Evaluation of Typhoid Conjugate Vaccine (TCV) Effectiveness Among Bangladeshi Children Using the Test-negative Design
Phase: PHASE3
Conditions: Typhoid Vaccination, Typhoid, Typhoid Fever
Sponsor: International Centre for Diarrhoeal Disease Research, Bangladesh

 
 
 
 
 
RRC APPLICATION FORM 
RESEARCH PROTOCOL  
Number: PR-24059 
Version No. 1.3 
Version date: 15-07-2024 
 
FOR OFFICE USE ONLY 
RRC Approval: 
 Yes 
 No 
Date:13-06-2024 
ERC Approval: 
 Yes 
 No 
Date:18-07-2024 
AEEC Approval: 
 Yes 
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Date:      
External IRB Approval  
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Name of External IRB: 
___________________________________________ 
Will the protocol be submitted for expedited review?  
 Yes   
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If yes, please check all that apply:  
  Outbreak investigation  
  Pilot Study 
  Secondary Analysis 
  Student protocol 
  Formative research 
  Observational study 
  Short surveys 
 
 Approved by external IRB (local or abroad) except Randomized Clinical Trial  
 Others (explain the justifications):_______________________________  
** Cover Letter to RRC/ERC Chairperson through SDD must be attached. 
Protocol Title:* (maximum 250 characters including space) Evaluation of typhoid conjugate vaccine (TCV) 
effectiveness among Bangladeshi children using the test-negative design 
Short Title: (maximum 100 characters including space) ZyVac-TCV Bangladesh study 
Key Words:* Bangladeshi population, conjugate vaccine, immunogenicity, safety, typhoid fever 
Name of the Research Division Hosting the Protocol:* 
  Health Systems and Population Studies Division 
(HSPSD) 
  Nutrition Research Division (NRD) 
  Infectious Diseases Division (IDD) 
 
 
   Maternal and Child Health Division (MCHD) 
   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 2019-22):* (check all that apply)  

 
   Reducing maternal, neonatal and child mortality and 
improving the well-being of women, children and 
adolescents 
   Preventing and treating maternal and childhood 
malnutrition 
   Detecting and controlling enteric and respiratory 
infections 
   Detecting and controlling emerging and re-emerging 
infections 
   Achieving universal health coverage 
   Achieving gender equality and promoting 
sexual and reproductive health and rights 
   Examining the health consequences of and 
adaptation to climate change 
   Preventing and treating non-communicable 
diseases 
   Others (specify) 
________________________________ 
 
 
 
 
 
Research Phase (4 Ds):* (check all that apply) 
   Discovery 
   Development 
 
   Delivery 
   Evaluation of Delivery 
Anticipated Impact of Research:* (check all that apply 
and please provide details below) 
   Knowledge Production 
   Capacity Building 
 
 
   Informing Policy 
   Health and Health Sector Benefits 
   Economic Benefits 
Knowledge gap: Typhoid fever is a systemic illness caused by Salmonella enterica serotype Typhi (Salmonella 
Typhi) and Paratyphi A, B, C. It is estimated to affect more than 9 million people with 110,000 deaths worldwide 
annually, primarily in low- and middle-income countries in Asia and Africa. A multi-center study was conducted 
in Bangladesh, Nepal, and Malawi between 2016 and 2018. In Bangladesh, the overall incidence of typhoid fever 
was 161/100,000 person-years of observation with the highest incidence in children aged 5-9 years. The study has 
also reported 39% multidrug resistance (resistance to chloramphenicol, ampicillin, and cotrimoxazole) Salmonella 
Typhi strains from Bangladesh, which is a major public health concern for treating typhoid patients as well as for 
reducing typhoid burden. Current efforts to control typhoid fever recommend the joint delivery of effective 
vaccines against typhoid and improvements in water, sanitation, and hygiene. The World Health Organization 
(WHO) has recommended typhoid conjugate vaccines (TCVs) to be introduced in the routine and catch-up 
vaccination with TCVs in countries with a high burden of typhoid fever. 
Here, we propose to conduct a prospective closed cohort, open-label, phase III effectiveness study to evaluate the 
effectiveness and immunogenicity of the Typhoid Vi Conjugate Vaccine I.P. manufactured by M/s. Zydus 
Lifesciences Limited. 
Capacity building: The study will generate data on the effectiveness and immunogenicity of the Typhoid Vi 
Conjugate Vaccine I.P. manufactured by M/s. Zydus Lifesciences Limited named ‘ZyVac® TCV’. It will increase 
the supply of vaccines and will meet the demand for vaccines in Bangladesh and the region.  
Informing policy: The result will be disseminated to the Communicable Disease Control (CDC), Directorate 
General Health Services (DGHS), Government of Bangladesh (GoB), WHO, and the global community. 
Health and health sector benefit: The study results will be used to inform the country decision-making for the 
introduction of typhoid vaccine into the routine expanded programme on immunization (EPI) in Bangladesh. 
Economic benefits: Typhoid burden has negatively impacted the global economy. In the developing world, this 
impact is felt even greater. A safe and immunogenic vaccine against typhoid fever could reduce the burden of 
typhoid fever in Bangladesh and globally. 

 
List of Abbreviations 
AE 
Adverse event 
AEFI 
Adverse events following vaccination 
AR 
Adverse reaction 
ALS 
Antibody in Lymphocyte Supernatant 
ASC 
Antibody secreting cell 
BSL2 
Biosafety level 2 
CRF 
Case report form 
CI 
Confidence interval 
CDC 
Communicable Disease Control 
CTL 
Cellular Technology Limited 
DSMB 
Data and Safety Monitoring Board 
DGDA 
Directorate General of Drug Administration 
DGHS 
Directorate General of Health Services 
DSCC 
Dhaka South City Corporation 
ELISA 
Enzyme-linked Immunosorbent Assay 
ERC 
Ethical Review Committee 
EDC 
Electronic data capturing 
EPI 
Expanded programme on immunization 
ELISPOT 
Enzyme-linked immunospot assay 
FACS 
Fluorescence-activated cell sorting 
GAVI 
Global Alliance for Vaccines and Immunisation 
GOB 
Government of Bangladesh 
GMT 
Geometric Mean Titre 
GCP 
Good Clinical Practice 
GIS 
Geographic Information System 
GPS 
Global Positioning System 
HH 
Household 
icddr,b 
International Centre for Diarrheal Disease Research, Bangladesh  
ID 
Identification number 
ICF 
Informed Consent Form  
ICH 
International Council on Harmonization 
IRB 
Institutional Review Boards 
IMP 
Investigational Medicinal Product 
IgG 
Immunoglobulin G 

 
 
 
 
 
 
ISM 
Independent safety monitor 
IVI 
International Vaccine Institute 
LMP 
Last menstrual period 
MDR 
Multidrug- resistant 
MoU 
Memorandum of understanding 
MTA 
Material transfer agreement 
NIBSC 
National Institute for Biological Standards and Control 
pCRF 
Paper Case report form 
PI 
Principal investigator 
PCR 
Polymerase chain reaction 
RT-PCR 
Real time polymerase chain reaction 
RRC 
Research Review Committee  
SAE 
Serious Adverse Event 
SQL 
Structured Query Language 
SOP 
Standard operating procedures 
STRATAA 
Strategic Typhoid alliance across Africa and Asia 
TCV 
Typhoid conjugate vaccine 
TND  
Test negative design 
TT 
Tetanus toxoid 
TyVAC 
Typhoid Vaccine Acceleration Consortium 
WHO 
World Health Organization 
Vi-PS 
Vi polysaccharide 
Vi-TT 
Vi polysaccharide  conjugated to tetanus toxoid  
Vi-rEPA 
Vi-polysaccharide conjugated with a recombinant exoprotein A  from 
Pseudomonas aeruginosa 
VVM 
Vaccine Vial Monitor 
XDR 
Extensively drug-resistant 

 
Project Summary 
[The summary, within a word limit of 300, should be stand alone and be fully understandable.] 
Principal Investigator: Dr. Farhana Khanam 
 
