 
Mason, PI.).  Nineteen volunteers (20 - 40 years old) participated in a phase 1b trial that was initiated on 31 
May 2010 [29].  Two months later, ten volunteers who had received the vaccine in phase 1b, in addition to 10 
naïve controls, were recruited for the phase 2b trial where they were challenged with a virulent S. sonnei 
strain 53G [29].  All volunteers resided in the Mahidol University Vaccine Trial Centre during the inpatient 
phases of both studies.  The volunteers were treated with ciprofloxacin for elimination of the vaccine strain 
on study day 8 in phase 1b and on study day 5 for the S. sonnei challenge strain in phase 2b. 
In the phase 1b trial, all volunteers ingested a bicarbonate buffer solution (2 g of sodium bicarbonate in 150 
ml of water) shortly before receiving the vaccine or placebo to neutralize gastric acidity.  Thirteen volunteers 
ingested 1.49 x 104 CFU of WRSS1 lot 0451 suspended in 30 ml of water, and 6 placebo control volunteers 
ingested an equal volume of water without vaccine.  All adverse events potentially related to ingestion of 
WRSS1 (or placebo) were mild, and there was no difference between the number of adverse events in 
vaccinees and placebo control groups (Table 4).  WRSS1 was detected by culture in the stools of 3 vaccinees 
(23%) and in none of the controls.  Nine of the 13 vaccinees (69%) had evidence of intestinal colonization 
with WRSS1 by culture or by PCR using primers that amplified a product which is unique to the vaccine.  
Antibody responses in 5 vaccinees (38%) were detected as IgA or IgG ELISA seroconversion against S. 
sonnei LPS.  ASC and antibody lymphocyte supernatant assays (ALS) increased the total to 7 immune 
responders (54%) [29]. No placebo controls had an immune response against S. sonnei LPS. 
Table 4.  Adverse Events (clinically related symptoms) in phase 1b trial of 1.5 x 104 CFU WRSS1 in 
Thai adults. 
Adverse Event 
Vaccines 
(n=13) 
Controls 
(n=6) 
Diarrheal stool 
2 
2 
Fever 
0 
0 
Nausea 
1 
0 
Abdominal pain 
1 
3 
Tenesmus 
0 
1 
Myalgia 
1 
0 
Headache 
1 
1 
Malaise 
0 
1 
Urticaria 
1 
0 
Eosinophilia 
0 
1 

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In the phase 2b challenge trial, 10 vaccinees and 10 controls received 1.7 X 103 CFU of virulent, wild-type S. 
sonnei 53G freshly harvested from TSA plates, diluted to the challenge dose and suspended in 30 ml of water 
after ingesting a bicarbonate buffer solution.  Five volunteers in the vaccine group and 7 volunteers in the 
control group had ≥ 4-fold increases in S. sonnei LPS-specific serum IgA and IgG after challenge with 53G.  
Shigella-related illness was determined by blinded external review of clinical record summaries.  Three 
vaccinees and 5 naïve controls experienced clinically relevant illness [29].  Although there was a trend 
toward vaccine protection in this phase 2b trial, there were insufficient clinical outcomes to achieve a 
statistically significant result. 
Preliminary data from a phase 1b trial conducted in Thai adults showed that a 104 CFU dose of WRSS1 lot 
0451 was well tolerated, but colonization and immune response data also suggested that this population was 
less responsive to the vaccine than were North American or Israeli volunteers.  For example, ingestion of a 
104 CFU dose of WRSS1 resulted in 23% fecal shedding in Thai volunteers versus 70% fecal shedding in 
North American and Israeli volunteers [27,28], and only half of Thai adults had a modest immune response 
against S. sonnei LPS while approximately 75 % of North American and Israeli volunteers had robust 
immune responses against WRSS1 [27,28]. These results probably reflect residual immunity against endemic 
S. sonnei in adult Thai volunteers.  A similar result was seen earlier with SC602, a live attenuated S. flexneri 
2a vaccine candidate, that was immunogenic for North American volunteers but did not colonize or induce 
immune responses in adults and older children in Bangladesh [30].  In previous studies, 105 CFU of WRSS1 
was more immunogenic than 104 CFU in North American and Israeli volunteers [27,28]. Perhaps higher 
doses or multiple doses of WRSS1 will be needed to induce immunity in Thai or Bangladeshi populations. 
Extensive clinical trials of WRSS1 in U.S., Israeli, and Thai adults suggest that this vaccine should undergo 
further clinical evaluation in less developed countries (e.g. Bangladesh), starting with a descending age study 
targeted toward 12-24 month-old infants who have yet to experience initial infections with S. sonnei. 
3.4.2.4 Preliminary Blinded results of Current Study (VAC 008/PR-12054) 
Per the current protocol, WRSS1 has been administered at the 104, 105, and 106 CFU dose levels in adults, 
ages 18-39 years and at 103, 104 105 and 106 CFU dose levels in children, ages 5-9 years old (Table 5). The 
vaccine has been safe thus far at all dose levels in both adults and children, with no vaccine-related serious 
adverse events reported to date. Overall there were fewer reactogenicity events reported in children as 
compared to adults and all reactogenicity events in children were reported as mild. Table 6 lists all solicited 
reactogenicity symptoms observed up to 6 days post-first vaccination and 3 days post-second and third 
vaccination for children (Note: the groups include both vaccinees and placebo recipients as the data remain 
blinded). It is important to note that all participants who experienced loose stool and/or diarrhea during the 
inpatient period tested negative for WRSS1 and other enteric pathogens.  
Table 5. Number of participants receiving live, attenuated S. sonnei vaccine or placebo by WRSS1 dose 
 
 
 
 
Number of participants 
Cohort 
WRSS1 
dose 
(CFU) 
No. 
of 
doses 
Schedule 
(day) 
Vaccine 
(N) 
Placebo 
(N) 
A1 
3 x 104 
1 
0 
10 
3 
A2 
3 x 105 
3 
0, 28, 56 
10 
3 
A3 
3 x 106 
3 
0, 28, 56 
10 
3 
B1 
3 x 103 
1 
0 
12 
4 
B2 
3 x 104 
3 
0, 28, 56 
12 
4 
B3 
3 x 105 
3 
0, 28, 56 
12 
4 
B4 
3 x 106 
3 
0, 28, 56 
12 
4 
Cohort A are adults 18-45 years old, cohort B are children 5-9 years old 

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Table 6.  Number (percent) solicited reactogenicity among children receiving WRSS1 or placebo 
WRSS1 dose cohort, WRSS1 dose/no. of participants 
B1 
B2 
B3 
B4 
3x103 CFU 
3x104 CFU 
3x105 CFU 
3x106 CFU 
Symptom† 
n=16 
n=16 
n=16 
n=16 
Abdominal Pain 
1 (6) 
1 (6) 
0 
3 (19) 
Abdominal Cramps 
1 (6) 
0 
0 
2 (13) 
Headache 
0 
0 
0 
2 (13) 
Light headedness 
0 
0 
0 
2 (13) 
Myalgia 
0 
0 
0 
2 (13) 
Fever 
0 
4 (25) 
0 
1 (6) 
Loose stools (outpatient) 
0 
1 (6) 
3 (19) 
1 (6) 
Nausea 
1 (6) 
0 
0 
1 (6) 
Arthralgia 
0 
0 
0 
1 (6) 
Vomiting 
1 (6) 
0 
0 
1 (6) 
Loose stools (inpatient) 
0 
1 (6) 
0 
0 
Diarrhea (inpatient) 
0 
1 (6) 
0 
0 
† all symptoms were rated as mild on a scale of mild, moderate or severe 
Note: no subject reported bloating, chills, constipation, diarrhea (outpatient), decreased 
appetite, dysentery, excess flatulence, malaise, reactive arthritis 
a Graded from the Reactogenicity Record during the Inpatient Phase. During the inpatient period, diarrhea is defined as 
1, grade 3-5 stool of >300 g; OR >3 grade 3-5 stools totaling >200 g during any 24-hour period within 72 hours after 
investigational product administration. 
b During the outpatient period diarrhea is defined as >3, grade 3-5 stools in a 24-hour period. 
c Severity determined on the basis of stool number, grading and stool weight during the inpatient period  
d Severity determined on the basis of stool number and grading only during the outpatient period 
None of the children excreted the vaccine even at a dose of 106 CFU, while 5 out of 10 adults (50%) excreted 
the vaccine strain at the 106 CFU dose.  
Immune responses as defined as a 4-fold increase in antibody titer were observed at all dose levels (Table 7).  
The highest dose level appeared to provide the highest number of responders, particularly to Invaplex which 
is a combination of LPS and the IpaB and IpaD protein antigens.  Adults demonstrated high-LPS specific IgA 
and IgG responses in ALS and serum, and moderate responses in stool at higher dose levels.   
 
 
 

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Table 7. Immune response to Shigella sonnei LPS and Invaplex antigen in children 5-9 years old after 
three doses (at day 63) of the live, attenuated S. sonnei vaccine or placebo by WRSS1 dose 
 
 
Cohort and WRSS1 dose (CFU) 
B2 
B3 
B4 
Immune 
Type of 
104 
105 
106 
measure 
specimen 
n=14 
n=16 
n=15 
LPS-specific IgA 
ALS† 
0 
1 (7%) 
3 (20%) 
Serum† 
2 (15%) 
4/ (27%) 
4 (27%) 
Stool 
4 (29%) 
7 (44%) 
4 (27%) 
Invaplex-specific IgA 
ALS 
0 
0 
3 (20%) 
Serum 
0 
1 (7%) 
5 (33%) 
Stool 
5 (36%) 
5 (31%) 
4 (27%) 
LPS-specific IgG 
ALS 
0 
0 
3 (20%) 
Serum 
0 
1 (7%) 
2 (13%) 
Invaplex-specific IgG 
ALS 
0 
0 
5 (33%) 
Serum 
0 
0 
1 (7%) 
(Note: presented groups include both vaccinees and placebo recipients as the 
data remain blinded).   
† Blood could not be obtained from one child each in B2 and B3 cohorts. 
 
In the cohort (n=15) that received the highest dose of 106 CFU (B4), 10 participants responded in either 
serum or ALS, 5 participants did not respond in either serum or ALS, and one subject was lost to follow up. 
Considering that 12 participants out of the 15 participants received the vaccine, a response rate in 10 (83%) 
would be quite good if all responders were in the vaccine group.  Of note, only 2 of 6 participants who 
responded in ALS had a serum response and of 6 participants who responded in serum, only 2 of those 
responded in ALS.  There were an additional 2 participants that had a 4-fold increase in immune response 
detected in the stool who did not respond in either serum or ALS (Table 8).   
Table 8.  Serum immune response to LPS or Invaplex after 3 doses of WRSS1 vaccine by dose cohort 
among children 5 to 9 years old, icddr,b (blinded) 
Study design 
No. of participants 
No. of participants with ≥ 4-
fold increase in titer 
Cohort 
WRSS1 
dose 
No. of 
doses 
Schedule 
(day) 
Vaccine 
Placebo 
Missing 
serology 
LPS 
Inva- 
plex 
Either 
antigen 
B2 
3 x 104 
3 
0, 28, 56 
12 
4 
2 
2 
2 
3 
B3 
3 x 105 
3 
0, 28, 56 
12 
4 
1 
6 
6 
8 
B4 
3 x 106 
3 
0, 28, 56 
12 
4 
1 
8 
10 
10 
Note:  IgA and IgG antibody to LPS and Invaplex antigens was measured by ALS and serum 7 days after each dose 
administration.  
 
 
 

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Table 9.  Fold increase in titer from pre-vaccination (day 0) to post third dose (day 63) for 
cohort B4 (children receiving 106 CFU WRSS1 or placebo).   
 
ALS 
Serum 
Stool 
ID 
IgA  
LPS 
IgA  
INV 
IgG  
LPS 
IgG 
INV 
IgA  
LPS 
IgA  
INV 
IgG  
LPS 
IgG 
INV 
IgA  
LPS 
IgA  
INV 
2120 
6 
6 
7 
20 
3 
2 
0 
1 
26 
9 
2141 
1 
2 
2 
8 
1 
1 
0 
1 
0 
0 
2142 
2 
2 
1 
10 
2 
2 
0 
1 
1 
1 
2148 
1 
1 
1 
1 
2 
1 
1 
1 
6 
6 
2149 
1 
1 
1 
2 
1 
1 
1 
2 
8 
5 
2153 
1 
1 
1 
1 
138 
253 
3 
1 
1 
1 
2072 
1 
1 
1 
1 
293 
160 
5 
3 
3 
3 
2112 
1 
1 
1 
1 
1 
1 
340 
1 
0 
1 
2160 
1 
1 
1 
2 
1 
159 
3 
8 
0 
0 
2165 
12 
5 
12 
3 
7 
4 
<4   
<4   
0 
0 
2166 
1 
1 
1 
4 
1 
1 
2 
2 
1 
1 
2167 
18 
11 
14 
4 
546 
4 
<4   
1 
17 
16 
Non responders 
 
 
 
 
 
 
 
 
2145 
1 
1 
1 
2 
1 
1 
2 
1 
0 
0 
2147 
1 
1 
1 
2 
0 
0 
1 
1 
0 
1 
2157 
1 
1 
1 
2 
1 
1 
1 
3 
2 
1 
2179 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
In summary, blinded data from VAC 008 reveal that WRSS1 vaccine was safe and immunogenic in adults at 
the highest dose level and in children at the two highest dose levels (3x105 and 106 CFU). Immune response 
was observed to both LPS and invaplex antigens in both blood and stool.  The safety and immunogenicity of 
the WRSS1 vaccine in 5 to 9 year old children at dose levels of up to 106 CFU, justifies moving to toddlers in 
this protocol. 
3.5 Rationale for Current Study and Planned Future Development 
Given the heterogeneous distribution of Shigella species and serotypes, any vaccination approach based on 
O-polysaccharide antigens must depend on multivalent or cross-protective strategies.  S. flexneri 2a and S. 
sonnei antigens would be the basis of any multivalent Shigella vaccine because these two components should 
cover 40% to 75% of Shigella infections in LDCs and over 90% in developed countries [2,4,20].  As an 
initial step to achieving a multivalent Shigella vaccine for optimal coverage, we have started with WRSS1, a 
S. sonnei vaccine with a long history of safety and immunogenicity.  
Previously, a S. flexneri 2a live vaccine candidate SC602 was tested in a randomized, double-blind, 
descending age inpatient and outpatient placebo-controlled trial in Bangladesh. The trial enrolled adults and 
children 8-10 years old [30]. SC602 showed 100% protection against shigellosis in US volunteers [31]. 
Bangladeshi volunteers were given a single oral dose of the vaccine (1 x 104, 1 x 105 or 1 x 106 CFU) and 
monitored for vaccine excretion and immunogenicity [30].  Adults were given 120 ml and children given 75 
ml of bicarbonate solution prior to vaccine administration. None of the volunteers had diarrhea and no 
significant or severe symptoms were noted in adults or children. In the initial inpatient trial of 20 adult 
volunteers, SC602 was isolated from 1/5 participants in the 1x105 dosing group, and 2/5 participants in the 
1x106dosing group. Two of 5 participants in the 6-log group also showed 4-fold increases in serum IgG anti-
LPS immune responses, even though pre-vaccination antibody titers were high in this population [19]. None 
of the children excreted the vaccine strain and no immune responses were noted. Lessons learned from the 
SC602 trial includes the need to further modify vaccine dose and regimen for endemic populations, as what 
has worked in naïve volunteers may not be suitable for endemic populations.  Some of the factors that affect 
colonization of live vaccines and immune responses in LDC’s include, but are not limited to, the actual dose 
given and the number of doses, volumes of buffer and buffer composition for vaccine delivery, particularly in 
children, preexisting background immunity, differences in gut physiology and microbial composition, role of 
maternal antibodies in breast milk, and macro and micronutrient deficiencies.  Some of these parameters can 