Research Protocol Title: Evaluation of typhoid conjugate vaccine (TCV) effectiveness among Bangladeshi 
children using the test-negative design 
Proposed start date: 01-08-2024                                                                   Estimated end date: 31-05-2026 
Background (brief): 
a. Burden: 
Typhoid fever is a systemic illness caused by the human-restricted pathogen Salmonella enterica serotypes Typhi 
(Salmonella Typhi) and Paratyphi A, B, C. It is estimated to affect more than 9 million people worldwide 
annually, with an estimated 110,000 deaths per annum, primarily in low- and middle-income countries in Asia 
and Africa. A multi-center study was conducted in Bangladesh, Nepal, and Malawi under the Strategic Typhoid 
Alliance across Africa and Asia (STRATAA) consortium. In Bangladesh, the overall incidence of typhoid fever 
was 161/100,000 person-years of observation with the highest incidence in children aged 5-9 years. The study 
has also reported 39% multidrug resistance (resistance to chloramphenicol, ampicillin, and cotrimoxazole) to 
Salmonella Typhi in Bangladesh which is a major concern for treating typhoid patients as well as for reducing 
the burden of typhoid fever. Another multicentre study was conducted in Bangladesh, Nepal, and Malawi under 
the Typhoid Vaccine Acceleration Consortium (TyVAC) using Typbar-TCV. The study result shows 85% 
(97·5% confidence interval [CI] 76 to 91, p<0·0001), 81.6% (95% CI, 58.8 to 91.8; P<0.001) , and 80.7% (95% 
CI, 64.2 to 89.6) effectiveness in Bangladesh, Nepal and Malawi respectively. 
 
b. Knowledge gap: 
The World Health Organization (WHO) has recommended typhoid conjugate vaccines (TCVs) to be introduced 
in the routine and catch-up vaccination with TCVs in countries with a high burden of typhoid fever. But, there is 
a global shortage of vaccine to introduce in the endemic settings. It highlights the uncertain pace of country 
introductions, and the challenges countries face to adopt the vaccine.  
c. Relevance: 
An effective vaccination program in the higher risk populations will be useful to apply the cost-effective control 
measures. So, the evaluation of new vaccine effectiveness will be crucial to reduce the demand uncertainty and 
the need to improve forecast accuracy as well as concerns to ensure industry can plan supply availability. 
d. Hypothesis (if any): 
The typhoid Vi-polysaccharide conjugate vaccine will be effective in preventing typhoid fever among 
Bangladeshi children. 
 
e. Objectives: 
Primary objective: 
To evaluate the effectiveness of typhoid conjugate vaccine (TCV) against blood culture-confirmed typhoid fever 
using the test-negative design 
 
• 
To evaluate the effectiveness of typhoid conjugate vaccine (TCV) against blood culture-confirmed 
typhoid fever stratified by age groups (6 months to <2 years, 2 to 4 years, 5 to 15 years) 
• 
To evaluate the effectiveness of typhoid conjugate vaccine (TCV) against blood culture-confirmed 
typhoid fever stratified by the period of follow-up (first and second six months) after vaccination 
• 
To determine the immunogenicity of TCV in a subset of participants, stratified by age groups (6 
months to <2 years, 2 to 4 years, 5 to 15 years) 
 
 

 
f. Methods: 
This is a prospective closed cohort, open-label, phase III effectiveness study using a test-negative design of a 
typhoid conjugate vaccine, ZyVac® TCV (purified Vi capsular polysaccharide of Salmonella Typhi conjugated 
to tetanus toxoid as carrier protein), manufactured by Zydus Lifesciences Limited. The study will be conducted 
in a closed cohort population among children aged 6 months to 15 years residing in wards 5, 6, 7, 48, 49, 50, 63, 
71, and 72 of Dhaka South City Corporation (DSCC). The targeted number of age-eligible children in the study 
area is ~92,000 among them ~60,000 will be vaccinated. A subset of the first 600 consenting participants will be 
selected by age strata (6 months to <2 years, 2-4 years, 5-15 years) for enrollment in the immunogenicity study 
with an additional three follow-up visits. Diary cards will be used to collect adverse events (AEs) following 
immunization (AEFI) data up to day 7 for a subset of active follow-up of the first 600 vaccinated participants. 
Participants not in this subset will be encouraged to go to the ‘Adverse Event Monitoring Cell’ at the Maniknagar 
field office. Data on serious adverse events (SAEs) will be reported for six months after vaccination. All study 
updates including AEs and SAEs will be reported to the data safety and monitoring board (DSMB) and sponsor. 
Passive surveillance for typhoid fever will be carried out in the Maniknagar field office and Mugda Medical 
College and Hospital in the catchment area among the age-eligible children. 
 
g. Outcome measures/variables: 
 
Primary outcome 
Blood culture-confirmed typhoid fever 
 
Secondary outcome  
Anti-Vi-IgG antibody concentration 
Blood culture-confirmed paratyphoid fever  
 
 
 
 
 
 
 
 

 
Description of the Research Project 
 
Hypothesis to be tested: 
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)  
 
The typhoid Vi-polysaccharide conjugate vaccine will be effective in preventing typhoid fever among Bangladeshi 
children. 
 
Specific 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. 
 
Primary objective: 
To evaluate the effectiveness of typhoid conjugate vaccine (TCV) against blood culture-confirmed typhoid fever using 
the test-negative design 
 
Secondary objective: 
• 
To evaluate the effectiveness of typhoid conjugate vaccine (TCV) against blood culture-confirmed typhoid 
fever stratified by age groups (6 months to <2 years, 2 to 4 years, 5 to 15 years) 
• 
To evaluate the effectiveness of typhoid conjugate vaccine (TCV) against blood culture-confirmed typhoid 
fever stratified by the period of follow-up (first and second six months) after vaccination 
• 
To determine the immunogenicity of TCV in a subset of participants, stratified by age groups (6 months to <2 
years, 2 to 4 years, 5 to 15 years) 
 
Background of the Project including Preliminary Observations: 
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.  
 
 
Typhoid fever is a systemic illness caused by the human-restricted pathogen Salmonella enterica serotypes Typhi 
(Salmonella Typhi) and Paratyphi A, B, C [1]. Patients with typhoid fever typically experience symptoms such as fever, 
malaise, abdominal pain, and alternating constipation and diarrhea. Untreated or improperly managed cases can lead to 
severe complications, including intestinal perforation, bleeding, and encephalopathy. Approximately 3% to 5% of 
patients may become long-term asymptomatic carriers without proper treatment. Among these carriers, about 90% are 
likely to develop gallstones or gallbladder disease. The carrier state of typhoid, whether accompanied by gallstone 
disease or not, is a risk factor for gallbladder cancer. The highest incidence and mortality from typhoid fever occur in 
the developing regions of South Asia, Southeast Asia, and Africa, largely due to inadequate sanitation and water supply 
[1-3]. Annually, it is estimated that typhoid fever affects over 9 million people and causes 110,000 deaths globally. The 
incidence rates of typhoid fever are categorized as low (less than 10 cases per 100,000 population per year), medium 
(10-100 cases per 100,000 population per year), high (more than 100 but less than 500 cases per 100,000 population 
per year), and very high (over 500 cases per 100,000 population per year). Central Africa reports the highest rates, 
followed by certain areas in Central, South, and Southeast Asia. A multi-center study by the Strategic Typhoid Alliance 
across Africa and Asia (STRATAA) consortium, conducted in Bangladesh, Nepal, and Malawi, found that in 
Bangladesh, the overall incidence rate was 161 cases per 100,000 person-years, with the highest rates in children aged 
5-9 years. The study also highlighted a concerning 39% prevalence of multidrug-resistant Salmonella Typhi in 
Bangladesh, posing significant challenges for treatment and efforts to reduce the typhoid burden [4]. The diagnostic 
challenges in accurately identifying acute typhoid fever cases and asymptomatic carriers of Salmonella Typhi causes 
the rise of multidrug-resistant (MDR) strains (resistant to chloramphenicol, ampicillin, and cotrimoxazole) and 

 
extensively drug-resistant (XDR) strains (additionally resistant to fluoroquinolones and third-generation 
cephalosporins), are exacerbating the complexity of typhoid fever management. 
 
Historically, control of typhoid fever has been achieved through enhanced sanitation and infrastructure improvements, 
which have successfully eradicated the disease as a major public health concern in most developed nations. Despite this 
success, high-incidence regions face significant financial and logistical challenges in adopting these measures. 
Therefore, incorporating an effective vaccination program aimed at the most vulnerable populations could be a valuable 
and cost-efficient supplement to existing control strategies. Since typhoid-causing organisms are exclusive to humans, 
global eradication is feasible, and a potent vaccine could significantly advance this goal. In 1999, the World Health 
Organization (WHO) endorsed the use of new-generation vaccines for school-aged children in light of the persistent 
typhoid burden and the emergence of antibiotic-resistant strains. The WHO also advised immunization in regions with 
elevated typhoid rates or significant antibiotic resistance. Nonetheless, apart from specific areas in China, Vietnam, and 
India, this guidance has not been widely implemented in countries where typhoid is endemic [5].  
 