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be tested using vaccine candidates, such as WRSS1, that has demonstrated good safety and immunogenicity 
in immunologically naïve volunteers.  
The proposed study builds upon successful preliminary observations with this vaccine in the US, Israel and 
Thailand and now in Bangladesh. While secondary objectives include studying the immunogenicity of the 
WRSS1 vaccine, the primary goal of the current trial is to establish a clear safety profile for the WRSS1 
vaccine in toddlers 12-24 months. Blinded data from the current trial in Bangladeshi adults and 5-9 year old 
children (See Section 3.4.2.4) suggest that 106 CFU would be the minimal threshold dose in these two groups.  
It is thought that the target population in the current study would have a lower immunity to the organism than 
seen in these older groups, so 106 CFU could be effective in this group without achieving a dose excess that 
may have safety considerations. This protocol proposes to evaluate WRSS1 in toddlers 12-24 months old. 
The primary and secondary hypotheses will remain the same as the previous study. The toddler study seeks to 
examine critical variables to maximize immune responsiveness in young children, such as buffer volumes, 
dose number and dose levels for improved vaccine delivery and increased immunogenicity, using approaches 
compatible with routine immunization practices.  WRSS1 vaccine is likely to have a more profound impact 
on the prevention of severe life threatening diarrhea in children than in adults, and therefore there is great 
urgency and interest in the prompt development of WRSS1 for use in infants and toddlers.  The WRSS1 
vaccine has been demonstrated to induce similar levels of immune responses to those observed during 
convalescence from natural infection.  
We know from previous human trials of virG-deleted vaccines that this strategy is powerful and that the loss 
of virG significantly attenuates the strain. This knowledge has come primarily from repeated clinical trials of 
live oral Shigella vaccine candidates (WRSS1, SC602, WRSd1) [27,31,32]. We have tested the WRSS1 
vaccine in an appropriate number of adults to ascertain maximally acceptable tolerability, followed by studies 
in 5-9 year old children. In this protocol we will proceed cautiously in toddlers by testing the lowest dose (103 
CFU) before moving up to higher doses (up to 106 CFU). Furthermore, all participants in the study will 
receive the first dose of vaccine as in-patients with the dual purpose of obtaining more accurate clinical 
assessments as well as promptly addressing any potential safety issues. If the first dose is well tolerated, the 
second and third doses will be given in the CTU on an out-patient basis. 
The overall long-term strategy for development of a live Shigella vaccine includes defining the formulation 
of buffers and vaccine vehicles that permit low-volume administration and maximum viability of the vaccine 
[33].  Ultimately, the number of needed doses and the identification of a maximally immunogenic, safe dose 
level of this WRSS1 prototype strain must be determined in the target population. Concurrent with this study, 
development and Phase 1 testing of a trivalent (S. flexneri 2a and 3a, S. sonnei) vaccine is being planned. 
Upon completion of this future US trial, the safety and immunogenicity of a novel complete, trivalent live 
attenuated vaccine in the target population will be evaluated for potential advancement to Phase 3 trials. 
3.5.1 Rationale for exploratory objectives 
A better understanding of the relationships between vaccination, immune response, and protection is of great 
interest to researchers. Most of the current vaccines work through antibodies in serum or on mucosal surfaces 
that prevent infection. Both concentration and functional characteristics of antibodies are important for this 
purpose. Vaccine-specific antibodies may correlate with protection or act synergistically with other immune 
functions. Cellular immune response kills or suppresses replication of intracellular pathogens and may also 
act in synergy with antibodies. For some vaccines, true correlates of protection are not known, but only 
surrogates (a useful measurement that is not functional in protection) are available. Though there are no 
definite immune correlates of protection for Shigella vaccines, it is generally believed that protection will 
require a humoral and a mucosal (as well as a memory) response since Shigella is a mucosal pathogen. The 
WRSS1 live, attenuated vaccine contains all the antigens of a wild type Shigella sonnei and induces a similar 
immune response. It is therefore important to characterize each of these arms of the immune response and 
explore for associations with the profile of the adaptive immunity in an attempt to derive correlates of 
protection. 
Functional activity of serum/plasma antibodies after vaccination  
Antibody-dependent, complement-mediated bactericidal killing confers protection against infectious 
microrganisms, and serum bactericidal antibody activity (SBA) has therefore been used as a test for the 
functional activity of serum antibodies and as a surrogate of protection for several vaccines. SBA assay has 

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been used to support meningococcal vaccine licensure [34]. Serum vibriocidal antibodies constitute the best 
correlate of protection against cholera and have been used extensively to monitor the immunogenicity of oral 
and subcutaneous cholera vaccines [35]. With the O-polysaccharide conjugate vaccine candidate, that has 
shown protection in Israeli field studies, protection is believed to be due to the high serum antibody titers to 
Shigella antigens that transfers over onto the mucosal epithelial layer in the gut and prevents the bacteria 
from invasion [36]. Whether it does so by simple binding, opsonophagocytosis and/or bactericidal activity is 
not clear. Therefore functionality of the serum immune response becomes important and the SBA assay is 
one measure of that parameter. Using a modified SBA assay for cholera, a serum shigellacidal activity for S. 
flexneri was previously established at icddr,b [37]. This assay will be appropriately modified to evaluate SBA 
activity in serum/plasma of toddlers immunized with WRSS1. The SBA activity will be correlated with the 
serum IgG and IgA titers to LPS and to Invaplex. 
Role of cytokines  
One of the key features of Shigella pathogenesis is the release of mature interleukin-1β (IL-1β) and 
interleukin-18 (IL-18) from the resident macrophages of the rectal mucosa, and subsequent production and 
secretion of a large array of proinflammatory cytokines and chemokines from the mucosal tissue [38]. The 
secretion of these cytokines is part of the activation of innate immunity that is triggered by the interaction of 
bacterial LPS with the TLR4/MD2/CD14+ receptor complex on macrophages and dendritic cells. In patients 
with acute Shigella infections, increased expression of proinflammatory cytokines in the rectal mucosa and 
stool and systemic dissemination in plasma correlated with the clinical severity of the disease [39,40]. 
Although specific antibodies participate in protection against natural Shigella infection, these observational 
studies suggest that cellular immunity also represents a dominant role that eventually leads to the elimination 
of Shigellae from the host.  
The profile of cytokines in serum/plasma, lymphocyte supernatant and stool samples would add great value 
and information not only to the characteristics of the innate immune response achieved in toddlers after oral 
vaccination with a live vaccine but may also explain the levels of humoral and mucosal antibody responses to 
Shigella antigens. 
3.6 Study Site 
Describe the availability of physical facilities at site of conduction of the study. If applicable, describe the use of Biosafety Level 2 and/or 3 laboratory 
facilities. For clinical and laboratory-based studies, indicate the provision of hospital and other types of adequate patient care and laboratory support 
services. Identify the laboratory facilities and major equipment that will be required for the study. For field studies, describe the field area including its 
size, population, and means of communications plus field management plans specifying gender considerations for community and for research team 
members. 
Recruitment, screening, and consenting will take place at the urban field icddr,b site in Mirpur, a suburb of 
Dhaka, Bangladesh. Each vaccine dose will be given in the Clinical Trials Unit (CTU) in the icddr,b campus 
(details of CTU is given under Facilities Available). All participants will gather in the Field Office, from 
where they will be brought to CTU by icddr,b transport. 
In cases where the subject may require hospitalization, the participant can be shifted directly to the Dhaka 
hospital at the main icddr,b campus.  
Vaccine preparation will take place at the INT Laboratory under BSL-2 conditions and transported to the 
CTU for dose administration within 2 hours of reconstitution according to the vaccine preparation SOP.  
4.0 RESEARCH DESIGN AND METHODS 
Describe the research design and methods and procedures to be used in achieving the specific aims of the research project. If applicable, mention the 
type of personal protective equipment (PPE), use of aerosol confinement, and the need for the use BSL2 or BSL3 laboratory for different part of the 
intended research in the methods.. Define the study population with inclusion and exclusion criteria, the sampling design, list the important outcome 
and exposure variables, describe the data collection methods/tools, and include any follow-up plans if applicable. Justify the scientific validity of the 
methodological approach (biomedical, social, gender, or environmental).  
Also, discuss the limitations and difficulties of the proposed procedures and sufficiently justify the use of them.  
This is a single site, double-blind, randomized, placebo-controlled, dose-escalation study that will test the 
WRSS1 vaccine in healthy toddlers (12-24 months old).  The study is designed as a dose escalation study 
comprising four cohorts. In each cohort, the first dose and immediate safety evaluation will be conducted at 
the icddr,b Inpatient Unit (Clinical Trials Unit, icddr,b main campus), where the participants will be admitted 
for observation for 24 hours post-vaccination. Follow-up visits for participants will take place on an 
outpatient basis at the Mirpur Field Office. Second and third vaccinations will take place on an outpatient 

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basis in the CTU. Before enrolling participants in subsequent cohorts to receive a higher vaccine dose, the 
safety data from the previous cohort(s) (through Study Day 7) will be evaluated and reviewed by the Internal 
Protocol Safety Team (IPST) comprised of the study physician, the Medical Monitor from CRO, the principal 
investigator, and the Medical Monitor from PVS. 
Toddlers (12-24 months old) 
This study will enroll a total of 64 toddler participants. 48 toddlers will be randomized to receive vaccine and 
16 will be randomized to recieve placebo. The participants will be divided into four separate ”dose” cohorts 
to be recruited step-wise. Each cohort will include 16 participants, randomized to receive three doses of 
WRSS1 vaccine (12 participants) or placebo (4 participants).  Within each dose level, the study will initially 
enrol a sentinel group of 8 children (6 vaccinees and 2 placebos) while the remaining 8 (6 vaccinees and 2 
placebos) will be enrolled after safety data through Day 7 is reviewed by the Internal Protocol Safety Team. 
The placebo preparation will be bicarbonate buffer. The number and allocation of participants are shown 
below. 
Table 10. Vaccine Dose and Subject Allocation By Cohort 
Dosing 
Group 
 
 WRSS1 vaccine 
particles/mL 
 Dosing schedule 
Number of participants 
Sub-groups 
Vaccine 
group 
Placebo 
group 
C1 
Group I 
3 x 103 
Day 0, 28, 56 
6 
2 
Group II 
3 x 103 
Day 0, 28, 56 
6 
2 
 
C2 
Group I 
3 x 104 
Day 0, 28, 56 
6 
2 
Group II 
3 x 104 
Day 0, 28, 56 
6 
2 
 
C3 
Group I 
3 x 105 
Day 0, 28, 56 
6 
2 
Group II 
3 x 105 
Day 0, 28, 56 
6 
2 
 
C4 
Group I 
3 x 106 
Day 0, 28, 56 
6 
2 
Group II 
3 x 106 
Day 0, 28, 56 
6 
2 
Participants in all 4 cohorts are scheduled to receive three doses of WRSS1 at an increasing dose as described 
in the table or placebo at Days 0, 28 and 56.  The first immunization will be given on an inpatient basis.  If 
after review by the Internal Protocol Safety Team the first dose appears safe and well tolerated, all 
subsequent doses will be administered on an outpatient basis in the CTU.   
For C1, C2 and C3, the Internal Protocol Safety Team will review the first dose data through Day 7 and 
decide to proceed to administration of next 2 doses as well as starting the next higher dose level. After the 
completion of  C2, the icddr,b DSMB will review all available safety data and decide whether to proceed to 
cohorts C3 and C4. At the end of dosing of C4, the DSMB will review the safety and available 
immunogenicity data for all cohorts.   
5.0 STUDY POPULATION 
Participants will be recruited via word of mouth and home visits from Field Staff, and will be screened 
according to the following criteria:  
5.1 Eligibility Criteria 
Inclusion criteria  
1. Male or female children aged between 12 and 24 months of age at the time of vaccination. 
2. General good health as determined by the screening evaluation no greater than 30 days before 
admission.  
3. Father or mother properly informed about the study, able to understand it and sign the informed consent 
form. 
4. Normal bowel habits (< 3 grade 1 or2 stools each day; ≥ 1 grade 1 or 2 stools every 2 days). 
5. Free of obvious health problems as established by medical history and clinical examination before 
entering into the study. 

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6. Parent or guardian available for the entire period of the study and reachable by study staff throughout the 
entire follow-up period. 
7. Signed Informed Consent from the parent.  
Exclusion criteria 
1. Presence of a significant medical condition that in the opinion of the Investigator precludes participation 
in the study. 
2. Infection with HIV. 
3. Presence in the serum of HAV or HEV antibody or HBsAg. 
4. History of congenital abdominal disorders, intussusceptions, abdominal surgery or any other congenital 
disorder. 
5. Participation in research involving another investigational product (defined as receipt of investigational 
product) 30 days before planned date of first vaccination or concurrently participating in another clinical 
study, at any time during the study period, in which the child has been or will be exposed to an 
investigational or a non-investigational product. 
6. Clinically significant abnormalities on physical examination. 
7. Clinically significant abnormalities in screening hematology, serum chemistry as determined by the PI 
or the PI in consultation with the Study Physician.  
8. History of febrile illness within 48 hours prior to vaccination. 
9. Prior receipt of any Shigella vaccine. 
10. Fever at the time of immunization. Fever is defined as a temperature ≥ 37.5C (99.5F) on axillary 
measurement. 
11. History of known shigellosis, chronic diarrhea/dysentery in the past 2 months. 
12. Current use of iron or zinc supplements within the past 7 days; current use of antacids (H2 blockers, 
omeprazol, OTC agents) or immunosuppressive drug. 
13. Allergy to  macrolid and penicillin classes of antibiotics 
14. Clinical evidence of active gastrointestinal illness. 
15. Prior receipt of a blood transfusion or blood products, including immunoglobulins. 
16. Presence of any significant disorder (cardiovascular, pulmonary, hepatic including hepatitis C, renal, 
gastrointestinal, endocrine, immunological, dermatological, neurological, cancer or autoimmune disease) 
as determined by medical history and/or physical examination which would endanger the participant’s 
health or is likely to result in non-conformance to the protocol.  
17. History of any neurologic disorders or seizures or family history of rheumatologic disorders.  
18. Acute disease at the time of enrolment. 
19. Medically significant malnutrition, defined as moderate malnutrition (wt-for-age z-score between           
-3.0 and -2.0) and severe malnutrition (wt-for-age z-score <-3.0 or edema). 
20. Any conditions which, in the opinion of the investigator, might jeopardize the safety of study 
participants or interfere with the evaluation of the study objectives. 
21. Receipt of antimicrobial drugs for any reason or a fever ≥ 38C within 7 days before vaccination. 
22. History of diarrhea during the 7 days before vaccination.  
23. Has any household member(s) who is immunocompromised or under the age of 1 year old. 
24. Culture or PCR positive for any Shigella strain 
6.0 RECRUITMENT, SCREENING, RANDOMIZATION, AND MASKING PROCEDURES  
6.1 Recruitment 
The icddr,b team will develop a plan in order to achieve enrollment of the specified number of children, and 
will track screened candidates along with enrolled participants.  The team will review this plan periodically 
throughout recruitment in order to determine the effectiveness of the plan. Potential alternates will be 
recruited in order to ensure robust enrollment in the case a participant is found to be ineligible the day before 
vaccination.  
Participants for this study will be recruited by word of mouth in the Mirpur field site. Upon identification of 
potential participants, field staff  will visit the households in Mirpur surveillance area to have preliminary 
discussions with parents regarding potential eligibility and participation in the study. The ERC and WIRB-
approved consent forms will be utilized by the field workers throughout the recruitment process to assist in 