The WHO recommended that endemic countries consider the programmatic use of vaccines, to target only the high-
risk groups and populations for most countries with the strategies of school- or community-based vaccination or as 
appropriate depending on the local context such as disease incidence, age-specific disease burden, and percent of school 
enrollment population. Vaccination is also recommended for outbreak control [6]. In response to an outbreak of 
XDR Salmonella Typhi, that occurred in November 2016, in Hyderabad, Pakistan, the Aga Khan University Karachi 
in collaboration with the Ministry of health Sindh had initiated a mass immunization campaign with Typbar-TCV 
vaccine targeting children aged 6 months to 10 years [7]. Pakistan was the first country to introduce the Typhoid 
Conjugate Vaccine (TCV) into its routine immunization schedule in 2019 with GAVI Support. Following Pakistan, 
several other countries including Liberia, Malawi, Nepal, Zimbabwe (GAVI Supported), and Samoa (without GAVI 
support) have also incorporated TCV into their routine immunization programs [8, 9]. Currently, GAVI approved the 
vaccination campaign in Bangladesh to cover children aged 9 months to <16 years. Considering the global incidence, 
a huge bulk of WHO-prequalified vaccines is needed to control the disease. Different vaccine manufacturers step up to 
mitigate the short supply of the vaccine. Moreover, the 62nd ICC approved the TCV vaccine application, and the request 
was sent to the Gavi Secretariat on September 13, 2022. Since the TCV is going to be rolled out in Bangladesh with 
Gavi's support soon, it would be considered unethical to conduct a randomized controlled trial for the TCV. The test-
negative design (TND) approach allows researchers to leverage existing data and infrastructure without the need for 
extensive experimental settings and additional resources. In India, there are four licensed vaccines for typhoid fever. 
The ZyVac® TCV is one of them which was manufactured by Zydus Lifesciences Limited, Ahmedabad, India. There 
are several preclinical and clinical trials conducted using this vaccine. Currently, the company has applied for WHO 
pre-qualification which requires an effectiveness study. The purpose of this study is to evaluate the effectiveness of the 
typhoid conjugate vaccine (TCV) among Bangladeshi children using the TND.   
Currently available typhoid vaccines 
 
Inactivated whole-cell vaccine 
This vaccine consists of heat-phenol-inactivated whole-cell Salmonella Typhi, which is injected subcutaneously in two 
doses four weeks apart. It had an efficacy of 51-67% in controlled trials. It was associated with a high degree of 
reactogenicity, causing fever and systemic symptoms in 9-34% of recipients leading to school absence in 2-17% of 
cases [10]. Due to these side effects, it has largely dropped out of mainstream use, however, it is still used in several 
developing countries.  
 
Vi Polysaccharide vaccine (Vi-PS) 

 
Developed in the 1980s this vaccine consists of purified virulence factor (Vi antigen) capsular polysaccharide (Vi-PS) 
that forms the capsule of, and is specific to, Salmonella Typhi. It elicits a T-cell independent antibody response, which 
means it has poor immunological memory and repeat doses do not result in an additional boosting response [11, 12]. 
Similar to other polysaccharide vaccines, the Vi-PS vaccine is poorly immunogenic and not licensed for use in children 
under 2 years old, presumably due to the absence of specific splenic marginal zone B-cells that are needed to produce 
an immunological response to polysaccharides. In clinical trials, efficacies were of 64-72% [13-15]. Additionally, 
protection is short-lived, lasting only 2-3 years [16, 17]. 
 
Live attenuated oral vaccine (Ty21a) 
Also developed in the 1980s, this is an attenuated strain of Salmonella Typhi (Ty21a) that has had many virulence genes 
mutated chemically, including the gene leading to failure to produce the Vi antigen. Ingestion of this strain induces 
local gut mucosal immunity as well as systemic antibody and cell mediated response [18, 19]. The strain is lyophilized 
and administered in either an oral enteric capsule or a liquid solution and requires 3-4 doses to induce effective 
protective immunity. Clinical trials performed in Chile and Indonesia demonstrated Ty21a vaccine had a protective 
efficacy of 67% and 53%, respectively [20, 21]. While the enteric-coated formulation is difficult to administer to young 
children, the alternative liquid formulation is better tolerated but may be less immunogenic in younger children [19, 
22].  Ty21a vaccine is not licensed for children under the age of 6 years.  
 
Vi-rEPA vaccine 
This vaccine was developed by the US National Institute for Health (US NIH) in 1994 utilizing Vi-polysaccharide 
conjugated with a recombinant exoprotein A from Pseudomonas aeruginosa (rEPA) [23]. A two-dose schedule six 
weeks apart was shown to be highly immunogenic with a protective efficacy of 91.1% in children aged 2 to 5 years in 
a trial in Vietnam [24]. More recently, a study has demonstrated its immunogenicity in infants [25]. However, the 
licensure of Vi-rEPA has been delayed due to a lack of regulatory precedent for the use of rEPA carrier-based vaccines. 
 
Vi antigen typhoid conjugate vaccine (Vi-TCV) 
Typhoid conjugate vaccines consist of Vi polysaccharide antigen linked to different carrier proteins that can extend 
protection and allow immunization of infants and toddlers, which unconjugated Vi (T-independent antigen) cannot do 
successfully. Vi-TCV (Typbar-TCV) is a newly available vaccine developed by Bharat Biotech International Limited, 
India, consisting of 25µg of Vi polysaccharide antigen conjugated to a nontoxigenic tetanus toxoid carrier protein. 
Similar to other vaccines which are designed to protect against encapsulated bacterial pathogens and are conjugated to 
tetanus toxoid carrier proteins, Vi-TCV induces a T-cell dependent response. It can therefore produce an immunogenic 
response in infants under 2 years of age and has the potential to generate a durable immune response via induction of 
immunological memory. Vaccines should be stored at 2–8°C. This was first licensed in India in 2013 for use in 
individuals from 6 months of age up to 45 years of age. A Phase III randomized controlled trial comparing Vi-TCV 
with Vi-PS demonstrated seroconversion to anti-Vi IgG in the 6 month to 2 year age group [26]. Additionally, a 
comparison of the sub-groups receiving boosters of either vaccine at two years demonstrated significantly higher anti-
Vi IgG titers in the Vi-TCV group compared to the Vi-PS group (mean titre of 1685.3 EU/ml [95% CI: 1468-1797] in 
Vi-TCV vs 445.6 EU/ml [95% CI: 323-615] in Vi-PS) [26]. Safety data from the same study demonstrated that Vi-
TCV was well tolerated by all age groups and that there were no differences in the number or variety of adverse events 
reported between the vaccine arms [27, 28]. The multicentre study was conducted in Bangladesh, Nepal, and Malawi 
under the Typhoid Vaccine Acceleration Consortium (TyVAC) using Typbar-TCV. The study result shows 85% 
(97·5% CI 76 to 91, P <0·0001), 81.6% (95% CI, 58.8 to 91.8; P<0.001) and 80.7% (95% CI, 64.2 to 89.6, P<0.001) 
effectiveness in Bangladesh, Nepal and Malawi respectively [29-31]. 
 
Peda-Typh (BioMed) 
Another Vi-TT vaccine, Peda-Typh (BioMed). It consists of Vi coupled to tetanus toxoid (TT). This vaccine has been 
licensed only in India for children over 3 months of age [32], but this has not been prequalified by the WHO and only 

 
limited clinical data are available to document its safety and immunogenicity. Each dose of the vaccine contains 5µg 
of Vi polysaccharide of Salmonella Typhi conjugated to 5µg of tetanus toxoid.  
 
Vi-CRM (TYPHIBEV Biological E) 
Vi-CRM is a typhoid conjugate vaccine (TCV) manufactured by Biological E and developed in collaboration with the 
GSK Vaccines Institute for Global Health. It contains Vi polysaccharide from Citrobacter freundii, which is clinically 
indistinguishable from the Vi polysaccharide from Salmonella Typhi, conjugated to a variant of diphtheria toxin 
(CRM197) as the carrier protein. TYPHIBEV was prequalified by WHO in December 2020. Results from a Phase 2/3 
study conducted in India demonstrated that the immune response profiles of TYPHIBEV are comparable to Typbar-
TCV [33, 34].  This vaccine is likely to be the vaccine given during the national vaccination campaign in Bangladesh. 
 
 
Results of clinical study of the trial vaccine, ZyVac® TCV 
 
Phase II/III clinical trial: In this study, the seroconversion rate (proportion of subjects achieving ≥4-fold increase in 
anti-Vi IgG antibody titre) at 6 weeks post-vaccination in the subjects aged 6 months to 45 years (overall population), 
6 months to < 18 years (pediatric cohort) and 18 to 45 years (adult cohort) was 94.8%, 93.1%, 96.6% respectively. The 
seroconversion rate reported with ZyVac® TCV was non-inferior to that reported with the comparator TCV. In the 
subjects who had received ZyVac® TCV, the pre-vaccination geometric mean titre (GMT) of anti-Vi IgG antibodies 
reported were 7.6 EU/ml, 5.7 EU/ml, and 10.0 EU/ml in the overall population, pediatric cohort, and adult cohort 
respectively, while the GMT of anti-Vi IgG antibodies reported at 6 weeks post-vaccination was 1121.0 EU/ml, 891.1 
EU/ml and 1411.0 EU/ml in the overall population, pediatric cohort and adult cohort respectively. There was a 
significant increase in GMTs at 6 weeks post-vaccination as compared to pre-vaccination GMTs (P<0.0001). Both the 
pre-vaccination and post-vaccination GMTs reported in the subjects who had received ZyVac® TCV were comparable 
to the respective GMTs reported in the subjects who had received the comparator TCV in this study [35].  
 
Phase IV clinical trial: In this study, the subjects who had received primary vaccination with TCV in the previous phase 
II/III clinical trial were followed up to 3 years of post vaccination. 77.2% of the subjects who had received ZyVac® 
TCV in the previous phase II/III clinical trial and enrolled in this study had anti-Vi IgG antibody titre above the protocol-
defined cut-off titre of 10 IU/ml when assessed using the first WHO International Standard for anti-typhoid capsular Vi 
polysaccharide IgG (human) (NIBSC code 16/138) which is equivalent to the proposed seroprotective cut-off titre of 2 
μg/ml as derived from the studies of other TCV. The baseline GMT of anti-Vi IgG antibodies (3 years after primary 
vaccination) reported in this study in the subjects who had received ZyVac® TCV as a part of previous phase II/III 
clinical trial was 140.8 EU/ml. This baseline GMT was significantly higher as compared to pre-vaccination GMT 
reported for the same subjects in the previous phase II/III clinical trial (P<0.0001). A total of 17 subjects who had 
baseline anti-Vi IgG antibody below the proposed seroprotective cut-off titre were administered booster vaccination 
with ZyVac® TCV. All the subjects followed up at 10 days and 28 days after booster vaccination achieved 
seroconversion (≥4-fold increase in anti-Vi IgG antibody titre). The GMTs of antibodies reported at 10 days and 28 
days after booster vaccination were 2306.9 EU/ml and 1900.5 EU/ml respectively. The GMTs of antibodies after 
booster vaccination were higher than those reported after primary vaccination in the previous phase II/III clinical trial 
[35].  
 
Phase III clinical trial: The study was conducted in healthy adults aged 45 to 65 years, the seroconversion rate 
(proportion of subjects achieving ≥4-fold increase in anti-Vi IgG antibody titre) at 4 weeks post-vaccination was 94.1%. 
The seroconversion rate reported with ZyVac® TCV was non-inferior to that reported with the comparator TCV. In the 
subjects who had received ZyVac®TCV, the pre-vaccination GMT of anti-Vi IgG antibodies reported was 8.0 EU/ml 
while the GMT of anti-Vi IgG antibodies reported at 4 weeks post-vaccination was 1378.3 EU/ml. There was a 
significant increase in GMTs at 4 weeks post-vaccination as compared to pre-vaccination GMTs (P<0.0001). Both the 

 
pre-vaccination and post-vaccination GMTs reported in the subjects who had received ZyVac® TCV were comparable 
to the respective GMTs reported in the subjects who had received the comparator TCV in this study [35]. 
 
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.  
 
Study design 
This will be a phase III effectiveness study of the typhoid conjugate vaccine (Vi-TCV) (ZyVac® TCV). The study will 
be conducted in a closed cohort population of children aged 6 months to 15 years residing in a selected geographical 
catchment area in Dhaka, Bangladesh using a test-negative design (TND). The TND is a particular type of case-control 
study including controls who present themselves with similar signs and symptoms (to those of the cases) in the same 
healthcare facilities. Individuals with the disease of interest are tested with a laboratory assay where test-positive and 
test-negative individuals are considered cases and controls, respectively.    
 
Study area 
The study will be conducted in wards 5, 6, 7, 48, 49, 50, 63, 71, and 72 of Dhaka South City Corporation, covering an 
area of ~10.33 km2. No TCV campaigns have been conducted in this area. According to the  Population and Housing 
Census 2022, the total population within the selected geographical catchment area is ~465,000 with households of 
~122,000.  The proportion of males is 54 percent and the average household size is ~3.8 people. Assuming ~20% of 
the population is from the 6-month to 15-year age group, the expected number of age-eligible children is ~92,000.  
 
Baseline mapping 
A map of the study area will be created based on open-source georeferenced satellite images (source: Esri, Maxar, 
Earthstar Geographics, and the GIS User Community). The satellite image of the study area will be printed by separating 
it into several small pieces and provided to the field team for data collection. In this process field team will visit the 
entire area according to the map and draw the structure/building, road, and other salient geographic features in the 
printed images.  A unique geographical information system (GIS) identification number will be assigned for all the 
buildings/structures, and submitted to the GIS team to develop the digital vector data (point, line, and polygon feature). 
The digital data of structure/building (polygon feature) centroid x and y coordinates will be used to represent real-world 
coordinate values or GPS values. 
 

 
 
Figure: Study catchment area with health facilities 
Baseline listing 
All households in the study area will be assigned a unique identification number and all household members will be 
enumerated. Information about the age-eligible children including name, age, sex, date of birth, and parent’s name will 
be obtained. In addition, the name of the HH head, address, and cell phone number of HHs will be collected. The 
previously assigned GIS ID will be incorporated into the baseline listing form and the newly identified structure will 
be updated in the GIS database. We will take photos of birth certificates or national immunization cards to confirm the 
age of participants if available. Verbal consent will be provided by either the head of the household or a key informant.  

 
Participant card for potentially eligible children for vaccination  
After preparing the baseline list, we will identify the age-eligible (6 months to 15 years) participants. Then we will 
prepare participant cards for all eligible children which will be provided before the vaccination. There will be a unique 
identification number on the participant card which will be used during vaccination. A master list of potential eligible 
participants will be prepared.      
Vaccination 
 
Inclusion criteria for vaccination:  
The participant must satisfy all the following criteria to be eligible for enrolment: 
 
• Participant was included in the baseline list of the study population  
• Participants living within the study catchment area at the time of vaccination 
•  Parent/guardian is willing and competent to provide informed consent (if the participant is 11 to 15  years of age, 
assent will also be sought) 
• Participants aged between 6 months to15 years (i.e. up to 15 years 364 days) at the time of vaccination 
• Apparently healthy (no complaints of febrile illness) on the day of vaccination 
• Parent/guardian confirms that their child will be willing and be able to comply with study requirements  
Exclusion criteria for vaccination:  
The participant will not be enrolled if any of the following criteria apply: 
• Has knowingly received a typhoid vaccine in the past 
• Known allergy to any vaccine in the past 
• Medical or social reasons that will prevent the participant from conforming to the study requirements as judged 
by a medical professional 
• Planning to move away from the catchment area within the next 12 months 
• 
Pregnant at the time of vaccination, as confirmed by a urine test (urine pregnancy test will be done in girls who 
are married)  
• 
Confirmed or suspected immunosuppressive or immunodeficiency disorder; or subjects on any 
immunosuppressive or immunostimulant therapy  
• 
Subject participated in another clinical study in the past 3 months  
 
Temporary exclusion criteria for vaccination:  
Participants will be temporarily excluded from being vaccinated if, at the point of vaccination, any of the following 
apply: 
• Receipt of any other vaccines in the last 30 days 
• Current temperature of at least 38oC or reported fever within 24 hours prior to vaccination 
• Use of antipyretics within 4 hours prior to vaccination 
• Unmarried girls between the ages of ≥12 to 15 years old whose first day of their last menstrual period (LMP) is 
more than 28 days ago or who do not know the date they last menstruated upon presentation 
 
If these apply, the participant will be temporarily excluded from vaccination until the temporary exclusion criteria no 
longer apply. Please note, for children with fever above 38oC this must be a minimum of 48 hours after the fever has 
resolved. Unmarried girls between the ages of ≥12 to 15 years whose first day of their LMP is more than 28 days or 
who do not know the date they last menstruated upon presentation will be asked to return after starting their next 

 
menstruation for vaccination. A re-assessment will be conducted to ensure these temporary exclusion criteria no longer 
exist. 
 