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their interactions with parents of potential participants. Parents will be given the informed consent form 
(ICFs) to keep overnight for further discussion with their families. Interested individuals will be invited to the 
Mirpur field office for further discussions where they will receive and sign the ICFs upon their consent.  
6.2 Screening  
Parents of potential participants will be made aware that the screening process may take several visits to 
complete.  Participants will be screened for eligibility based on a screening questionnaire listing the inclusion 
/ exclusion criteria. A signed, dated informed consent document must be obtained by the PI or designee 
before initiating any study specific procedure. The study medical officers will discuss the informed consent 
document in detail with the parents. Group discussions may also take place to orient parent(s) to the study; 
however individual discussions will commence prior to obtaining consent signatures. Informed consent 
documents will be signed once the toddler’s parent has willingly agreed to participate in the study and prior 
to initiation of any screening procedures and data collection. A literacy rate of 40% is expected in the Mirpur 
surveillance area with Bangla being the most commonly spoken language. An impartial witness will be 
present during the informed consent discussions with illiterate parent(s). A person who can only write his/her 
name and cannot read or write in Bangla will be considered as illiterate for the study purposes. The consent 
will outline study procedures, participant expectations, potential risks and benefits, as well as outline the 
compensation plan for their participation. Participants will be compensated based on the number of visits 
they are able to complete. Alternates will also be compensated accordingly.   
According to protocol procedures, a medical history/exam and a series of clinical laboratory tests may be 
completed to rule out occult illness.  These laboratory tests may include, but are not limited to hematology, 
serum chemistries, hepatitis A and E antibodies, HBsAg and HIV in serum. For detecting Shigella spp, stool 
will be cultured and bacterial DNA will be extracted from stool for ipa PCR.  
During screening, participants will be assigned a study number which will be documented on that 
participant’s source documents. If the participant qualifies for the study, he/she will be enrolled into the 
electronic data system with his/her study ID number. Only participants who have met all of the inclusion 
criteria and none of the exclusion criteria will be officially enrolled in the study and entered into the EDC 
database.   
6.3 Randomization 
Randomization will occur manually on the day participants are to receive their first study vaccination, after 
confirmation of eligibility and immediately prior to immunization. Subjects who replace those who vomit the 
first dose within 30 minutes after the administration of study vaccine/placebo will be administered the same 
vaccine/placebo assigned to the subjects being replaced.  
Participants will be assigned to a coded treatment assignment provided by Emmes, and each participant’s 
treatment assignment will be entered into the Emmes AdvantageEDC
SM system after product administration 
has occurred. The unblinded study pharmacist or member of the IP formulation team, who is not involved in 
other study activities (including assessment of safety), will be provided with the treatment assignment codes 
for preparation of the vaccine or placebo to be given to each participant. The unblinded study pharmacist (or 
designee) will maintain the treatment code list in a secure place.  
6.4 Masking procedures 
WRSS1 vaccine and placebo will be provided to participants by randomization. This process will be blinded 
to both the participants and the investigators and the study personnel who are involved in the clinical and 
laboratory evaluation of participants. Only study personnel who will not be involved in these evaluations will 
be assigned to prepare and distribute the vaccine or placebo to each participant according to the 
randomization code. These staff members will not reveal the randomization code to any other study staff 
member or participant.  The vaccine preparation staff will disguise the appearance of the vaccine and placebo 
according to the vaccine preparation SOP. The sealed randomization code will be kept by the designated 
unblinded staff in a locked, secure location.  In case of an SAE or any unexpected circumstance that requires 
breaking a code, the randomization code will be opened by the local DSMB for that particular participant to 
assess the clinical conditions and necessary steps.   

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7.0 STUDY PROCEDURES/EVALUATIONS 
7.1 Clinical Definitions  
Grading system for stools  
Grade 1: hard (normal) 
Grade 2: soft (normal)  
Grade 3: thick liquid   
Grade 4: opaque watery liquid   
Grade 5: clear watery   
Diarrhea:   
 
Diarrhea is defined as ≥ three grade 3-5 stools in a 24-hour period. 
 
If a subject meets the definition of diarrhea, the start of the diarrhea episode will be the time of the 
first grade 3-5 stool in the episode of diarrhea and the end of the diarrhea episode will be the time of 
the last grade 3-5 stool. 
 
Diarrhea severity will be determined on the basis of stool number and grading. 
7.1.1 Grading system for diarrhea  
Mild Diarrhea  
 
A case of diarrhea as defined above, which does not meet the case definition of moderate or severe 
diarrhea as defined below. 
 
Moderate Diarrhea  
 
A case of diarrhea as defined above, with 4-5 grade 3-5 stools in 24 hours and/or moderate 
dehydration  
 
Severe Diarrhea  
 
A case of diarrhea as defined above, with >6 grade 3-5 stools in 24 hours and/or severe dehydration 
Dysentery: > grade 3 stools with gross blood (confirmed by study personnel) 
Shigellosis: Temperature > 38.3°C (100.94°F), diarrhea and/or dysentery, and one or more severe intestinal 
symptom, and shedding of any Shigella spp. 
Dehydration: Classification of the severity of dehydration will follow WHO guidance per icddr,b standard of 
care.   
Grading the severity of child adverse events: toxicity grading scale for abnormal values will be based on 
appendices 2 and 3.  
7.1.2 Inpatient Phase (All cohorts – Day 0 to Day 1) 
Participants will be monitored daily during the inpatient phase of the study via examination by a study 
clinician, solicitation of daily progress reports, monitoring of gastrointestinal and systemic signs and 
symptoms, reviewing medical conditions, and noting AEs.  Vital signs will be recorded two times daily, and 
more often as appropriate, based on the clinician’s judgment.  If a participant develops moderate or severe 
diarrhea, vital signs will be measured as necessary for clinical management according to the judgment of the 
clinician. In case of emergency children will be brought to the ICU of Dhaka Hospital.   
All stools passed during the inpatient phase will be collected in disposable plastic containers for grading the 
stool consistency. Frequency of defecation on each day will also be recorded. At least one stool specimen 
from each participant will be used for culture and PCR for WRSS1. If no stool is passed during the day, 2 
rectal swabs will be obtained (2 swabs in BGS for culture and PCR testing). Swabs will be discarded with 
other biohazardous waste. If a participant meets the protocol definition of diarrhea, those stool specimens will 
also be cultured. In addition, all stools passed by study participants while inpatients will be visually inspected 
for the presence of frank blood.  Only stools with apparent frank blood will be tested using microscopy to 
confirm the presence of blood.  Only those stools testing positive by microscopy will be recorded as positive 
for blood. 

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7.1.3 Outpatient Phase  
On Day 28 and Day 56, all participants in Cohorts C1, C2, C3 and C4 will return to the CTU for the second 
and third dose of vaccine or placebo (assuming the initial dose is deemed safe).  On Days 7, 35, 63, and 84, 
participants will return to the Field Office for follow-up visits as described in Section 8.6.  
7.2 Concomitant Medications/Treatments 
Current use of immunosuppressive drugs and antimicrobial agents is unacceptable for participation in this 
study as indicated in the exclusion criteria.  
Oral rehydration solution (ORS) is not considered a concomitant medication. Parents will be encouraged to 
give ORS to participants with diarrhea; or other replacement fluids may be given when needed. 
Antimicrobial drugs for treatment of shigellosis and for long-term shedding of WRSS1 as per protocol 
Section 7.2.1, will also not be considered as concomitant medication. The medicine and treatment to be used 
in the study will be based on the judgment of the attending clinicians.   
7.2.1 Antimicrobials 
Pivmecillinam will be administered to participants who meet the criteria for early antibiotic treatment or who 
are still shedding vaccine at Day 84 or after. Standard of care followed in icddr,b’s Hospital will be employed 
for treatment of all patients. Standard antibiotic treatment at the Dhaka Hospital of icddr,b for dysenteric 
patients is Pivmecellinam; patients with severe watery diarrhea will receive Azithromycin. Pivmecillinam 
will be administered orally at 15 mg/kg/dose 4 times a day for 5 days when necessary. Alternatively, 
Azithromycin will be given at 10 mg/kg once daily for 5 days.  
These antibiotics are usually well tolerated, but side effects may occur such as diarrhea, nausea, vomiting, 
abdominal pain/discomfort, headache and dizziness. In rare cases, severe side effects may also happen such 
as nerve damage in arms or legs or allergic reactions that, if severe, can cause difficulty in breathing.  There 
may be other risks or side effects which are currently unknown. 
Criteria for early antibiotic treatment for all participants: 
1. Participants meeting the criteria for shigellosis (temperature ≥ 38.3°C, and diarrhea and/or dysentery, 
and one or more severe reactogenicity symptoms, and shedding WRSS1) 
2. Participants with >6 diarrheal stools per day, or >3 dysenteric stools per day, or require intravenous 
hydration, with or without fever  
3. Any volunteer still shedding vaccine at day 84 will be treated with antibiotics. 
7.2.2 Antipyretics 
In the event that a participant develops a fever ≥38.0°C as confirmed by two independent axillary temperature 
measurements 10 minutes apart, 15 mg of acetaminophen per 1 kg child weight will be administered orally 
every 6 hours until the fever is resolved, not to exceed 90 mg/kg within a 24 hour period.  
7.3 Laboratory Evaluations 
7.3.1 Clinical Laboratory Evaluations  
During screening, and at day 7, blood samples will be collected for clinical laboratory evaluations. Per 
icddr,b DSMB recommendation for protocol VAC 008/PR 12054,  in those children who have a gradeable 
decrease in hemoglobin (Hb) level on day 7 after the 1st dose, a repeat measurement will be obtained on 
follow-up days, as appropriate, utilizing scheduled immunogenicity testing time points, if possible, to 
minimize venipuncture episodes. Samples will be packaged according to SOPs and picked up by laboratory 
staff.  Standard clinical laboratory tests for the purpose of inclusion and exclusion of potential participants 
and for safety monitoring will be carried out by the Clinical Laboratory Services. 
7.3.2 Research Laboratory Evaluations 
For the first dose of each cohort and throughout the inpatient period, all research samples will be collected at 
the CTU and sent to the Immunobiology, Nutrition and Toxicology (INT) Laboratory for testing. For the 
second and third doses of each cohort, all specimens will be collected at the CTU; throughout all outpatient 
follow-up visits, all research samples will be collected at the urban field site in Mirpur, Dhaka. Samples will 
be packaged and transported via official transport to the INT Lab.  Immunologic assays will be carried out at 

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the INT Laboratory, will be performed as per written SOPs, and will be a part of the study documentation. 
Culture and PCR detection of WRSS1 in stool will be done in Enteric Microbiology Laboratory.  The assays 
will occur as outlined in the Study Event Schedule.  For immunologic assay, samples will be stored so that all 
specimen time points can be tested together for each individual. 
7.3.2.1 Systemic immune response to WRSS1  
The systemic immune response to WRSS1 will be evaluated by assessing the IgA, IgG and IgM antibody 
responses to S. sonnei 2a LPS and Invaplex in serum/plasma samples at Days 0, 7, 35 and 63. Invaplex is a 
mixture of purified S. sonnei LPS and purified protein antigens IpaB and IpaC. Serotype-speciﬁc LPS from 
the Walter Reed Army Institute will be used to coat the ELISA plates. A ≥4-fold rise in serum/plasma 
antibody titers will be considered signiﬁcant. 
A serum bactericidal assay method can reliably measure functional activity of antibodies in vitro. The killing 
capacity of serum/plasma (or serum/plasma bactericidal antibody) as an immunological correlate for 
efficiency/ effectivity of the vaccine will be evaluated in toddlers at Days 0, 7, 35 and 63 using the method 
described previously37. The titer of the serum will be defined as the reciprocal of the last dilution in which no 
growth is evident by visual inspection. A ≥4-fold increase in antibody titers from baseline is defined as 
seroconversion. 
Concentration of cytokines will be measured in serum/plasma using a multiplex platform that will allow 
simultaneous profiling of multiple cytokines and chemokines in a single sample. Serum/plasma samples from 
Days 0, 7, 35 and 63 will be used for this purpose. 
7.3.2.2 Mucosal immune response to WRSS1 (ALS, Blood) 
The mucosal immune response to WRSS1 will be evaluated by assessing specific IgA, IgG and IgM antibody 
responses to S. sonnei 2a LPS and Invaplex using the ‘antibodies in lymphocyte supernatant’ (ALS) assay on 
culture supernatants of cultured peripheral blood mononuclear cells from different study days (Days 0, 7, 35, 
and 63) before and after vaccination to determine LPS- and Invaplex-specific IgA, IgG and IgM responses 
from circulating lymphocytes. Concentration of cytokines will be measured in ALS supernatant using a 
multiplex platform 
 
7.3.2.3 Mucosal immune response to WRSS1 (Fecal IgA and cytokines) 
The mucosal immune response to the WRSS1 will be evaluated by assessing Fecal IgA antibody responses to 
S. sonnei 2a LPS and Invaplex in ELISA assays at Days 0, 7, 28, 35, 56, 63, and 84 according to the method 
described earlier35. Pro-inflammatory cytokines (IL-1β, IL-8, TNF-α, IFN-γ etc.) will be measured in stool 
extracts using commercially available kits. 
7.3.2.4 Intestinal Colonization of WRSS1 (Stool Culture, PCR) 
The shedding of WRSS1 vaccine in stool will be evaluated by stool culture and PCR at Days 0 (after 
vaccination), 1, 7, 28, 35, 56, 63 and 84. 
8.0 STUDY SCHEDULE 
8.1 Screening, Visit 01 (Days -30 to -9) 
The following procedures will be completed during screening, between Days -30 and -9, to determine study 
eligibility and may occur over potential multiple screening visits.  The study PI or designee must also obtain 
a signed study specific ICF from the participants prior to screening initiation. Additional screening visits may 
be scheduled for any follow-up as needed, but are not required.  At the screening visit, the PI or her designee 
will provide parents of prospective participants with a detailed description of the study objectives and study 
participation requirements, as well as the potential health risks and benefits associated with study 
participation, including knowledge of diarrhea and Shigella bacteria. One or 2 additional participants will be 
recruited and screened for each group of 8 children in the case that another volunteer is found ineligible on 
admission / vaccination day (Visit 02, Day 0) or vomits in the 30 minute post-vaccination period. Alternates 
will be duly compensated for their time.  
The following procedures will be conducted at the Screening Visit(s):  

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 
Provision of written study description and ample time to read the ICF; signing the ICF 
 
Assessment for study eligibility via a review of inclusion and exclusion criteria 
 
Medical interview to collect relevant information on the participant’s past and concomitant medical 
illness and medications  
 
Physical examination: Head/ears/eyes/nose/throat (HEENT), skin, lymph nodes, respiratory (lung), 
cardiovascular (heart), abdomen, neurological and musculoskeletal system.  Weight and height will be 
measured at screening only for assessment of participant BMI 
 
Vital signs: heart rate, axillary temperature and respiratory rate   
 
Baseline hematology: Total and differential Cell Count and hemoglobin 
 
Baseline clinical chemistry: Creatinine, AST,ALT, bilirubin, GT 
 
Screening serology: anti-HAV, anti-HEV and HBsAg 
 
HIV test in serum 
 
Stool culture and PCR for presence of any Shigella strain obtained between Day -30 to -9.  
 