Verification of the potentially eligible children 
All the households that have age-eligible children will be informed to visit the nearest vaccination centre. If a 
participant's birth certificate or national immunization cards were not provided during the baseline listing, we will 
notify their parents or guardians to bring them during vaccination. After arrival at the vaccination centre, the 
participant and vaccination ID will be verified with the listing database and the master list. Then the screening will be 
carried out according to inclusion/exclusion criteria. 
Informed consent 
The latest approved version of written informed consent/assent will be presented to the participant’s parent/guardian in 
the local language detailing no less than the exact nature of the study, what it will involve for the participant, the 
implications and constraints of the protocol, the known side effects and any risks involved in taking part. It will be 
clearly stated that the participant’s parent/guardian is free to withdraw their child from the study at any time for any 
reason without prejudice to future care, without affecting their legal rights, and with no obligation to give the reason 
for withdrawal. The participant and their parents/guardians will be allowed as much time as they wish to consider the 
information, within the recruitment period, and the opportunity to question the investigator or other independent parties 
to decide whether they will participate in the study. Written informed consent/assent will then be obtained, with 
additional opt-in consent for blood specimen, using participants’ parent/guardian's dated signature or thumbprint and 
dated signature of the person who presented and obtained the informed consent. Information sheets will be given to 
children aged 11 -15 years in a language that they can understand. The person obtaining consent will record informed 
assent on the consent form, for children aged 11 and older. The person who obtained the consent must be suitably 
qualified and experienced and have been authorized to do so by the Principal Investigator (PI). A copy of the signed 
informed consent will be given to the participant. The original signed form will be retained at the study site. A third 
party will act as a witness for the parent/guardian to attest that the information in the consent form and any other written 
information was accurately explained to, and understood by the parent/guardian and that informed consent was freely 
given by the parent/guardian. The witness will also sign and date the consent form. 
Screening and eligibility assessment 
An informed written consent will be obtained after confirming the participants by checking the database. After proper 
consenting process, a temperature will be taken and parents will be asked about antipyretic use and timings of previous 
vaccination to assess temporary exclusion criteria. A brief medical history will be taken and eligibility will be assessed 
against inclusion and exclusion criteria. Participants with temporary exclusion criteria will be informed and asked to 
return 48 hours after the fever has resolved or 30 days after the last vaccination was given for repeat assessment and re-
consent. Unmarried girls between the ages of ≥12 to 15 years whose first day of their LMP is more than 28 days or who 
do not know the date they last menstruated upon presentation will be asked to return after starting their next 
menstruation, for vaccination. A member of the study team will collect/edit demographic information (including age 
and address) and participant contact details by screening case report form (CRF). After completion of the eligibility 
checking, a sticker will be allocated for the participant which includes the information of vaccination ID and 
temperature; these will be used in the vaccine accountability log.  
 
Vaccination  
The vaccination team will finally check the identification and then administer the vaccine. After vaccination, 
information will be recorded in the vaccination CRF. If the participant is included in the immunogenicity study, a blood 
sample will be taken by a suitably trained staff member before vaccination. After administration of the study vaccine, 
participants will be asked to wait at least 30 minutes for any immediate hypersensitivity reactions or any other adverse 

 
events (AEs). The female participants of reproductive age should have been advised not to be pregnant within three 
months after the vaccination. 
Investigational Medicinal Product (IMP) 
Purified Vi capsular polysaccharide of Salmonella typhi conjugated to tetanus toxoid as carrier protein (Typhoid Vi 
Conjugate Vaccine I.P.). Trade name: ZyVac® TCV, Zydus Lifesciences Limited.  
Each 0.5ml vaccine dose contains: 
 
• 
Purified Vi-capsular polysaccharide of S.typhi 25 μg  
• 
conjugated to tetanus toxoid (carrier protein) 16 to 50 μg  
• 
2-Phenoxyethanol (as preservative) 2.50 mg  
• 
Isotonic buffer solution q.s.  
The vaccine is packaged as a pre-filled 2.5ml 5-dose vial. It will be administered as an intramuscular injection preferably 
in the anterolateral aspect of the middle thigh for younger children, or the upper arm for older children with aseptic 
precautions. 
Supply 
The Vi-TCV vaccine (ZyVac® TCV) will be provided by Zydus Lifesciences Limited.  
Storage  
The Vi-TCV study vaccine will be stored at 2º to 8º C in a temperature monitored icddr,b  cold room facilities, when 
not in use for daily activities. The vaccines will be stored in temperature-monitored refrigerators or cool boxes, when 
in use for daily activities. Each vial will be labeled with a “vaccine vial monitor”(VVM); a temperature-sensitive dot 
that provides an indication of the cumulative heat to which the vial has been exposed. It warns the end-user when 
exposure to heat is likely to have degraded the vaccine beyond an acceptable level and should not be used.  
 
Special precautions for storage  
Store at 2°C to 8°C. 
Do not freeze. Discard if frozen. 
Keep out of reach of children. 
Shake gently before use. 
Do not use the vaccine after the expiration date shown on the label 
 
Accountability of the study vaccine 
The vaccines will be shipped to a central storage facility in Bangladesh and passed through customs. It will then be 
transported and distributed to local vaccination sites whilst maintaining the cold chain (aiming for a temperature 
between 2º-8ºC). The number of doses of study vaccines that are received, used, and wasted will be documented daily 
during the study and checked weekly.  
 
Adverse events following immunization 
After administration of the study vaccine, participants will be observed closely for at least 30 minutes for any immediate 
hypersensitivity reactions or any other adverse events (AEs). All details will be recorded in the CRF.  
 
Adverse events follow up (Day 7, -1/+7):  
A subset of 600 participants will be selected for active surveillance follow-up and the immunogenicity sub-study. A 
diary card will be given to each participant on the day of vaccination. Participants will be contacted by telephone or in-
person after seven days of vaccination for follow-up and to record any adverse events following immunization (AEFI). 

 
The selection of these participants will be age-stratified (6 months to <2 years, 2-4 years, and 5-15 years) with an 
allocation of 200 in each age stratum.  
Parents of participants not in this subset will be encouraged to go to the ‘adverse event monitoring cell’ at the 
Maniknagar field office which will have a medical doctor and also available on-call 24 hours a day for adverse event 
monitoring throughout the whole vaccination period + 7 days.  
Information will include: 
• 
Verbal reconfirmation of consent for participation in the study. 
• 
Report from parent/guardian of adverse events related to vaccination and use of medications following 
vaccination.  
• 
After administration of TCV, solicited local AEs (pain, redness, swelling and induration) and solicited systemic 
AEs (fever, nausea, vomiting, diarrhea, headache1, malaise1, myalgia1, arthralgia1, abnormal crying2, loss of 
appetite2, drowsiness2 and irritability2 (1: applicable for subjects aged ≥2 years; 2: applicable for subjects <2 
years of age)) will be recorded for 7 days after vaccination. Unsolicited (other) AEs will be recorded for 28 
days after vaccination. 
• 
Reiteration of contact details and instructions to attend the Maniknagar field office in case of fever (≥2 days 
and/or temperature ≥38oC at presentation). 
Safety reporting: 
Definitions 
Below are the various categories of adverse events following immunization (AEFIs). 
Adverse event (AE) 
Any untoward medical occurrence in a participant to whom a medicinal product 
has been administered, including occurrences that are not necessarily caused by 
or related to that product. 
Adverse reaction (AR) 
 
An untoward and unintended response in a participant to an investigational 
medicinal product which is related to any dose administered to that participant. 
The phrase "response to an investigational medicinal product" means that a 
causal relationship between a study medication and an AE is at least a 
reasonable possibility, i.e. the relationship cannot be ruled out. 
All cases judged by either the reporting medically qualified professional or the 
sponsor as having a reasonable suspected causal relationship to the study 
medication qualify as adverse reactions. 
Serious adverse event 
(SAE) 
A serious adverse event is any untoward medical occurrence that: 
• results in death 
• is life-threatening 
• requires inpatient hospitalization or prolongation of existing 
hospitalization 
• results in persistent or significant disability/incapacity 
• consists of a congenital anomaly or birth defect. 
Other ‘important medical events’ may also be considered serious if they 
jeopardize the participant or require an intervention to prevent one of the above 
consequences. 

 
NOTE: The term "life-threatening" in the definition of "serious" refers to an 
event in which the participant was at risk of death at the time of the event; it 
does not refer to an event which hypothetically might have caused death if it 
were more severe. 
Causality 
The relationship of each adverse event to the study medication must be determined by a medically qualified individual 
according to the following definitions: 
 
Related: There is suspicion that there is a relationship between the study vaccine and the AE (without determining the 
extent of probability); there is a reasonable possibility that the study vaccine contributed to the AE 
 
Not related: There is no suspicion that there is a relationship between the study vaccine and the AE; there are other 
more likely causes and administration of the study vaccine is not suspected to have contributed to the AE 
Procedures for recording adverse events 
From vaccination through day 7 
After administration of TCV, solicited local AEs (pain, redness, swelling and induration), solicited systemic AEs (fever, 
nausea, vomiting, diarrhea, headache1, malaise1, myalgia1, arthralgia1, abnormal crying2, loss of appetite2, 
drowsiness2 and irritability2 (1: applicable for participants aged ≥2 years; 2: applicable for participants <2 years of 
age)) for 7 days after vaccination. Unsolicited (other) AEs will be recorded for 28 days after vaccination.    
For injectable vaccines, the local signs and symptoms to be documented usually include, as a minimum, pain, redness, 
and swelling at the injection site in all age groups. Measuring devices of various types may be used to record the extent 
of redness and swelling. Fever will be documented using digital thermometers and axillary temperature will be 
determined. For subjective symptoms (for example, fatigue and myalgia) following scoring system will be considered. 
 
The following scoring system will be used for grading of severity: 
Parameter 
Mild 
Moderate 
Severe 
Potentially life 
threating 
Injection site pain  
 
(pain without touching)  
Or  
Tenderness (pain when 
area is touched) 
Pain/tenderness  
causing no or 
minimal  
limitation of use of  
limb 
Pain/tenderness  
limiting use of 
limb OR  
Pain/tenderness  
causing greater 
than minimal 
interference with 
usual social & 
functional 
activities 
Pain/tenderness  
causing inability to  
perform usual 
social &  
functional 
activities 
Pain/tenderness 
causing  
inability to perform 
basic  
self-care function 
OR  
Hospitalization 
(other than 
emergency room 
visit) indicated for 
management of 
pain/tenderness 
Fever 
                                              
37.7 – 38.6°C 
38.7 – 39.3°C 
 
39.4 – 40.5°C 
 
> 40.5°C 
Malaise 
Symptoms causing 
no  
or minimal 
interference with 
usual social & 
Symptoms causing  
greater than 
minimal 
interference with 
usual social & 
Symptoms causing  
inability to perform 
usual social & 
functional 
activities 
Incapacitating 
fatigue/  
malaise symptoms 
causing inability to 

 
functional 
activities 
functional 
activities 
perform basic self-
care functions 
Myalgia  
 
Muscle pain 
causing no or 
minimal 
interference with 
usual social & 
functional 
activities 
 
Muscle pain 
causing greater 
than minimal 
interference with 
usual social & 
functional 
activities 
 
Muscle pain 
causing inability to 
perform usual 
social & functional 
activities 
 
Disabling muscle 
pain causing 
inability to perform 
basic self-care 
functions 
 
All adverse events related to vaccination, as judged by a medically qualified investigator, occurring during the first 7 
days post-vaccination that are observed by the study team/investigator or reported by the participant’s parent/guardian, 
will be recorded on the CRF. The information will be collected both passively and actively.  
For active surveillance of a subset of 600 participants, information will be actively collected via phone calls or home 
visits. Parents of participants not in this subset will be encouraged to go to the ‘Adverse Event Monitoring Cell’ at the 
Maniknagar field office which will have a medical doctor and also available on-call 24 hours a day for adverse event 
monitoring throughout the whole vaccination period + 7 days.  
The following information will be recorded: description, date of onset and end date, severity, assessment of relatedness 
to study medication, and action taken. Follow-up information should be provided as necessary. 
 
The severity of events will be assessed on the following scale:  1 = mild, 2 = moderate, 3 = severe, 4 = Potentially life-
threatening 
 
All Serious Adverse Events (SAEs) observed by the Investigators, members of the study team, or reported by the 
parent/guardian will be recorded on CRFs. 
 
Throughout the study period  
 
Serious Adverse Events (SAEs), as judged by a medically qualified investigator, observed by the investigator, or 
members of the study team, or reported by the parent/guardian, will be recorded on the CRF.   
The following information will be recorded: description, date of onset and end date, severity, assessment of relatedness 
to study medication, and action taken. Follow-up information should be provided as necessary. 
 
All SAEs will be recorded in the CRFs, for the entire study duration. Participants with SAEs will be followed by a 
medically qualified investigator either until resolution or the event is considered stable.   
Reporting procedures for serious adverse events  
The forms developed by the icddr,b institutional review board (IRB) will be used for reporting all SAEs.  
All SAEs occurring within the 6 months post-vaccine administration will be reported to the Data Safety Monitoring 
Board (DSMB), Directorate General of Drug Administration (DGDA), the PI, and the other study investigators within 
24 hours of the site study team becoming aware of the event. A more detailed report form will be completed and sent 
within the shortest period of the initial report, to all parties mentioned above. Additional and further requested 
information (follow-up or corrections to the original case) will be detailed in subsequent safety report forms. All SAEs 
must be reported to the study sponsor within 7 days. Summary reports will be submitted to the icddr,b IRB, and DGDA 
at the end of the study.  

 
All SAEs will be reported to sponsor at below mentioned email ID:   
drugsafety@zyduslife.com,  Pavankumar.Daultani@zyduslife.com  
Indication for antipyretics and analgesics 
 
Antipyretics will be used in case of fever (axillary temperature >38◦C/100.4◦F or feels hot to touch) following 
vaccination. Analgesics will be used in case of pain, or discomfort after getting vaccinated. 
 
Safety monitoring committee 
A DSMB will also be formed to oversee the safety component of the study. The DSMB members will not be involved 
in the study in any way. In addition, a physician with relevant study-related or therapeutic expertise will be identified 
as an independent safety monitor (ISM). The ISM will not be an investigator for this study. Under any circumstances, 
the identified ISM would be requested to carry out an independent assessment of the child presented with adverse 
events. The assessment details will be reported to the PI and DSMB. 
Pregnancy follow-up  
Follow-up on pregnancy outcomes will be conducted for all married girls identified during routine follow-up as either 
being pregnant at the time of vaccination or becoming pregnant within three months of vaccination. This follow-up will 
include monthly visits until the end of the pregnancy, with the final contact occurring after delivery or pregnancy 
outcome. Starting from the eighth month of gestation, additional weekly follow-ups will be conducted via phone calls. 
We will record pregnancy-related information considering it as a medically important event.   
 
Immunogenicity study 
A subset of 600 participants will be enrolled in the immunogenicity sub-study and will have an additional three face-
to-face follow-up visits at the field office at one month (D30/+7 days), at 6 months (D180+/-14 days), and at one year 
(D365+/-30 days) after vaccination. The first 200 participants from each age stratum (6 months to <2 years, 2 to 4 years, 
5 to 15 years) who give their consent will be selected and enrolled for the immunogenicity sub-study.  
 
At these additional visits, the following procedures will be performed: 
• 
Confirmation of continued participation in the study 
• 
Collection of blood (3ml for 6 months to 4 years and 5ml for 5 to 15 years )  
• 
Confirmation of contact details and reiteration of instructions to attend the health care facilities in case of fever 
for  ≥2 days and/or temperature ≥38°C at presentation. 
The above information will be recorded in the immunogenicity CRF. Parents/guardians of participants enrolled in the 
immunogenicity study will be given details of the follow-up appointments. 
 
Surveillance for enteric fever 
 
Surveillance for enteric fever will be undertaken at the Maniknagar field office and Mugda Medical College and 
Hospital for all age-eligible (from 6 months to 15 years) residents recorded in the baseline list. Participants with a 
subjective history of ≥2 days of fever and/or a temperature of ≥38°C at presentation will be enrolled in the surveillance. 
A blood sample (3ml) will be collected for microbiological culture. The participants will receive appropriate clinical 
management. Blood culture-positive patients will be visited at home to confirm their identity, and collect information 
about their illness and patients will be asked to visit the field office if their condition is not improved. The treatment 
will be adjusted as necessary, based on laboratory testing.  
Study staff will visit households, included in the baseline listing, periodically, to remind them to seek treatment at the 
Maniknagar field office if their children (both vaccinees and non-vaccinees) develop a fever. 