For continued eligibility prior to vaccination on Day 0, another stool specimen will be obtained at days -7 
to -5 for culture and PCR for presence of any Shigella strain. If a stool is not obtained between Day -7 
and -5, rectal swab will be obtained on Day -5 in Buffered Glycerol Saline (BGS) for culture and PCR 
testing and the results back prior to vaccination on Day 0. 
8.2 Day of First Vaccination (Visit 02) 
Day 0 is the day of the first study product administration (WRSS1 or placebo).  The following procedures 
will be conducted at Day 0 before vaccination in the Field Office: 
 
Assessment of continued eligibility by assessment of relevant inclusion/exclusion criteria 
 
Physical examination: HEENT, skin, lymph nodes, respiratory (lung), cardiovascular (heart), abdomen, 
neurological and musculoskeletal system  
 
Vital signs (heart rate, axillary temperature and respiratory rate); body weight will be measured  
 
Interim medical interview  
Inpatient Admission 
Eligible participants will be admitted to the CTU Inpatient Unit on the day of administration of WRSS1 or 
placebo.  As stated above, alternates will be brought along to replace any participants who are determined to 
be ineligible on the day of vaccination or vomits in the 30 minutes post-vaccination period. Those alternates 
will be duly compensated for their extra time.  
 
Admission to the CTU Inpatient Unit and overview of logistics during inpatient stay 
 
Inpatient unit orientation and review of requirements, expectations, and evacuation procedures 
Review of proper stool collection, hand-washing technique and hygiene procedures 
 
Vital signs (heart rate, axillary temperature and respiratory rate) prior to vaccination, 30 minutes post-
vaccination, and once later in the day will be measured. Body weight will be measured. 
 
Serum sample collected for assessing IgG, IgA and IgM antibody titers to S. Sonnei LPS and invaplex. 
 
Whole blood sample collected for ALS  
 
Stools collected, graded, and visually inspected for blood.  Microscopy will be performed if blood is 
suspected. Frequency of defecation will also be recorded. 
 
Stool sample for immunogenicity will be collected from Day -1 to Day 0 prior to vaccination.   
Vaccination 
Participants will be required to fast for 60 minutes before and after ingestion of the investigational product.  
To maintain the blind, staff who will not be involved in the clinical evaluation of the participants and 
laboratory evaluation of specimens will be responsible for randomization and labelling vaccine/placebo cups 
with treatment number.  

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If a participant vomits in the 30 minute post-vaccination period, the participant will remain in the study for 
safety follow-up evaluations required on Days 0 to 1 (inpatient period), 7, 28, 35, 56, 63, 84 and Day 224,  
and will not be re-dosed. If such an event occurs, an alternate participant may be dosed at the discretion of the 
IPST.  
The following procedures will be conducted:  
 
Preparation of WRSS1 and placebo  
 
Participant randomization 
 
Dosing of WRSS1 or placebo 
 
Stool sample collected after vaccination will be assessed for shedding. In addition to routine culturing at 
study-defined time points, cultures will be performed on stools that meet the protocol-definition of 
diarrhea 
8.3 Inpatient Period (Visit 03) 
Participants will remain in the CTU Inpatient Unit overnight for post-vaccination observation. Vital signs 
will be measured at least 2 times in the day.  
The following procedures will be conducted on Day 1: 
 
Physical examination including monitoring for intussusception and interim medical interview to collect 
any new AEs or assess ongoing AEs  
 
Vital signs twice a day: heart rate, axillary temperature and respiratory rate.  If the participant is febrile, 
additional vital signs will be measured,if needed. Body weight will be measured. 
 
Stools collected, graded, and visually inspected for blood.  Microscopy will be performed if blood is 
suspected.  Frequency of defecation will be recorded on each day. 
 
Stool sample collected for shedding. If no stool is obtained during the day, 2 rectal swabs will be 
obtained in BGS (for culture and PCR testing). In addition to routine culturing at study-defined time 
points, cultures will be performed on stools that meet the protocol-definition of diarrhea. 
Additional medically indicated clinical investigations will be performed based on study physician’s 
judgment. A study physician and 2 nurses will be present 24 hours/day.  
8.4 Discharge (Visit 03) 
Routine discharge is scheduled for the day after dosing i.e. on Day 1 (24-hr after vaccination).  Vital signs 
and physical assessment at discharge will be recorded in the source documents and in the electronic CRF.  
Guardians/mothers of participants will be educated about and given a simple Memory Aid to document 
frequency of defecation and stool grade each day up to the next 3 days for determination of diarrhea. 
Guardians/mothers will be informed that  field staff will visit their home for 3 days to monitor their health, 
including taking their temperature, reviewing their Memory Aid, and recording any symptoms on a source 
document. The field staff will be responsible for that source document and bring it to and from the 
participant’s home. Symptoms/signs solicited by the field staff and recorded on the source document will 
include the following: diarrhea, temperature, abdominal pain, nausea, vomiting, irritability, decreased 
activity, loss of appetite and excessive flatulence. 
8.5 Outpatient Visits   
On Day 7 after dosing, all participants will return to the Mirpur field office for follow-up visit as described in 
section 8.5.2. On Days 28 and 56, participants will return to the CTU for the second and third doses of 
WRSS1 or placebo (assuming the initial dose is deemed safe).  The visits on Days 7, 35, 63, and 84 are for 
safety and immunogenicity assessment.  The visit on Day 224 is for long term safety follow-up.  
All mothers/guardians of study participants will be encouraged (but not required) to give them food 
approximately 70-80 minutes before dosing.  Unscheduled visits may be completed at any time during the 
study for safety assessment and follow-up, based upon the clinical judgment. 
8.5.1 Outpatient Vaccinations (Visit 05 and Visit 07) 
The following procedures will be conducted on Days 28 and 56: 

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 
Assessment of continued eligibility by review of relevant inclusion/exclusion criteria  
 
Interim medical interview to collect any new AEs or to assess ongoing AEs and concomitant medications 
review 
 
Physical examination prior to vaccination: HEENT, skin, lymph nodes, respiratory (lung), cardiovascular 
(heart), abdomen (including monitoring for intussusception), neurological and musculoskeletal system  
 
Vital signs: heart rate, axillary temperature and respiratory rate and body weight will be measured. 
 
Dosing of WRSS1 or placebo. If a participant vomits within 30 minutes after receiving his/her second 
dose, he/she will remain in the study and complete all follow-up safety assessments and receive the third 
dose at the discretion of the IPST 
 
Stool sample collected for immunogenicity and shedding. Study staff will visit participants at their home 
in the morning for specimen collection and transport back to the lab for fecal IgA processing, culture, and 
PCR. Stool specimens may be no more than 3 hours old. If no stool is available in the morning, the 
guardians may bring in the sample when they visit CTU for completion of their outpatient vaccination.   
If no stool is available before vaccination or the specimen is received outside of 3 hours from collection, 
2 rectal swabs will be obtained in BGS (for culture and PCR testing) 
 
Guardians/mothers of participants will be educated about and given a Memory Aid to track stool 
frequency and grade each day up to the next 3 days for the determination of diarrhea  
 
Guardians/mothers will be informed that a field staff will visit their home for the next 3 days to monitor 
the health of the child, including taking their temperature, reviewing their Memory Aid and recording any 
symptoms on a source document. The field staff will be responsible for that source document and bring it 
to and from the participant’s home and icddr,b (symptoms/signs collected daily for 3 days post-
vaccination). 
8.5.2 Outpatient Assessments (Visit 04, 06, 08, 09) 
The following procedures will be conducted on Day 7 for all participants:  
 
Physical examination : HEENT, skin, lymph nodes, respiratory (lung), cardiovascular (heart), abdomen 
(including monitoring for intussusception), neurological and musculoskeletal system  
 
Vital signs: heart rate, axillary temperature and respiratory rate and body weight will be measured 
 
Clinical Chemistry: Creatinine, AST/ALT, bilirubin, GT  
 
Hematology: Total and differential Cell Count and hemoglobin 
 
Interim medical interview of guardians/mothers to collect any new AEs, to assess ongoing AEs, and to 
review concomitant medications  
 
Stool sample collected for immunogenicity and shedding. Study staff will visit participants at their home 
in the morning for specimen collection and transport back to the lab for fecal IgA processing, culture, and 
PCR. Stool specimens may be no more than 3 hours old. If no stool is available, the guardian may bring 
in the sample when they visit the Mirpur field site for completion of their child’s outpatient follow-up 
visit.   If no stool is available before the child returns home or the specimen is received outside of 3 hours 
from collection, 2 rectal swabs will be obtained in BGS (for culture and PCR testing). 
 
Whole blood sample collected for ALS; plasma from whole blood will be saved for ELISA  
The following procedures will be conducted on Day 35 (Visit 06) and Day 63 (Visit 08) 
 
Physical examination : HEENT, skin, lymph nodes, respiratory (lung), cardiovascular (heart), abdomen 
(including monitoring for intussusception), neurological and musculoskeletal system 
 
Vital signs: heart rate, axillary temperature and respiratory rate and body weight will be measured 
 
Interim medical interview to collect new AEs, assess ongoing AEs and review concomitant medications 
 
Stool sample collected for immunogenicity and shedding. Study staff will visit participants at their home 
in the morning for specimen collection and transport back to the lab for fecal IgA processing, culture, and 
PCR. Stool specimens may be no more than 3 hours old.  If no stool is available, the participant may 
bring in their sample when they visit the Mirpur field site for completion of their outpatient follow-up 

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visit.  If no stool is available before the child returns home or the specimen is received outside of 3 hours 
from collection, 2 rectal swabs will be obtained in BGS (for culture and PCR testing).  
 
Whole blood sample collected for ALS; plasma from whole blood will be saved for ELISA.   
The following procedures will be conducted on Day 84 (Visit 09): 
 
Physical examination : HEENT, skin, lymph nodes, respiratory (lung), cardiovascular (heart), abdomen 
(including monitoring for intussusception), neurological and musculoskeletal system 
 
Vital signs: heart rate, axillary temperature and respiratory rate and body weight will be measured 
 
Interim medical interview to collect information about any new chronic health conditions or serious 
health events  
 
Stool sample collected for immunogenicity and shedding. Study staff will visit participants at their home 
in the morning for specimen collection and transport back to the lab for fecal IgA processing, culture, and 
PCR. Stool specimens may be no more than 3 hours old.  If no stool is available, the guardians may bring 
in their sample when they visit the Mirpur field site for completion of their child’s outpatient follow-up 
visit.  If no stool is available before the child returns home or the specimen is received outside of 3 hours 
from collection, 2 rectal swabs will be obtained in BGS (for culture and PCR testing). 
8.6 Early Termination  
 
A participant may be withdrawn from the trial if any of the following reasons apply.  However, any 
participant who has received WRSS1 vaccine will be encouraged to complete antibiotic treatment or 
ascertainment of clearance of infection and remain in the study for periodic safety evaluations for the 
duration of the study. An enrolled/vaccinated participant may be withdrawn from the study for any of these 
reasons.  
a) Parent of participant withdraws consent. 
b) Investigator or PVS Medical Officer decides that termination is necessary in the event of illness, for 
participant safety, or for social reasons. 
c) If the participant needs treatment that is not allowed in this study. 
d) Investigator or PVS Medical Officer decides that termination is necessary to protect the integrity of 
the study or achieve the objectives of the study.  
e) Sponsor terminates the study. 
f) If the child fails to follow instructions or miss study visits. 
A volunteer may be withdrawn from receiving further vaccinations, but encouraged to complete safety-
related follow-up. If the volunteer agrees, other study procedures (e.g., blood sampling for measuring levels 
of antibodies) may be continued.  Withdrawal from further vaccination will occur for: 
Safety-related reasons at the discretion of the PI, Study Physician, Emmes Medical Monitor, PVS Medical 
Officer, IPST, or the participant.  
A participant may be discontinued from receiving further vaccinations, but encouraged to complete safety-
related follow-up for the following reasons: 
(a) A participant vomits in the 30 minute post-vaccination period during 1st vaccination 
(b) Occurrence of a severe reactogenicity event. 
An excessive number of withdrawals may affect the scientific validity of the study; therefore unnecessary 
withdrawal will be avoided whenever possible.  Should withdrawals occur, efforts will be made to ensure 
participant safety and to continue and complete safety monitoring as thoroughly as possible.  In case of 
participant withdrawal, for whatever reason, a final trial evaluation must be completed stating the reasons.  
Withdrawals due to non-attendance must be followed-up by the investigator to the extent possible to obtain 
the reason for non-attendance.  

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9.0 STUDY INTERVENTION/INVESTIGATIONAL PRODUCT  
9.1 WRSS1 
9.1.1 Biological Name and Structure 
The vaccine will consist of reconstituted lyophilized ampoules of WRSS1 (lot# 0451) containing live 
attenuated S. sonnei bacteria. The vaccine was manufactured in June 1997 and stored at -80C at the WRAIR 
Pilot Bioproduction Facility in Silver Spring, Maryland, under current good manufacturing practices. The 
viability and stability of WRSS1 has been tested periodically since the date of manufacture as shown in the 
table below. Viability results are averages of at least 2 vials. Stability results are from colony immunoblots 
using an IpaB monoclonal antibody. The presence of the engineered virG deletion allele in WRSS1 has also 
been confirmed via PCR. 
 
Date of testing 
Viability (CFU/vial) 
Stability (% Form I CFU) 
1997 Jul 1 
1.8×1010 
92% 
2002 Jun 4 
3.2×109 
ND 
2003 Jan 29 
1.6×1010 
ND 
2004 Feb 6 
1.6×1010 
ND 
2004 Oct 7 
1.3×1010 
ND 
2006 Jan 5 
5.0×109 
ND 
2009 Jun 23 
1.7×1010 
96% 
2009 Dec 30 
1.4×1010 
97% 
2010 Jun 2 
2.4×1010 
97% 
2012 Feb 7 
3.0×1010 
95% 
2013 Jun 24 
1.4×1010 
93% 
2014 Jun 23 
6.0×109 
94% 
2015 Jul 15 
2.7×109 
93% 
ND = not determined 
So far, no changes in viability or stability of the vaccine has been noted since its manufacture. Prior to 
vaccination in Bangladesh, vaccine vials will be shipped on dry ice to the icddr,b INT Laboratory and 
maintained at -65C to -85C until use in the trial.  
During the trial, the vaccine vial will be thawed and reconstituted with 5 mL Sterile Water for Injection 
(SWI) and dilution in saline to achieve the target concentration for dosing. 1 mL of vaccine in saline with the 
specific CFU dose will be added to the designated volume of saline. Table 11 outlines target volume and 
buffer volume for vaccine administration for toddlers.  
Table 11: Vaccine and Placebo Volumes 
 
WRSS1 dose 
volume (ml) 
Saline 
volume (ml) 
NaHCO3 
buffer (ml) 
Total 
volume 
Placebo 
N/A 
5 ml 
10 ml 
15 ml 
Vaccinee 
1 ml 
4 ml 
10 ml 
15 ml 
9.1.2 Chemistry, Manufacturing, and Control 
S. sonnei WRSS1 vaccine Lot No. 0451 was manufactured by Walter Reed Army Institute for Research 
(WRAIR) under cGMPs.  A cryovial of PCB Lot No. 0433 was added to 1 liter of Luria Bertani (LB) 
medium (10 g tryptone, 5g yeast extract, 10 g NaCl) with 0.2% glucose and was shaken at 37 0C for 7.5 hrs; 
the one-liter culture was used to inoculate 30 liters of LB medium with glucose in a 40 liter production 
fermenter.  When an OD600 of 1 - 2 (1.4 x 109 CFU/ml) was attained, the temperature was set to 4°C and the 
culture was harvested by continuous flow centrifugation.  The cell pellet was resuspended in 2 liters of 
cryopreservative consisting of 7.5% Dextran T10, 2% sucrose, and 1.5% glycerol in Dulbecco’s phosphate 
buffered saline (PBS) [final concentration 3.7 x 1010 CFU/ml]. 