 
 
Eligibility criteria for enrollment in the passive surveillance  
 
• 
Participants information, available in the listing database   
• 
Parent/guardian is willing and competent to provide informed consent. For participants aged 11 to 15 years, 
informed assent will also be considered   
• 
Participants from the study area who experienced a fever lasting for ≥2 days and/or temperature ≥38oC at 
presentation [4, 30]  
• 
Participants aged 6 months to 15 years on the first day of the vaccination campaign 
 
Flow diagram 
 
 
 
Laboratory procedures 
 
Passive surveillance samples 
Microbiological culturing of blood samples collected from enrolled participants will be carried out using BACTEC or 
BacT/ALERT systems in the icddr,b Clinical Microbiology, Immunology and Molecular Diagnostic Laboratory. The 
sensitivity and specificity of the blood culture method are 59% and 100% respectively [36]. Organisms from the 
suspected colonies will be identified by the biochemical tests and final identification will be done by slide agglutination 
test with Salmonella-specific antisera (Denka Sieken, Tokyo, Japan). The pattern of antibiotic susceptibility of 
Salmonella Typhi and Salmonella Paratyphi causing enteric fever will be assessed by the disk diffusion method on a 
Mueller-Hinton agar plate with Oxoid disks containing ampicillin, cotrimoxazole, chloramphenicol, azithromycin, 
ceftriaxone, ciprofloxacin, nalidixic acid, and cefixime. The procedure will be carried out following GCP guidelines 
and standard operating procedures. The results of these tests will be recorded in the participant CRF. The isolated strain 
will be preserved at the Mucosal Immunology and Vaccinology Laboratory for further analysis.    
 
Immunogenicity study samples 

 
Blood samples taken for the immunogenicity sub-study will be transported to the icddr,b laboratory daily. The samples 
will be processed and stored by trained study staff, following standard operating procedures (SOP). The serum/plasma 
will be stored and used to measure the anti-Vi-IgG antibody using a commercially available Enzyme-linked 
Immunosorbent Assay (ELISA) kit (VaccZyme, The Binding Site). This assay will be performed according to the 
manufacturer’s instructions. Laboratory processes will be conducted at the Mucosal Immunology and  Vaccinology 
Laboratory at the icddr,b.  
Withdrawal of participants  
Participants’ parents/guardians can withdraw consent at any point. The investigator may also discontinue a participant 
from the study at any time if the investigator considers it necessary for any reason including: 
• 
Ineligibility (either arising during the study or retrospectively having been overlooked at screening) 
• 
Significant protocol deviation 
• 
Significant non-compliance with treatment regimen or study requirements 
• 
An adverse event or disease progression resulting in the inability to continue to comply with study procedures 
and follow-up 
Depending on which aspects participants wish to withdraw from, the withdrawal will result in the cessation of any 
follow-up calls, visits, or blood tests (as applicable to the subset). No further treatment is required in the study, so no 
additional action will need to take place.  Participants’ parents/guardians will have the choice when withdrawing, to 
withdraw from active study procedures only (follow-up calls and visits) but remain in passive surveillance for the 
primary outcome, (allowing us to access their hospital records and blood test results), or withdraw from all study 
contact. Data and blood samples collected prior to the time of participant withdrawal will be kept and analyzed as part 
of the study data. A participant who withdraws from the study has the option to re-engage at a future date if they choose 
to do so. All participants who withdraw from the study will be given information on how to re-engage with the study if 
they so choose. Reasons for withdrawal from the study, if known, will be recorded in the participant's CRF. 
 
Source documentation  
Each participant will have a complete source documentation of records including CRF, lab reports, and test results for 
the entire study period. 
 
Data management 
The investigators will populate the content of participants’ CRFs and all the study data will be recorded directly into an 
Electronic Data Capture (EDC) system (e.g. Octalsoft). Electronic devices and hard copies of CRFs will be used to 
collect and record data. Data will be collected online and uploaded to a cloud server regularly when electronic devices 
are brought back to the central field office, and reliable internet is available. Paper CRFs (pCRFs) will also be available 
as backup, in case of interruption of online data collection, pCRFs will be used for collecting data and data will then be 
entered into the EDC system when feasible. 
Baseline listing data will be collected offline on an electronic device with an internally designed Android application 
database SQLite and SQL Server. The system will have some logical checks incorporated in the system and it will be 
checked before saving to the server. Data will be checked periodically using the SQL scripts for detecting errors. Errors 
will be corrected by the user and transferred to the server again. An audit trail of correction will always be available in 
the system after correction of data. Data management staff will keep backups from the SQL server database regularly 
and store the backup on the desktop, laptop, and other removable devices.  
 
All other CRFs will be designed and maintained on EDC system, named Octalsoft, a secure web application for building 
and managing online surveys and databases. Quality control checks of the system will be performed on a regular basis. 
Both the EDC system (CRF data) and internally designed Android application will use a relational database via a secure 
web interface with data checks applied during data entry to ensure data quality.  

 
 
All participants will be identified by a unique trial-specific number of and/or codes; this will not include any identifiable 
information. Some personal information will be saved on the Android application designed by icddr,b. Only site 
research staff and sponsor data managers will have access to view the personal information.  
 
Trial staff will have access to both the internally designed Android application and Octalsoft via a unique username and 
password. Each trial staff member will have an appropriate level of access to CRFs and collected data, according to 
their roles and responsibilities within the trial.  
 
All participant data will be stored and maintained on local servers in icddr,b as mentioned above for the duration of the 
trial. Anonymized data will be shared with the sponsor in India. At the end of the trial, all individually identifiable data 
will be removed, and fully anonymized data will be retained for further analysis.  
 
 
 
 
 
 

 
Study timeline 
 
Activities 
Months 
1 2 3 4 5 6 7 8 9 
1
0 
1
1 
1
2 
1
3 
1
4 
1
5 
1
6 
1
7 
1
8 
1
9 
2
0 
2
1 
2
2 
Agreement  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Protocol 
development 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Regulatory 
approvals 
including RRC, 
ERC, and DGDA 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Staff recruitment 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
SOP and training 
materials 
development 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Training for 
vaccination and 
surveillance 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Listing of 
interested children 
for vaccination in 
the study area 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Vaccination 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
AEFI recording  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
SAE recording 
and reporting 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Diary card review 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Patient enrollment 
in the passive 
surveillance 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Patient 
management 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Sample collection 
for imunogenicity 
and passive 
surveillance 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Laboratory 
analysis 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Data cleaning     
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Development of 
statistical analysis 
plan       
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Data analysis    
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Clinical study 
report (CSR) 
development   
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Manuscript 
writing and 
submission 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Dissemination of 
findings in 
national and 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  

 
international 
community 
Sample Size Calculation and Outcome (Primary and Secondary) Variable(s) 
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. 
 
A sample size of 515 (103 cases and 412 controls) will be required in this case-control test-negative design study 
assuming vaccine effectiveness of 80%, vaccine coverage of 65% in the target age-eligible population (6 months to 15 
years), 4 controls per case, precision width of ± 10% (20%) and type 1 error rate of 0.05. Considering the incidence rate 
for typhoid fever of 230 per 100,000 person-years among the non-vaccinated population aged 6 months to 15 years, the 
target number of vaccinations is ~60,000.   
 
To achieve the target number of vaccinees aged 6 months to 15 years representing ~31% of the total population), the 
required age-eligible children and population of all age groups will be ~92,000 and ~297,000 respectively.  
 
 
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. 
 
Focal time  
Focal time will be defined as the date of onset of symptoms leading to presentation for care.  
 
Culture-positive case 
Eligible febrile patients who will be positive by blood culture for Salmonella enterica serotype Typhi and symptoms 
developed >14 days after vaccination, or, if unvaccinated, >14 days after the midpoint of the vaccination period. 
 
Culture-negative control 
Eligible febrile patients who will be negative by blood culture for Salmonella enterica serotype Typhi and symptoms 
developed >14 days after vaccination, or, if unvaccinated, >14 days after the midpoint of the vaccination period. 
Given that the sensitivity of the blood culture method is  ~59% [36], we will apply the clinical rule, defined in a previous 
analysis for blood culture-negative typhoid patients, to exclude clinical typhoid patients from the analysis [37]. This 
exclusion will be helpful for a better evaluation of the magnitude of protection conferred by the vaccine in the proposed 
study. 
 