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9.1.3 Final container and composition  
S. sonnei WRSS1 is formulated lyophilized in 50 ml serum vials with gray split rubber stoppers sealed with 
aluminum crimps.  Each vial was filled with 5.0 g +5% of S. sonnei WRSS1 vaccine prior to lyophilization. 
One ml, prior to lyophilization, contained 3.7 x 1010 CFU of S. sonnei WRSS1 in Dulbecco’s PBS containing 
7.5% Dextran T10, 2% sucrose, and 1.5% glycerol. Reconstituted vaccine in 5 ml SWI will yield 1-2 x 1010 
CFU per vial or 2-3 x 109 CFU/ml.  
Vials are labeled:  
 
 
 
 
 
 
 
 
Shigella sonnei Strain WRSS1  
BPR No.:  BPR-228-00  
 
Lot No.:  0451 
Contents:  5.0 g + 5% (lyophilized) 
Date of Mfg:  27 Jun 97 
Caution:  New Drug Limited by Federal Law for Investigational Use Only 
Storage:  -80 + 10C 
Manufactured By:  WRAIR, Washington D C.  20307 
 
Labelled vials are sampled for QC release and stored at -80C + 10C  
9.2 Preparation, Administration and Dosage of Study Investigational Product, and Buffer 
9.2.1 WRSS1 
Lyophilized, frozen vials of WRSS1 Lot 0451 will be transported on dry ice to the INT Laboratory for 
storage at -80C ± 10C until the day of vaccination.  On the day of vaccination the vaccine vials will be 
removed for preparation and placed on ice in a laminar flow hood and allowed to thaw for 30 minutes before 
adding 5 ml of sterile water for injection (SWI). The vaccine is allowed to rehydrate for another 15 minutes 
on ice, with intermittent swirling of the suspension to ensure homogeneous mixing of the vial contents. The 
reconstituted vaccine will then be diluted in sterile saline to arrive at the desired concentration and the diluted 
vaccine will be kept on ice until vaccination. Time of reconstitution will be noted. The dilutions will be 
titered and replica spread plate quantitative cultures will be made of the inoculum dilutions before and after 
vaccination to confirm viability and dose. 
One ml of the vaccine solution in saline at the appropriate concentration will be placed into an additional 4 
ml of sterile saline.  Ten ml of a freshly prepared bicarbonate solution (2 g in 150 ml of SWI) will be placed 
in a second cup.   
All participants will fast for 60 minutes prior to and after receiving the vaccine. Care must be taken to ensure 
that the time between reconstitution and administration of vaccine to the participants does not exceed 2 hours. 
Further details for vaccine preparation, administration, and dispensation are provided in the study-specific 
SOP.  
9.2.2 Placebo 
Placebo recipients will receive the designated volume of bicarbonate buffer plus the designated volume of 
sterile saline solution according to the Table 11.  
9.2.3 Sodium Bicarbonate  
Participants randomized to the vaccine and placebo group will receive the same amount of bicarbonate buffer 
as mentioned in table 11. Further details for buffer preparation, administration, and dispensation are provided 
in the study-specific SOP. 
9.3 Accountability Procedures for the Investigational Product(s)  
The PI will ensure that the investigational product supplies are stored as specified in the protocol and in a 
secured area, with access limited to authorized study personnel.  The investigator will maintain accurate 
records of the receipt of all investigational products, including date received, manufacture or expiration date, 
amount received, and disposition.  A record will be maintained that includes the dispensation date, amount of 
investigational product dispensed, initials, and identification number.  The investigational product will be 
administered only at the specified institution.  The investigator will keep an inventory of the study materials, 
will hold the amount of product needed, and will be responsible for adequate storage and dispensing of the 

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vaccines.  Investigational product management will be delegated by the investigator to designated study staff 
at the INT Laboratory.  
9.4 Assessment of Participant Compliance with Investigational Products  
A study staff member or designee will witness the ingestion of WRSS1 or placebo. 
10.0 ASSESSMENT OF SAFETY  
10.1 Adverse Events  
The investigator is responsible for accurate documentation of all AEs according to the detailed guidelines set 
out below.  The guardians will be instructed to contact the investigator immediately should they observe any 
signs or symptoms in their child perceived as significant during the study period. Approximately 1 month 
after administration of the last dose of investigational product, all participants will be seen at the Mirpur Field 
Office for a final outpatient visit to close out any existing adverse events and identify and track any new 
chronic health conditions or SAEs.   
10.1.1 Adverse Event Definitions (http://www.ecfr.gov/cgi-bin/text- idx?SID=e3eba258602082611 
eadeafcee788296&mc=true&node=pt21.5.312&rgn=div5#sp21.5.312.b) 
Adverse event is any untoward medical occurrence in humans, whether or not considered drug related, that 
occurs during the conduct of a clinical trial. Any change in clinical status, ECGs, routine labs, x-rays, 
physical examinations, etc., that are considered clinically significant by the study investigator, is considered 
an AE.  
This definition includes any noxious, pathological or unintended change in anatomical, physiological or 
metabolic functions, as indicated by physical signs, symptoms and/or clinically significant laboratory 
abnormalities occurring in any phase of the clinical study, whether associated with the study vaccine or 
placebo. 
This definition also includes an exacerbation or worsening of pre-existing conditions or events, intercurrent 
illnesses, injuries, or vaccine or drug interaction, or worsening of abnormal clinical laboratory values.  
Suspected adverse reaction is any adverse event for which there is a reasonable possibility that the drug 
caused the adverse event. A reasonable possibility implies that there is evidence that the drug caused the 
event. 
Adverse reaction is any adverse event caused by the drug.  
Serious Events (Serious Adverse Events, Serious Suspected Adverse Reactions Or Serious Adverse 
Reactions)  
A Serious Adverse Event, including serious suspected adverse reaction or serious adverse reaction as 
determined by the Investigator or the sponsor, is any event that results in any of the following outcomes: 
1. Inpatient hospitalization or prolongation of existing hospitalization  
2.    Life-threatening AE (Life-threatening means that the study participant was, in the opinion of the 
investigator or sponsor, at immediate risk of death from the reaction as it occurred.) 
3. Persistent or significant incapacity or substantial disruption of the ability to conduct normal life 
functions  
4. Congenital abnormality or birth defect 
5.    A medically important event that may jeopardize the participant or may require intervention to 
prevent one of the other outcomes listed above.  Medical judgment should be exercised in deciding 
whether an AE is serious in other situations.  Important AEs that are not immediately life-
threatening or do not result in death or hospitalization, but may jeopardize the participant or may 
require intervention to prevent one of the other outcomes listed in the definition above, should be 
considered as serious.  (Examples of such cases are intensive treatment in an emergency room or at 
home for allergic bronchospasm; blood dyscrasia or convulsions that do not result in 
hospitalization; developments of drug dependency, overdose, or drug abuse.) 
6.    Death 

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Unexpected Adverse Event 
An adverse event is “unexpected” when its nature (specificity) or severity is not consistent with applicable 
product information, such as safety information provided in the Investigator’s Brochure or if not available, 
the investigational plan, or the protocol.   
 
10.1.2 Reactogenicity 
Reactogenicity events are AEs that are commonly seen with vaccines and/or known to occur for WRSS1 and 
similar enteric vaccines and will be collected in the source documents and eCRFs using a grading scale based 
on functional assessment or magnitude of reaction. Reactogenicity are solicited AEs that occur within 72 
hour after WRSS1 vaccination and will be collected on the appropriate source document and recorded on a 
reactogenicity eCRF.  
Based on prior studies of WRSS1 vaccine, it is anticipated that WRSS1 will be well-tolerated.  However, 
there is the possibility that this vaccine may result in symptoms similar to infection with Shigella, including 
moderate to severe diarrhea (which, though unlikely, could lead to dehydration and the need for oral or 
intravenous rehydration).  Other symptoms consistent with shigellosis that may occur are listed in the table 
below. 
Table 12.  Reactogenicity events to be solicited daily during the first 72-hours post-vaccination 
Systemic symptoms 
GI symptoms 
fever 
abdominal pain  
dysentery 
decreased appetite 
nausea 
bloating 
irritability 
vomiting 
excess flatulence 
decreased activity 
loose stool 
constipation 
 
diarrhea 
 
The grading for reactogenicity events are given in Appendix 3. 
 
Systemic and intestinal reactogenicity symptoms and severity grading are as follows:   
Study Specific Definitions 
Systemic symptoms – fever, decreased appetite, irritability, decreased activity 
Intestinal symptoms – abdominal pain, nausea, vomiting, loose stool, diarrhea, dysentery, bloating, excess 
flatulence, constipation 
 
10.1.3 Guidelines for Determining Causality of an Adverse Event  
The Investigator should consider the following question when assessing causality of an AE to study drug:  
Is there a reasonable possibility that the drug caused the event? 
Reasonable possibility implies there is evidence that the study product caused the reported event. An 
affirmative answer designates the event as a suspected adverse reaction. 
 
10.1.4 Follow-up of Adverse Events and Assessment of Outcomes  
The Investigator should monitor for AEs until Day 224 for all participants. For reporting and follow-up of an 
SAE, see protocol section 10.1.8. 
 
10.1.5 Treatment of Adverse Events 
Treatment of any AE is at the sole discretion of the investigator and according to the best treatment currently 
available. The applied measures should be recorded in the CRF of the participant.  The PI may also refer a 
participant for appropriate care, if applicable. PATH will provide payment for any harm caused by 
participation in the study.  

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10.1.6 Recording Adverse Events 
Any research related injuries and adverse events will be followed periodically until the events are resolved or 
become stable. Follow-up process can be both telephone and/or asking the mothers/guardians to visit the 
study site with their children, which depends on their conveniences and/or the severity of the particular event 
per clinical judgment of the study physician and PI.  Whenever possible, the investigators will record the 
diagnosis, if available.  If no diagnosis is available, the investigators will record each sign/symptom as 
individual AEs.  The nature of each event, date of onset, end date, outcome, severity and relationship 
(causality) to WRSS1 should be established.  AEs already documented in the eCRF, i.e., at a previous 
assessment and not resolved, should be reviewed at subsequent follow-up assessments.  If resolved, 
documentation in the eCRF should be completed.  The entire duration of AEs will be recorded.  AE severity 
will be recorded as the worst grade for the duration of each illness/symptom.  Once the sign/symptom 
resolves for 24 hours or greater, the AE will be resolved.  If the sign/symptom reoccurs after 24 hours, then it 
will be documented as a new event, unless clinician has judged these as constituent components of the same 
event.  
10.1.7 Reporting Serious Adverse Events  
All SAEs (irrespective of the causality) are to be reported to the DSMB of icddr,b Ethical Review Committee 
(ERC), Sponsor, medical monitors of Sponsor and Emmes within 24 hours of the study team becoming aware 
of the event.  
All SAEs must be documented on an icddr,b  Serious Adverse Event Form as well as entered into the 
electronic SAE Reporting System.  A complete written report will follow the initial notification.  
The PI will provide the SAE form to the Sponsor who will decide whether or not the SAE meets FDA and 
WIRB criteria for immediate reporting.  The investigator will update the Sponsor, Sponsor MM, and Emmes 
MM with new information as available.   
The Sponsor MM and Emmes MM are required to review all unanticipated events involving risk to 
participants or others, serious adverse events and all participant deaths associated with the protocol and will 
provide a written report.  At a minimum, the Sponsor MM and Emmes MM must comment on the outcomes 
of the event or problem and, in case of a serious adverse event or death, comment on the relationship to 
participation in the study.  The Sponsor MM and Emmes MM must also indicate whether he/she concurs with 
the details of the report provided by the principal investigator.  
All unanticipated events involving risk to participants or others, serious adverse events related to 
participation in the study, and participant deaths related to participation in the study should be promptly 
reported by phone to the following Medical Monitors: 
Tushar Tewari, MD 
PVS Medical Officer 
PATH Vaccine Solutions 
Phone: 91.11.2653.0080 
Fax: 91.11.2653.0089 
Email: ttewari@path.org  
Robert Lindblad, MD 
Emmes Medical Monitor 
The Emmes Corporation 
Phone: 301-251-1161 
Fax: 301-251-1355 
Email: rlindblad@emmes.com  
The Investigator is required to submit a completed SAE Report form via the electronic SAE reporting system 
within 24 hours of recognition of the event as an SAE. All additional follow-up information for the SAE must 
be entered or uploaded into the electronic SAE reporting system within 24 hours upon receipt of new 
information, and entered into the electronic SAE Reporting System. 
Sites will be trained in reporting AEs and SAEs. Notification of reported events will be generated at the time 
of entry into the data system and all SAEs will be promptly reviewed by the medical monitor within the data 
system. Summary review of all reported AEs and SAEs will be compiled on a weekly basis to identify any 
safety trends. Review of safety lab results will also be conducted on a weekly basis. All SAEs that meet 
expedited reporting criteria (suspected adverse reactions that are serious and unexpected)  will be submitted 
to FDA via phone/fax /e-mail within 7 days  followed up with a written report within 15 days of the event, if 
life threatening or result in death or  as a written report within 15 days for the remainder.  Written reports will 
use the MedWatch 3500A form.  Follow-up information on a Safety Report will be submitted as soon as the 

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information becomes available.  Any series of events that as a group are considered "unexpected" would be 
submitted within 15 days of that determination being made by the sponsor. Annual reports would be 
submitted in accordance with IND regulations. 
To the IRB 
The Investigator is responsible for submitting the safety report (initial and follow up SAE reports) or other 
safety information (e.g. revised Investigator’s Brochure) to the icddr,b ERC and DMSB  according to icddr,b 
requirements, and for retaining a copy in the site’s study file. The Investigator is also responsible for 
notifying the Western Institutional Review Board (WIRB), PVS’ designated Independent Ethics Committee, 
according to WIRB guidelines outlined below: 
WIRB Guidelines 
All SAEs will be reported to WIRB according to WIRB guidelines and using the Unanticipated Problems that 
are Adverse Events form.  
WIRB Phone: 800-562-4789, Fax: 360-252-2498.   
An unanticipated problem is defined as any incident, experience, or outcome that meets all of the following 
criteria: 
 
Unexpected (in terms of nature, severity, or frequency) given (a) the research procedures that are 
described in the protocol-related documents, such as the IRB-approved research protocol and informed 
consent document, or the Investigator Brochure; and (b) the characteristics of the subject population 
being studied; 
 
Related or possibly related to participation in the research (possibly related means there is a reasonable 
possibility that the incident, experience, or outcome may have been caused by the drugs, devices or 
procedures involved in the research); and 
 
Suggests that the research places participants or others at a greater risk of harm (including physical, 
psychological, economic, or social harm) than was previously known or recognized.  
Investigators are required to report adverse events that fit the above criteria within 10 working days of the 
time the investigator becomes aware of them. 
10.1.8 Follow-up of Serious Adverse Events 
All SAEs must be documented and followed up until the event resolves, subsides, or stabilizes or the study 
participant is lost to follow-up. Continued follow-up of SAEs is at the discretion of the PI according to good 
clinical management, or until the participant is referred to another institution for continued care. All SAEs 
will be reported to the DSMB of icddr,b; the PI will follow-up with the DSMB with any updates accordingly. 
WIRB expects reports of unanticipated problems to include a corrective action plan to address the issue, or 
written justification for why none is provided, on the Unanticipated Problems that are Adverse Events form.  
10.2 Safety Oversight - Data Safety Monitoring Board (DSMB) 
All clinical investigations (research protocols testing biomedical and/or behavioural intervention(s)) should include the Data and 
Safety Monitoring Plan (DSMP). The purpose of DSMP is to provide a framework for appropriate oversight and monitoring of 
the conduct of clinical trials to ensure the safety of participants and the validity and integrity of the data. It involves involvement 
of all investigators in periodic assessments of data quality and timeliness, participant recruitment, accrual and retention, 
participant risk versus benefit, performance of trial sites, and other factors that can affect study outcome. 
A Data and Safety Monitoring Board (DSMB) will be formed by the Ethical Review Committee to evaluate 
and assess the safety of study participants. Moreover, there will be an Internal Protocol Safety Team (IPST) 
comprising of the study physician, the Medical Monitor from Emmes, the principal investigator, and the 
Medical Monitor from PVS.  
In each cohort, the first vaccine dose will be conducted at the icddr,b Inpatient Unit (Clinical Trials Unit, 
icddr,b main campus), for immediate safety evaluation where the participants will be admitted for 
observation for 24 hours post-vaccination. This will help in clinical assessments accurately and promptly, 
addressing any potential reactogenicities. Clinical signs and symptoms, including daily stool output and 
temperature will be monitored. Second and third vaccinations within each cohort will take place on an 
outpatient basis. Before enrolling participants in subsequent cohorts to receive a higher vaccine dose, the 
safety data from the previous cohort(s) (through Study Day 7) will be evaluated and reviewed by the Internal 

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Protocol Safety Team (IPST). This data include, but is not limited to, physical examinations, vital signs and 
solicited reactogenicity symptoms.  
The DSMB will be composed of 3-5 members, two of which are nominated by the ERC chairperson. The PI 
also has the option of nominating 1-2 members with therapeutic expertise. The DSMB will convene three 
times during the study period, at the beginning before the trial is started, after completion of vaccination in 
cohorts 1 and 2 and at the end of the trial. IPST will review the safety of each cohort and have the option of 
convening a DSMB meeting if there is a safety issue that requires additional interpretation.  The PI can also 
request a DSMB meeting depending upon the study complexity or in light of safety concerns. The DSMB 
will advise the PI of its findings and provide recommendations. The DSMB will review all unanticipated 
problems involving risk to the participants or others, serious adverse events, and all participant deaths 
associated with the protocol. The PI will inform the Emmes Medical Monitor and the PVS Medical Monitor 
in detail about the discussions of those meetings. 
10.3 Stopping Rules 
The following study halting rules refers to suspected adverse reactions and will automatically pause or halt 
further vaccinations within any given cohort: 
1. One or more participants with a serious AE (SAE) occurring at any time following vaccination that is 
related to the WRSS1 vaccine  
2. One or more participants experience systemic allergic reaction (i.e., bronchospasm, allergy-related 
edema/angioedema, hypotension or anaphylaxis) associated with administration of WRSS1 vaccine 
3. One or more participants experience unexpectedly severe disease (i.e., hypotension (disproportionate to 
volume loss), renal dysfunction, or altered mental state (e.g., somnolence) after vaccine administration. 
4. Severe diarrhea occurring in >20% of participants at a given dose level. 
5. 20% or more of the participants present a vaccine related unanticipated moderate or greater AE, grade 3 
fever or grade 3 laboratory abnormalities. 
The study PI, with the assistance of the IPST, DSMB and Data Management group at Emmes will be 
responsible for determining if the halting rules are met. During the safety data assessment period, which may 
require unblinding of safety data by the DSMB committee as deemed necessary, no new participants will be 
vaccinated. The DSMB will be responsible for recommending whether or not to re-start the study. An outline 
of the extent of the safety assessment conducted and justification for the decision taken will be documented 
in the study file. Ongoing participants will receive their subsequent vaccinations at the discretion of the PI 
and IPST. In the event that there is an acute safety issue, the Sponsor MM may suspend enrolment/treatment, 
if warranted, pending review by the DSMB. 
10.4 Six Month Follow-up Safety Surveillance 
At Day 224  2 weeks, participants will be visited by study staff to inquire about post-study chronic health 
conditions, serious health events, or hospitalizations.  If three attempts to reach the participant are 
unsuccessful, then no more visits are required and this fact will be documented in the volunteer’s source 
documents and on the eCRF page.  This long-term follow-up information will be summarized in the final 
clinical study report. 
11.0 MONITORING BY SPONSOR’S REPRESENTATIVE 
During the study, the investigators will maintain complete and accurate documentation for the study, 
including records detailing the progress of the study for each participant, laboratory reports, eCRFs, signed 
ICF for each study participant, drug disposition records, correspondence with the IRB, the study monitor and 
the sponsor, AE reports and information regarding participant discontinuation and completion of the study.  
All required study data will be recorded clearly and accurately by authorized study personnel in the 
participant’s source documents, and required information will be entered into appropriate eCRFs.  Only 
designated study site personnel shall record or change data in the eCRFs.  During the study, the PI will be 
responsible for ensuring the quality of data recorded in the eCRFs as per written eCRF completion 
guidelines. Training of study personnel on GCP conduct of the trial, including study procedures and data 
management responsibilities, will be provided by the PI and Emmes prior to study initiation and throughout 
the trial, as needed.  

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11.1 Study Monitoring 
Sponsor monitoring responsibilities will be provided by Emmes as outlined in the Study Monitoring Plan.  
Monitoring will be conducted prior to beginning, at initiation, during, and at closeout of the study by the 
study monitor.   
During the course of the study, the monitor will visit the clinical site at regular intervals to verify compliance 
to the protocol; completeness, accuracy, and consistency of the data; and adherence to local regulations and 
requirements, including GCP on the conduct of clinical research.  The monitor should have access to 
participant medical records and other study-related records needed to verify the entries on the eCRFs. 
The PI and the monitor must agree to cooperate to ensure that any problems detected in the course of these 
monitoring visits, including eCRF completion and query resolution, are resolved.  
To ensure the quality of clinical data across all participants at the site, a clinical data management review will 
be performed on participant data received at Emmes.  During this review, participant data will be checked for 
consistency, omissions, and any apparent discrepancies.  In addition, the data will be reviewed for adherence 
to the protocol and GCP.  To resolve any questions arising from the clinical data management review 
process, data queries and/or site notifications will be sent to the site for completion and return to Emmes. 
Essential documents must be filed in the site study file on an ongoing basis.  Monitoring visits will be 
performed according to Study Monitoring Plan. 
11.2 Independent Auditing 
PVS or PVS representatives may audit the study to ensure that study procedures and data collected comply 
with the protocol and applicable SOPs at icddr,b and Emmes, and that data are correct and complete. The PI 
will permit auditors (employees of the Sponsor or an external company designated by the Sponsor) to verify 
source data validation of the regularly monitored clinical study.  The auditors will compare the entries in the 
eCRFs with the source data, and evaluate the study site for its adherence to the clinical study protocol and 
GCP guidelines and applicable regulatory requirements. 
11.3 Regulatory Agency Auditing 
The PI must be aware that representatives from regulatory authorities or the IRB may wish to inspect the 
eCRFs and associated study records.  The PI will notify the Sponsor within 24 hours following contact by a 
regulatory agency.  The PI and study coordinator must make the relevant records available for inspection and 
will be available to respond to reasonable requests and audit queries made by authorized representatives of 
regulatory agencies.  The PI will provide the Sponsor with copies of all correspondence that may affect the 
review of the current study or his/her qualification as an investigator in clinical studies conducted by the 
Sponsor.  The Sponsor will provide any needed assistance in responding to regulatory audits or 
correspondence. 
12.0 SAMPLE SIZE CALCULATION AND OUTCOME PRIMARY AND SECONDARY 
VARIABLES   
Clearly mention your assumptions. List the power and precision desired. Describe the optimal conditions to attain the sample size. 
Justify the sample size that is deemed sufficient to achieve the specific aims. 
 
12.1 Introduction 
This study is a double-blind, randomized, placebo-controlled, dose-escalation clinical trial to examine the 
safety, tolerability and immunogenicity of WRSS1.  The primary objective of the study is to evaluate the 
safety and tolerability of the vaccine; the secondary objective is to evaluate vaccine immunogenicity.     
Participants will be randomized separately for each cohort, at a vaccine: placebo ratio of 12:4. The 
randomization code will be generated and maintained by Emmes.  
12.2 Sample Size  
The chosen sample sizes (12 vaccinees and 4 placebo subjects/dose group) will not allow for detailed 
statistical comparisons across the dose groups and rigorous statements about the safety or immunogenicity of 

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the vaccines, it will provide preliminary safety information that may support testing the product in additional 
larger cohorts in the target population – children 12-24 months of age. 
The goal of the safety evaluation is to identify safety concerns associated with product administration. The 
ability of the study to detect SAEs can be expressed by the true rate above which at least 1 SAE would likely 
be observed and the true event rate below which no events would likely be observed.  
The active arms in which 48 participants will receive vaccine, will allow for the recognition of unacceptable 
toxicity rates (SAEs) occurring at a frequency of 10% or higher.  For instance, the probability of observing 
one event among thirty participants is very high (close to 90%) if the true attack rate of that event is 10%.   
12.3 Data Analysis 
Describe plans for data analysis, including stratification by sex, gender and diversity. Indicate whether data will be analysed by the 
investigators themselves or by other professionals. Specify what statistical software packages will be used and if the study is blinded, 
when the code will be opened. For clinical trials, indicate if interim data analysis will be required to determine further course of the 
study. 
For Primary Safety Endpoints 
All visits after the participants have been exposed to the study product will be included in the primary 
analysis of safety.  To assess safety, the number and the percentages of participants experiencing at least one 
AE, and the number and percentage of participants experiencing each specific AE will be tabulated by study 
arm.  Overall summaries by arm include: 1) any solicited reactogenicity; 2) any unsolicited AEs; 3) any 
SAEs, and 4) any unsolicited AEs and SAEs judged as having a reasonable possibility that the study product 
caused the event. 
Proportions will be used to assess the tolerability to the study vaccine using only the participants randomized 
to the vaccine arm.  Tolerability measures will be tabulated by study group along with their corresponding 
95% confidence intervals. 
For Secondary Immunology Endpoints: 
Immunologic response will be presented for each parameter (e.g. IgA, IgG, and  IgM antibody responses) and 
defined visits using descriptive statistics. Changes from baseline to each visit will be presented.  Repeated 
measure analysis of titer values will be conducted to assess treated dose and temporal differences. The 
number and percentage of participants with a positive immune response will be presented by dose group and 
analyzed with repeated measures models using generalized estimating equations. Attempt will be made to 
recruit equal number of male and female participants in the study so that separate analysis for males and 
females can be conducted and gender-specificity in the immune responses to the vaccine can be analyzed. 
Review of immunogenicity data will not be done until after specimens are collected at Day 63.   
Analysis of immunology data will include: 
 
Prevalence of 4-fold increases of anti-LPS and anti-invaplex IgG, IgA and IgM in ALS and 
serum/plasma over baseline (prevaccination) titers. Prevalence of 4-fold increase in LPS and invaplex-
specific IgA in stool over baseline titers. 
Analysis of vaccine shedding data will include: 
 
Prevalence and distribution of vaccine shedding as determined by quantitative culture and PCR  
For exploratory Endpoints 
• 
Prevalence of 4-fold increase in SBA antibody titers from baseline (seroconversion);  
• 
The cytokine data will be analyzed by generalized estimating equation model between placebo and 
vaccine groups, (cytokine assessment at (baseline and post vaccination days) and the interaction between two 
groups). 
12.3.1 General Analytic Plan Associated Assessment of Safety and Immunogenicity 
When the use of descriptive statistics to assess group characteristics or differences is required, the following 
methods will be used: for categorical variables, the number and percent in each category; for continuous 
variables, mean (with standard deviation) or median (with quartiles), and range (minimum, maximum).   

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Within-arm assessment of the change from the baseline measurement to a follow-up measurement will be 
analyzed using McNemar’s test (for categorical response variables), or the paired t-test or Wilcoxon signed-
ranks test (for continuous variables) or generalized estimating equation model.   
12.3.2 Final Analysis Plan 
A thorough Statistical Analysis Plan will be developed before any data reports are produced. The analysis of 
safety will include inpatient and outpatient observations and interviews with participants through Day 84. 
Analysis of immunogenicity will include all participants who provide specimens through Day 84.  The 
following specific outcomes will be analyzed: 
 
Rate of occurrence of reactogenic side effects and AEs.  Rates and 95% CIs will be described.   
 
Rate of seroconversion to LPS and invaplex by IgG, IgA and IgM ELISA at multiple time points.  A 
response for serum/plasma antibodies will be defined as a four-fold rise after immunization over 
baseline (prevaccination) titers.   
 
Prevalence of 4-fold increases of anti-LPS and anti-invaplex IgG, IgA and IgM in ALS over baseline 
titers. 
 
Rate of fecal response to LPS and invaplex by IgA ELISA at multiple time points.  Fecal antibody 
responses will be defined as a four-fold rise for specific IgA or a four-fold rise of the ratio of specific 
over total IgA over baseline titers at any time point after immunization. 
 
Comparison of cytokine response at multiple time points between vaccine and placebo groups. 
 
Rate of SBA seroconversion at multiple time points. 
13.0 DATA SAFETY MONITORING PLAN (DSMP) AND DATA MANAGEMENT  
The PI is responsible for ensuring the accuracy, completeness, and timeliness of the data reported.  Data 
collection is the responsibility of the clinical trial staff at the study site under the supervision of the study PI.  
Emmes is responsible for electronic data management, quality review, final safety analysis, and reporting of 
the study data.  
13.1 Database Development  
Case Report Form 
The eCRFs will be developed by Emmes and approved by PATH Vaccine Solutions.  
Completion of CRFs 
Electronic Data Capture (EDC) will be the method of data collection in this study.  Data for each participant 
will be recorded in the eCRF and verified by the Investigator.  It is the Investigator’s responsibility to ensure 
the accuracy, completeness, legibility, and timeliness of the data reported in the participant’s eCRF and any 
supporting documentation.   
Source documentation supporting the eCRF data should indicate the participant’s participation in the study 
and should document the dates and details of study procedures, AEs and the participant’s status.  The 
Investigator at icddr,b will maintain all information in the eCRFs and all source documents that support the 
data collected from each participant. 
The Investigator or designated representative should complete the eCRFs as soon as possible after 
information is collected.  Completed eCRFs must be submitted for each screened participant who signs the 
study specific ICF and is vaccinated.  The PI/Investigator will retain all essential documents and a CDROM 
copy of the eCRF data. 
Provider of Data Management 
Data Management activities will be provided by Emmes and according to SOPs. 
Details of Data Management 
A Data Management Plan (DMP) will be written by Emmes and contain all study-specific requirements.   
 

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Coding 
Coding of the AE and concomitant medication data will be done by Emmes. The most recent versions of the 
coding dictionaries will be used: MedDRA for coding of the AEs and WHO Drug for the coding of the 
concomitant medications.  Once all coding has been completed, listings with the coded data will be provided 
to the Sponsor for review and approval by the Sponsor Medical Monitor. 
13.2 Data Validation 
Emmes will confirm that all data entered into the database are correct and consistent with CRFs.  
13.2.1 Source Data Verification 
For source data verification (SDV), the monitor (on behalf of the study sponsor) must have direct access to 
source documents that support the data recorded, e.g. medical records, original laboratory records and ICFs. 
If source data are electronic, these data must be printed, signed and dated by Investigator and stored in the 
Investigator study file. Clinical laboratory data will remain in study participant records. Essential documents, 
including ICFs, must be filed and kept in the Investigational Study File (ISF) on an ongoing basis. 
13.2.2 Definition of Source Data  
Source data are all information in original records or certified copies of original records of clinical findings, 
observations, or other activities in a clinical study.  Source data are contained in source documents.   
 
13.2.3 Definition of Source Document 
Original source documents, data and records, e.g., hospital records, medical notes, laboratory notes, 
evaluation checklists, pharmacy dispensing records, records kept at the pharmacy and at the laboratory, 
documentation of shipments etc.   
13.3 Database Locking Procedures 
 
A final database lock, for the primary analysis will occur after all participants have completed all 
follow-up visits and a case by case review of the severity of any AEs has been performed and 
finalized.  
 
Remaining immunology data will be maintained in a separate immunology database.  
13.4 Record and Specimen Archival  
The Investigator is responsible for retaining all essential documents listed in the International Conference on 
Harmonization (ICH) Good Clinical Practice (GCP) Guideline.  Study documents will be retained for a 
minimum of 2-years after the last approval of a marketing application in an ICH region and until there are no 
pending or contemplated marketing applications in an ICH region or at least 2 years have elapsed since the 
formal discontinuation of clinical development of the investigational product. No records will be destroyed 
without the written consent of the IND sponsor, PVS. It is the responsibility of the Sponsor to inform the 
investigator when these documents no longer need to be retained.  
13.5 Screen Failures 
If a participant signs the study specific ICF but is not randomized because of non-eligibility (a screen failure), 
the reason for his/her non-eligibility should be entered in the medical records/notes/charts.  Also, a screening 
log must be kept.  Screen failure data will not be recorded in the eCRFs.   
13.6 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 participant, the investigator, or the study 
site staff. As a result of deviations, corrective actions are to be developed by the site and implemented 
promptly. Major deviations from the protocol, as defined as deviations that impact participant safety or 
endpoints, will be promptly reported to the Sponsor, WIRB, and DSMB. Minor deviations from the protocol 
will be recorded on a source document and corresponding eCRF, and included in the annual report. Examples 
of minor deviations are events outside the control of the investigators (e.g., participant missed visit window 
or study parameters that are not part of the primary endpoint was not performed at a visit).  

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14.0 OBLIGATIONS AND ROLES OF THE SPONSOR, INVESTIGATOR AND STUDY 
PERSONNEL  
This study will be conducted according to Good Clinical Practices and in accordance with all federal 
regulations regarding the protection of human participants in research including US 21 CFR Part 50 and US 
21 CFR Part 312.  The investigators agree to perform the research in strict accordance with this protocol, the 
International Conference on Harmonization (ICH) Guideline for Good Clinical Practice (E6). 
In addition, the investigator must follow local and institutional requirements including, but not limited to, 
investigational product, clinical research, informed consent and Investigational Review Board (IRB) 
regulations.  The Sponsor will provide notification to the investigator of protocol and amendment approvals 
by regulatory authorities when applicable.  
The MM will be responsible for reviewing all serious and unexpected adverse events and providing an 
unbiased written report of the event. 
15.0 ETHICS/PROTECTION OF HUMAN PARTICIPANTS  
Describe the justifications for conducting this research in human participants. If the study needs observations on sick individuals, 
provide sufficient reasons for using them. Indicate how participants’ rights will be protected, and if there would be benefit or risk to 
each participants of the study. Discuss the ethical issues related to biomedical and social research for employing special procedures, 
such as invasive procedures in sick children, use of isotopes or any other hazardous materials, or social questionnaires relating to 
individual privacy. Discuss procedures safeguarding participants from injuries resulting from study procedures and/or interventions, 
whether physical, financial or social in nature. [Please see Guidelines] 
15.1 Clinical Development Plan  
The proposed study builds upon successful preliminary observations with this vaccine in the US, Israel and 
Thailand (see WRSS1 Investigator’s Brochure) and in older endemic populations (adults and children in 
Bangladesh). Upon establishing a safety profile in the first two age groups (adults and children), and with 
concurrence with the DSMB, a separate protocol has now been developed to evaluate the WRSS1 vaccine in 
toddler group (12-24 months old). The new protocol seeks to examine critical variables to maximize immune 
responsiveness in young children, using approaches compatible with routine immunization practices.   
The overall long term strategy for development of a live Shigella vaccine includes defining the formulation of 
buffers and vaccine vehicles that permit low-volume administration and maximum viability of the vaccine, 
establishing the number of needed doses, and identifying a maximally immunogenic, safe dose level of this 
WRSS1 prototype strain in the target population. WRAIR is currently developing the S. flexneri vaccine 
strains to produce a broadly protective multivalent oral shigella vaccine. 
15.2 Ethical Conduct of the Study 
This study will be conducted using Good Clinical Practice (GCP) and in accordance with the World Medical 
Association (WMA) Declaration of Helsinki and all US federal regulations regarding the protection of human 
participants in research, including The Nuremberg Code, The Belmont Report, US 21 CFR Part 50 and US 
21 CFR Part 312 - Protection of Human Participants and 32 CFR 219 (The Common Rule).  
The investigator agrees to conduct the research in strict accordance with this protocol, the ICH Guideline for 
GCP, as well as in conformity with any US federal and Bangladeshi provincial or local regulations regarding 
the conduct of clinical studies.  The sponsor and investigator must comply with all applicable regulations.  In 
addition, the investigator must follow local and institutional requirements including, but not limited to 
Investigational Medicinal Product (IMP), clinical research, Informed Consent Forms (ICF) and Institutional 
Review Board (IRB) regulations.  
The Sponsor will provide notification to the investigator of protocol and amendment approvals by regulatory 
authorities.  Except where the investigator's signature is specifically required, it is understood that the term 
"investigator" as used in this protocol and on the electronic Case Report Forms (eCRFs) refers to the 
investigator or appropriate study personnel that the investigator designates to perform a certain duty.  
Nevertheless, the investigator alone is ultimately responsible for the conduct of all aspects of the study.  Sub-
investigators or other appropriate study personnel are eligible to sign for the investigator on designated 
eCRFs.  

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15.3 Institutional Review Board 
The icddr,b Research Review Committee (RRC); and icddr,b Ethics Review Committee (ERC) and Western 
Institutional Review Board (WIRB) will review the protocol.  
15.4 Informed Consent Process  
The study staff will discuss the informed consent document in detail with the parents of potential participant. 
Group discussions may also take place to orient participant to the study; however individual discussions will 
commence prior to obtaining consent signatures. Before any study-related activities and in agreement with 
applicable regulatory requirements, the Investigator must ensure that the parents of participants are fully 
informed about the aims, procedures, potential risks, and potential benefits of the study.  The parents will be 
given the written, IRB approved ICF, allowed ample time to read the consent, encouraged to ask questions 
about the study, have the questions answered and then be given time to decide if they would allow their 
children to participate in the study.  It will be emphasized that participation is voluntary, and that the parents 
of the participant has the right to withdraw their children from the study at any time without prejudice. 
The Investigator must obtain voluntary, personally signed and dated informed consent from parents before 
any study-related procedure.  The original, signed ICF must be kept in the site study file.  
15.5 Risk/Benefit  
The WRSS1 vaccine has been administered previously to participants at doses equal to or higher than the 
doses to be used in this study. Participants may experience fever and/or diarrhea post-vaccination, and 
possibly dehydration requiring oral or intravenous rehydration. Nausea, abdominal cramps and malaise are 
also possible post-vaccination. There is a theoretical risk of an oral vaccine causing intussusceptions which 
requires emergency management. There is also a rare risk that an oral live attenuated vaccine may cause 
seizures or reactive arthritis. Good phlebotomy practices will be performed during blood draws, to minimize 
the risk to the participant.  Participants and staff will be trained in proper hand washing techniques.   
The results of this study will provide additional information for obtaining potential immune responses in 
endemic populations in an effort to overcome mucosal administration issues such as enteropathy. This 
information will contribute to the scientific enteric vaccine field overall and potentially inform public health 
professionals for establishing immunization schedules and policies for at risk populations.   
16.0 USE OF ANIMALS 
Describe if and the type and species of animals to be used in the study. Justify with reasons the use of particular animal species in 
the research and the compliance of the animal ethical guidelines for conducting the proposed procedures. 
No animals will be used in this study.  
17.0 PUBLICATION POLICY 
PATH Vaccine Solution (PVS) has registered this trial on www.ClinicalTrials.gov accordingly to the trials 
registration policy (NCT01813071). Upon abstract and/or manuscript development by the investigators from 
icddr,b, PVS must review and approve any abstract and/or manuscript prior to publication as stated in the 
clinical trial agreement. Sub studies, if any, will have to be concurred by icddr,b and PVS.  
18.0 FACILITIES AVAILABLE 
Describe the availability of physical facilities at site of conduction of the study. If applicable, describe the use of Biosafety Level 2 
and/or 3 laboratory facilities. For clinical and laboratory-based studies, indicate the provision of hospital and other types of adequate 
patient care and laboratory support services. Identify the laboratory facilities and major equipment that will be required for the study. 
For field studies, describe the field area including its size, population, and means of communications plus field management plans 
specifying gender considerations for community and for research team members. 
The urban field site in Mirpur, a suburb of Dhaka in Bangladesh will be used for recruitment, screening, and 
consenting, outpatient dosing and/or assessment.  A team of field staff will be involved in initial selection of 
participants and visit participants’ home. One/two physicians and 1/2 nurses will be present during screening, 
and consenting, outpatient vaccination and/or assessment.  The data entry of outpatient dosing and/or 
assessment will be done in level 8 of the main icddr,b building.  The Mirpur site has been used for intensive 
studies of cohorts of children including epidemiology of amoebiasis using molecular techniques; Phase1/2 
studies of new vaccines for enterotoxigenic E. coli and rotavirus, and community-based interventions for 

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malnutrition. Children will be brought to the ICU of Dhaka Hospital in case of emergency when beds in the 
ICU are available.  
For each cohort, the first dose of vaccine and subsequent inpatient follow-up period will take place at the 
icddr,b Clinical Trials Unit (CTU) located on the icddr,b main campus and is about 150 yds from the main 
building. One full-time physician and 2 nurses will be available at the CTU during the entire period of 
inpatient phase. Another 3-4 physicians and nurses can be called from the main hospital during the inpatient 
days in CTU. icddr,b can provide the services of security guards, attendants, cook etc. The CTU has 18 beds 
(9 for females and 9 for males), patient exam room for vital signs recording and observation, dining area, 
recreation area, kitchen and wash rooms for the participants on the same floor. The sample processing room, 
and storage of study medication (refrigerator and ambient storage space available), document storage 
cabinets, store room, and data entry room are also located inside the CTU. The data entry for the Inpatient 
duration will be done at the CTU.  The participants are provided with individual lockers for storing their 
belongings during the admission to the facility (total 18 lockers available).The participants are provided with 
dress gowns at the time of admission to the facility and are not allowed to go out of the facility during the 
inpatient phase. There is a separate recreation room for the volunteers with facilities of TV, magazine and 
indoor games. The CTU also has a separate dining area for the volunteers, which is close to the kitchen. Fire 
extinguishers are available to deal with any fire emergencies. A list of all emergency contact numbers are 
available in the patient exam room. A physician and 2 nurses will be available at the CTU during the entire 
period of inpatient phase. The study volunteer, in case of an emergency, will be directly transferred to the 
Dhaka hospital located on the ground floor of the icddr,b’s main building on campus. An emergency trolley 
(Crash cart) will be available throughout the inpatient period, equipped with an ambu bag, laryngoscope, 
intubation tubes, emergency medicines, ECG machine, oxygen cylinders and oxygen masks, nebulizer, 
infusion equipment, and disposable syringes. 
Laboratory analysis of specimens for screening and safety will be carried out in the Clinical Hematology and 
Microscopy Lab, the Clinical Biochemistry Lab, and the Clinical Microbiology and Immunology Lab under 
the Clinical Laboratory Services. Analysis of immunology specimens, and stool culture/PCR will take place 
at the icddr,b INT Laboratory and Enteric Microbiology Laboratory, respectively, under the direction of the 
PI, Dr. Rubhana Raqib.  
Vaccine preparation will take place at the INT Laboratory under BSL-2 conditions and transported to the 
CTU for dose administration within 2 hours of reconstitution according to the vaccine preparation SOP.  

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19.0 Literature Cited 
Identify all cited references to published literature in the text by number in parentheses. List all cited references sequentially as they 
appear in the text. For unpublished references, provide complete information in the text and do not include them in the list of 
Literature Cited. There is no page limit for this section, however, exercise judgment in assessing the “standard” length. 
 
1. Ahmed D, Hoque A, Elahi MS, Endtz HP, Hossain MA (2012) Bacterial aetiology of diarrhoeal 
diseases and antimicrobial resistance in Dhaka, Bangladesh, 2005-2008. Epidemiol Infect 
140: 1678-1684. 
2. von Seidlein L, Kim DR, Ali M, Lee H, Wang X, et al. (2006) A multicentre study of Shigella 
diarrhoea in six Asian countries: disease burden, clinical manifestations, and microbiology. 
PLoS Med 3: e353. 
3. Trofa AF, Ueno-Olsen H, Oiwa R, Yoshikawa M (1999) Dr. Kiyoshi Shiga: discoverer of the 
dysentery bacillus. Clin Infect Dis 29: 1303-1306. 
4. Kotloff KL, Winickoff JP, Ivanoff B, Clemens JD, Swerdlow DL, et al. (1999) Global burden of 
Shigella infections: implications for vaccine development and implementation of control 
strategies. Bull World Health Organ 77: 651-666. 
5. Johanson E WT (2009) Why children are still dying and what can be done. World Health 
Organization. 
6. Rahman M, Shoma S, Rashid H, El Arifeen S, Baqui AH, et al. (2007) Increasing spectrum in 
antimicrobial resistance of Shigella isolates in Bangladesh: resistance to azithromycin and 
ceftriaxone and decreased susceptibility to ciprofloxacin. J Health Popul Nutr 25: 158-167. 
7. Zafar A, Hasan R, Nizami SQ, von Seidlein L, Soofi S, et al. (2009) Frequency of isolation of 
various subtypes and antimicrobial resistance of Shigella from urban slums of Karachi, 
Pakistan. Int J Infect Dis 13: 668-672. 
8. Mandomando I, Sigauque B, Valles X, Espasa M, Sanz S, et al. (2007) Epidemiology and clinical 
presentation of shigellosis in children less than five years of age in rural Mozambique. 
Pediatr Infect Dis J 26: 1059-1061. 
9. Vu DT, Sethabutr O, Von Seidlein L, Tran VT, Do GC, et al. (2004) Detection of Shigella by a 
PCR assay targeting the ipaH gene suggests increased prevalence of shigellosis in Nha 
Trang, Vietnam. J Clin Microbiol 42: 2031-2035. 
10. Wang SM, Ma JC, Hao ZY, Zhang ZY, Mason C, et al. (2010) Surveillance of shigellosis by 
real-time PCR suggests underestimation of shigellosis prevalence by culture-based methods 
in a population of rural China. J Infect 61: 471-475. 
11. Lindsay B, Ochieng JB, Ikumapayi UN, Toure A, Ahmed D, et al. (2013) Quantitative PCR for 
detection of Shigella improves ascertainment of Shigella burden in children with moderate-
to-severe diarrhea in low-income countries. J Clin Microbiol 51: 1740-1746. 
12. Liu J, Kabir F, Manneh J, Lertsethtakarn P, Begum S, et al. (2014) Development and assessment 
of molecular diagnostic tests for 15 enteropathogens causing childhood diarrhoea: a 
multicentre study. Lancet Infect Dis 14: 716-724. 
13. Platts-Mills JA, Babji S, Bodhidatta L, Gratz J, Haque R, et al. (2015) Pathogen-specific 
burdens of community diarrhoea in developing countries: a multisite birth cohort study 
(MAL-ED). Lancet Glob Health 3: e564-575. 
14. I.J TKAaA (2012) Population genetics and molecular epidemiology of Shigella spp. In: Faruque 
SM, editor. Foodborne and Waterborne Bacterial Pathogens: Epidemiology and Molecular 
Biology. UK: Horizon Scientific Press Ltd. 
15. Khatun F, Faruque AS, Koeck JL, Olliaro P, Millet P, et al. (2011) Changing species distribution 
and antimicrobial susceptibility pattern of Shigella over a 29-year period (1980-2008). 
Epidemiol Infect 139: 446-452. 
16. Das SK, Ahmed S, Ferdous F, Farzana FD, Chisti MJ, et al. (2013) Changing emergence of 
Shigella sero-groups in Bangladesh: observation from four different diarrheal disease 
hospitals. PLoS One 8: e62029. 

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17. Holt KE, Baker S, Weill FX, Holmes EC, Kitchen A, et al. (2012) Shigella sonnei genome 
sequencing and phylogenetic analysis indicate recent global dissemination from Europe. Nat 
Genet 44: 1056-1059. 
18. Checkley W, Buckley G, Gilman RH, Assis AM, Guerrant RL, et al. (2008) Multi-country 
analysis of the effects of diarrhoea on childhood stunting. Int J Epidemiol 37: 816-830. 
19. Lorntz B, Soares AM, Moore SR, Pinkerton R, Gansneder B, et al. (2006) Early childhood 
diarrhea predicts impaired school performance. Pediatr Infect Dis J 25: 513-520. 
20. Pack R, Wang Y, Singh A, von Seidlein L, Pach A, et al. (2006) Willingness to be vaccinated 
against shigella and other forms of dysentery: a comparison of three regions in Asia. Vaccine 
24: 485-494. 
21. Venkatesan MM, Ranallo RT (2006) Live-attenuated Shigella vaccines. Expert Rev Vaccines 5: 
669-686. 
22. Hartman AB, Venkatesan MM (1998) Construction of a stable attenuated Shigella sonnei 
DeltavirG vaccine strain, WRSS1, and protective efficacy and immunogenicity in the guinea 
pig keratoconjunctivitis model. Infect Immun 66: 4572-4576. 
23. Barnoy S, Baqar S, Kaminski RW, Collins T, Nemelka K, et al. (2011) Shigella sonnei vaccine 
candidates WRSs2 and WRSs3 are as immunogenic as WRSS1, a clinically tested vaccine 
candidate, in a primate model of infection. Vaccine 29: 6371-6378. 
24. Bedford L, Fonseka S, Boren T, Ranallo RT, Suvarnapunya AE, et al. (2011) Further 
characterization of Shigella sonnei live vaccine candidates WRSs2 and WRSs3-plasmid 
composition, invasion assays and Sereny reactions. Gut Microbes 2: 244-251. 
25. Hale TL, Oaks EV, Formal SB (1985) Identification and antigenic characterization of virulence-
associated, plasmid-coded proteins of Shigella spp. and enteroinvasive Escherichia coli. 
Infect Immun 50: 620-629. 
26. Sansonetti PJ, Kopecko DJ, Formal SB (1981) Shigella sonnei plasmids: evidence that a large 
plasmid is necessary for virulence. Infect Immun 34: 75-83. 
27. Kotloff KL, Taylor DN, Sztein MB, Wasserman SS, Losonsky GA, et al. (2002) Phase I 
evaluation of delta virG Shigella sonnei live, attenuated, oral vaccine strain WRSS1 in 
healthy adults. Infect Immun 70: 2016-2021. 
28. Orr N, Katz DE, Atsmon J, Radu P, Yavzori M, et al. (2005) Community-based safety, 
immunogenicity, and transmissibility study of the Shigella sonnei WRSS1 vaccine in Israeli 
volunteers. Infect Immun 73: 8027-8032. 
29. Bodhidatta L, Pitisuttithum P, Chamnachanan S, Islam D, Venkatesan MM, et al. (2011) Safety, 
immunogenicity and efficacy of WRSS1 oral live attenuated Shigella sonnei vaccine in Thai 
adults. Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand. 
30. Rahman KM, Arifeen SE, Zaman K, Rahman M, Raqib R, et al. (2011) Safety, dose, 
immunogenicity, and transmissibility of an oral live attenuated Shigella flexneri 2a vaccine 
candidate (SC602) among healthy adults and school children in Matlab, Bangladesh. 
Vaccine 29: 1347-1354. 
31. Coster TS, Hoge CW, VanDeVerg LL, Hartman AB, Oaks EV, et al. (1999) Vaccination against 
shigellosis with attenuated Shigella flexneri 2a strain SC602. Infect Immun 67: 3437-3443. 
32. McKenzie R, Venkatesan MM, Wolf MK, Islam D, Grahek S, et al. (2008) Safety and 
immunogenicity of WRSd1, a live attenuated Shigella dysenteriae type 1 vaccine candidate. 
Vaccine 26: 3291-3296. 
33. Chandrasekaran L, Lal M, Van De Verg LL, Venkatesan MM (2015) A study of different 
buffers to maximize viability of an oral Shigella vaccine. Vaccine 33: 6156-6160. 
34. (1976) World Health Organization technical report series. Geneva, Switzerland: World Health 
Organisation. 594 594. 
35. Johnson RA, Uddin T, Aktar A, Mohasin M, Alam MM, et al. (2012) Comparison of immune 
responses to the O-specific polysaccharide and lipopolysaccharide of Vibrio cholerae O1 in 
Bangladeshi adult patients with cholera. Clin Vaccine Immunol 19: 1712-1721. 

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36. Cohen D, Ashkenazi S, Green MS, Gdalevich M, Robin G, et al. (1997) Double-blind vaccine-
controlled randomised efficacy trial of an investigational Shigella sonnei conjugate vaccine 
in young adults. Lancet 349: 155-159. 
37. Rahman MJ, Sarker P, Roy SK, Ahmad SM, Chisti J, et al. (2005) Effects of zinc 
supplementation as adjunct therapy on the systemic immune responses in shigellosis. Am J 
Clin Nutr 81: 495-502. 
38. Salgado-Pabon W, Konradt C, Sansonetti PJ, Phalipon A (2014) New insights into the crosstalk 
between Shigella and T lymphocytes. Trends Microbiol 22: 192-198. 
39. Raqib R, Lindberg AA, Wretlind B, Bardhan PK, Andersson U, et al. (1995) Persistence of local 
cytokine production in shigellosis in acute and convalescent stages. Infect Immun 63: 289-
296. 
40. Raqib R, Wretlind B, Andersson J, Lindberg AA (1995) Cytokine secretion in acute shigellosis 
is correlated to disease activity and directed more to stool than to plasma. J Infect Dis 171: 
376-384. 
 
 

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APPENDIX 1: Time and Event Schedule 
 
Screen 
Admission 
and 1st Dose 
Inpatient 
Discharge 
F/U Visit 
2nd Dose 
F/U Visit 
3rd Dose 
F/U Visit 
F/U Visit 
F/U Visit 
Visit Number 
Visit 01 
Visit 02 
Visit 03 
Visit 04 
Visit 05 
Visit 06 
Visit 07 
Visit 08 
Visit 09 
Visit 10 
Study Day 
Day -30 to -9 
Day 0 
Day 1 
Day 7 
Day 28 
Day 35 
Day 56 
Day 63 
Day 84 
Day 224 
Visit Window 
-30 to -9 days 
0 to 1 days 
0 
Visit 02 + 
6-8 days 
Visit 02 + 
27-29 days 
Visit 05 + 
6-8 days 
Visit 05 + 
27-29 days 
Visit 07 + 
6-8 days 
Visit 07 + 
27-29 days 
Visit 09 + 126-
154 days 
Informed Consent  
X 
Inclusion/Exclusion Criteria 
X 
X 
X 
X 
Medical History 
X 
Physical Exam 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Vital Signs1  
X 
X 
X 
X 
X 
X 
X 
X 
X 
Screening serology2 
X 
Hematology  
X 
X 
Clinical Chemistry 
X 
X 
Admission and familiarization  
X 
Randomization 
X 
WRSS1 Vaccine Dosing 
X 
X 
X 
Discharge from Inpatient Unit 
X 
Interim Medical Interview, Concomitant 
Medications review, and AE Review 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
Reactogenicity Assessment 
X 
X 
X 
X 
Field staff Visits  
X 
X 
X 
X 
X 
Stool Culture3 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Volunteer Memory Aid 
X 
X 
X 
Stool for IgA  (ELISA) 
X 
X 
X 
X 
X 
X 
X 
Stool Grading, frequency of defecation 
X 
X 
X 
X 
Stool for Shedding4 
X 
X 
X 
X 
X 
X 
X 
X 
Antibiotic Treatment  
X 
Serum/plasma Sample for Serology 
(ELISA) 
 
X 
 
X 
 
X 
 
X 
 
 
Whole Blood for ALS 
X 
X 
X 
X 
SAE/AE follow-up  
  
X 
X 
X 
X 
X 
X 
X 
 X 
X 
Total Blood Volumes (mL)5 
3.5 
5 
  
5 
  
5 
  
5 
  
  
 

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Purple shading indicates stool specimen collection  
 
Blue shading indicates vaccination time points 
 
Orange shading indicates blood specimen collection 
1 If a vital sign needs to be repeated it should be obtained within approximately 30 minutes of the original reading.  Only the vital sign that needs to be repeated will be repeated.  
Both the original and repeat measurements will be recorded in the paper source documents.  The second measurement will be input on the eCRF in the database. 
2 Screening serology will include anti-HAV, anti-HEV and HBsAg 
3 Stool culture for presence of any Shigella strain should also be obtained between Days -7 and -5 for continued eligibility prior to vaccination on Day 0 
4 Any participant found to still be shedding any vaccine strain at V84 will be treated with antibiotic therapy. 
5 Blood draw volumes for participants will not exceed 5 ml/kg per draw or 10 ml/kg in any 8 week period 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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APPENDIX 2: Clinical Toxicity Grading – CHILDREN (12-24 months) 
During screening, participants will have blood drawn to determine if any clinical laboratory abnormalities exist that would preclude study participation. Participants 
that have 2 mild (grade 1) abnormalities may be included if the Study Physician and Principal Investigator determines that their participation will not present undue 
risk to the participant. (Mild decreases in both hemoglobin and hematocrit will be counted as 1 abnormality). Participants with more than 2 mild abnormalities may be 
included in the study only with the consensus of the principal investigator and either the medical monitor of CRO or the study sponsor. This consensus will be 
documented with a note in the participant’s chart by the PI or designee. 
 
Solicited Lab AEs 
Grade 1 – Mild 
Grade 2 – Moderate 
Grade 3 - Severe 
Grade 4 – Potentially Life 
Threatening 
Hemoglobin  (gm/dl) 
9.5 - 10.9 
8.0 - 9.4 
6.5 – 7.9 
< 6.5 
Leukocytes (cells/mm3) 
 
 
 
 
Decreased 2,000-2,500 
1,500-1,999 
1,000-1,499 
< 1,000 
Increased 13,501-15,000 
15,001-20,000 
20,001-25,000 
 
Neutrophils (cells/mm3)* 
1,500-1,999 
1,000-1,499 
500-999 
<500 
Eosinophil (cells/mm3) – 
absolute values 
0.6 to 1.5 x 10 9/L 
1.5 to 5 x 10 9/L 
> 5 x 10 9/L 
>100.0 x 109/L 
Lymphocytes (cells/mm3) 
750-1,000 
500-749 
250-499 
<250 
Platelets/mm3 
100,000-125,000 
50,000-99,900 
25,000-49,999 
< 25,000 
Creatinine (mg/dl) 
1.1 – 1.3 x ULN 
1.4 – 1.8 x ULN 
1.9 – 3.4 x ULN 
≥ 3.5 x ULN 
ALT (SGPT) 
1.25 – 2.5 x ULN 
2.6 – 5.0 x ULN 
5.1 – 10.0 x ULN 
> 10.0 x ULN 
AST (SGOT) 
1.25 – 2.5 x ULN 
2.6 – 5.0 x ULN 
5.1 – 10.0 x ULN 
> 10.0 x ULN 
Bilirubin (Total) 
1.1 – 1.5 x ULN 
1.6 – 2.5 x ULN 
2.6 – 5.0 x ULN 
> 5.0 x ULN 
Gamma GT 
>ULN – 2.5 x ULN 
>2.5 – 5.0 x ULN 
>5.0 – 20.0 x ULN 
>20.00 ULN 
* Neutrophils include band cell (stab form) at all ages & a small number of metamyelocytes and myelocytes in the first few days of life. In healthy adults, 
1-5% bands are normally seen 
Source:                  
1. Wintrobe’s Clinical Hematology 12th Edition. 
2. Dacie and Lewis Practical Hematology 10th Edition. 
3. McPherson RA, Matthew R. Pincus MR. Henry's Clinical Diagnosis and Management by Laboratory Methods. 22nd ed. Philadelphia: Elsevier Saunders; 
2011. 254-5. 
4. Common Terminology Criteria for Adverse Events according to the National Cancer Institute, National Institutes of Health, U.S. Department of Health and 
Human Services. 
 
 

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APPENDIX 3: Grading for Reactogenicity events – CHILDREN (<36 months) 
Event 
Grade 1 – Mild 
Grade 2 – Moderate 
Grade 3 - Severe 
Grade 4 – Potentially Life 
Threatening 
General Severity Grading 
Event that is easily 
tolerated 
Event that interferes with 
daily activity, requires 
medication/therapy, or a 
change in 
medication/therapy 
Event that prevents daily 
activity and requires 
medical intervention 
Event that requires 
emergency room (ER) visit 
or hospitalization 
Systemic Reactogenicity 
Grade1 – Mild 
Grade 2 – Moderate 
Grade 3 - Severe 
Grade 4 – Potentially Life 
Threatening 
Fever (Axillary) 
≥100.4°F and ≤ 101.1°F 
(38.0-38.4°C) 
>101.1°F and ≤ 102.0°F 
(38.5-38.9°C) 
> 102.0°F – 104°F 
(39.0°C - 40°C) 
> 104.0°F 
(40°C) 
Loose stool 
Transient or intermittent 
episodes of unformed stools 
OR Increase of ≤3 stools 
over baseline per 24-hour 
period 
Persistent 
episodes 
of 
unformed to watery stools 
OR Increase of 4 to 6 stools 
over baseline per 24-hour 
period 
 
Increase of ≥7 stools per 24-
hour period OR IV fluid 
replacement indicated 
Life-threatening 
consequences 
(e.g.,hypotensive shock)) 
 
Abdominal pain 
Mild abdominal pain, No 
interference with activity 
Moderate abdominal pain, 
Some interference with 
activity  
Severe abdominal pain, 
prevents daily activity 
 
Nausea/Vomiting 
No interference with activity 
of 1-2 episodes/24 hours 
Some interference  
with activity or > 2  
episodes/24 hours 
Prevents daily  
activity, requires  
outpatient IV  
hydration 
ER visit or  
hospitalization for  
hypotensive shock 
Anorexia 
Loss of appetite without 
alteration in eating habits 
Oral intake altered without 
significant weight loss or 
malnutrition; oral nutritional 
supplements indicated 
Associated with significant 
weight loss or malnutrition 
(e.g., inadequate oral caloric 
and/or fluid intake); IV 
fluids, tube feedings or TPN 
indicated 
 
Bloating/abdominal  
Asymptomatic 
Symptomatic, but not 
Symptomatic, interfering 
 

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Distension/ excess flatulence 
interfering with GI function 
with GI function 
Constipation  
Occasional or intermittent 
symptoms; occasional use of 
stool softeners, laxatives, 
dietary modification, or 
enema 
Persistent symptoms with 
regular use of laxatives or 
enemas indicated 
Symptoms interfering with 
daily activities; obstipation 
with manual evacuation 
indicated 
 
Irritability  
Easily consolable 
Requiring increased 
attention 
 
Inconsolable 
 
Decreased activity 
Symptom present, but 
activity level unchanged 
Activity reduced, but still 
able to function 
Activity markedly reduced, 
bed ridden 
 
Reference. Terminology Criteria for Adverse Events (TCAE), In Trials of Adult Pancreatic Islet Transplantation, Version 5.0 (03 August 2011)