Matching of cases and controls 
A statistician, blinded to the vaccination status of participants will select blood culture-negative controls (after 
excluding clinical typhoid patients, vide supra) against each case. Matching of controls for each case will then be done 
considering the age groups at focal time (6 months to <2 years; 2 to 4 years; and 5 to 15 years) and date of presentation. 
Controls will be randomly selected on the day of the presentation; if matched controls will not be available on that day, 
the selection period will be widened for up to a maximum of ±7 days. 
 
Statistical analysis  
For the evaluation of vaccine protection, a conditional logistic regression model will be used, considering matched case-
control status as the outcome and vaccination status and selected covariates as independent variables, taking non-receipt 
of vaccine as the referent category for assessment of vaccine protection. Odds ratios for the associations between 
vaccination and typhoid fever will be estimated by exponentiation of the coefficient for vaccination from the fitted 
model, and the standard error of the coefficient will be used to calculate 95% confidence intervals for the estimated 
effect. Protective effectiveness will be calculated as [(1 – odds ratio) X 100%]. The threshold of statistical significance 
will be considered at P <0.05.  

 
 
Analysis of immunogenicity and safety data  
Geometric Mean Titres (GMT) and seroconversion rate (≥4-fold rise of anti-Vi-IgG antibody titre induced on day 30 
compared to day 0 before vaccination) of anti-Vi-IgG antibody titre will be calculated. Analysis of covariance will be 
used to adjust for imbalances in baseline titres.  
 
In addition, descriptive analyses of safety data will be done for AEFI or SAEs cases. 
 
Shell table for effectiveness of the typhoid conjugate vaccine against culture-confirmed typhoid cases  
 
Overall period 
Typhoid Cases 
Matched controls 
Vaccine effectiveness (VE) 
(95% CI) 
Vaccinees 
n/N (%) 
Non-
vaccinees 
n/N (%) 
Vaccinees  
n/N (%) 
Non-
vaccinees 
n/N (%) 
Crude 
VE 
P 
value 
Adjusted 
VE 
P 
value 
All ages 
6 months - <2 
years 
2 - 4 years 
5 - 15 years 
First six months 
All ages 
Second six months 
All ages 
 
 
 
Data Safety Monitoring Plan (DSMP) 
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. 
 
Trial Monitoring  
The study will be conducted by the currently approved protocol, ICH GCP, relevant regulations, and standard operating 
procedures. Data will be evaluated for compliance with the protocol and accuracy of source documents. 
Monitoring will be performed by representatives of the icddr,b, and sponsor according to the principles of ICH GCP. 
The monitoring team will be independent and will not take part in any other study-related procedures. 
 Monitoring shall be carried out by the sponsor’s Clinical Research Associate/Study Monitor or by personnel designated 
by sponsor. The frequency and methodology of conducting periodic monitoring visit(s) shall be further described in the 
study specific monitoring plan. 
Quality Assurance  
A quality assurance audit of this trial will be conducted by the sponsor or sponsor’s designee. The quality assurance 
auditor will have access to all medical records, the investigator’s trial related files and correspondence, and the informed 
consent form documentation that is relevant to this clinical trial. 
 
Serious breaches 
A serious breach is defined as “A breach of GCP or the study protocol which is likely to affect to a significant degree  

 
(a) The safety or physical or mental integrity of the participants of the study; or 
(b) The scientific value of the study. 
If a serious breach is suspected, the DSMB, IRB, and Sponsor must be contacted within one working day.  
 
Protocol deviations  
A protocol deviation is any noncompliance with the clinical study protocol, GCP, or site SOP requirements. The 
noncompliance may be either on the part of the participant, the PI, or the study site staff. As a result of deviations, 
corrective actions are to be developed by the site and implemented promptly.  
These practices are consistent with ICH E6:  
• Compliance with Protocol 
• Quality Assurance and Quality Control 
• Noncompliance 
It is the responsibility of the site to exercise continuous vigilance to identify and report deviations. All deviations must 
be promptly reported via the appropriate CRF. All deviations from the protocol must be addressed in study participant 
source documents. A completed copy of reportable protocol deviation forms must be maintained in the regulatory file, 
as well as in the participant’s source document. Protocol deviations must be sent to the icddr,b Ethical Review 
Committee (ERC) as per its guidelines. The site PI/study staff will be responsible for knowing and adhering to the 
icddr,b ERC requirements. 
 
 
Ethical Assurance for Protection of Human rights 
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] 
 
Ethical and regulatory considerations 
 
Declaration of Helsinki 
The Investigator will ensure that this study will be conducted in accordance with the principles of the Declaration of 
Helsinki.  
 
Guidelines for Good Clinical Practice (GCP) 
The investigators will ensure that this study will be conducted in accordance of Good Clinical Practice and other relevant 
regulations. All personnel participating in the study will receive online GCP training to ensure their proficiency in these 
standards.  
 
Approvals 
The study protocol, informed consent, assent forms, and participant information sheet will be submitted to the Research 
Review Committee (RRC) and the Ethical Review Committee (ERC) of icddr,b for their approval. 
The Principal Investigator (PI) at icddr,b will submit and, where necessary, obtain approval from the above parties for 
all substantial amendments to the original approved documents. 
 
Reporting 
The PI will submit the reports to all committees (RRC, ERC, and sponsor) at the end of the study. 

 
 
Participant confidentiality 
The study staff will ensure that the participants’ anonymity is maintained.  The participants will be identified only by a 
participant/vaccination ID number on all study documents and any electronic database, including the CRF. All 
documents will be stored securely and only accessible by study staff and authorized personnel. The study will comply 
with the icddr,b data policy, which requires data to be anonymized as soon as it is practical to do so, and local 
regulations. 
Expenses and Benefits 
There will not be any payments or reimbursements made to participants, as an incentive for participant recruitment. It 
is anticipated that the provision of vaccination will be enough incentive to reach the necessary sample size. Local 
hospitals, clinics, and vaccination points are being used to deliver all study vaccines, which will not add additional 
travel or expense to participants and their families. The study will cover the costs of standard care treatment for 
participants as part of the study, including the cost of tests, antibiotics, and/or other prescribed medications, and in-
patient hospital stays and care, if medically necessary.   
 
Other ethical considerations      
All efforts will be made to conduct the research in a way that is sensitive to the Bangladeshi culture and social values. 
Trained study staff will be present at all times during the consent process, and the participant study-related materials 
(information sheet, consent forms, etc) will be printed in the local language. 
The study will be conducted based on the ethical requirements of the icddr,b. Children aged 6 months to 15 years have 
been selected for the study because of the substantial burden of the disease in both mortality and morbidity, with 
increased demand for the vaccines.   
All samples will be kept for a minimum of 5 years after the end of the study. Storage of these samples may also allow 
important future research to be done without needing to take new samples from the Bangladeshi healthy population.  
Potential participants or their parents/guardians will be notified that they will be able to refuse to have the relevant 
biological samples stored, without influencing participation in the study or the clinical care of their child. They will 
also be informed that should they no longer wish for their samples to be retained they may request their destruction.  
 
Use of Animals 
Describe if and the type and species of animals to be used in the study. Justify with reasons for the use of 
particular animal species in the research and the compliance of the animal ethical guidelines for conducting the 
proposed procedures. 
 
Not applicable. 
 
 
Collaborative Arrangements 
Describe if this study involves any scientific, administrative, fiscal, or programmatic arrangements with other 
national or international organizations or individuals. Indicate the nature and extent of collaboration and include 
a letter of agreement between the applicant or his/her organization and the collaborating organization. 
 
This project will be a collaborative study between icddr,b, and Zydus Lifesciences Limited. MTA, MoU, and other 
agreements are under process for this collaboration. After finalization, these documents will be submitted to the IRB. 
 

 
 
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. 
 
Existing field, hospital, laboratory, and office facilities are adequate and are outlined: 
 
Study field office 
The Maniknagar field office is located in the Dhaka Metropolitan area, approximately 9-10 km from the icddr,b Dhaka 
Hospital, and has been used for field studies since 2022. Dhaka South City Corporation is densely populated with 
approximately 3.5 million individuals. This research office has enjoyed a long-standing relationship with this stable 
community. The Maniknagar field office is composed of two floors, with each floor consisting of 3 rooms; 
approximately 3000 square feet of research space. This dedicated office building is situated within the same 
neighborhood as the study population. The facility contains a participant waiting area, an examination room, a staff 
work and file room, a specimen processing area, and a meeting room. The specimen processing area contains a 
refrigerator with 24-hour power backup. The building has 24-hour security coverage and internet service. The 
infrastructure of the facility has made it possible to carry out vaccine and other clinical studies. 
 
Laboratory facilities 
The Mucosal Immunology and Vaccinology Laboratory (MIVL) at the icddr,b is a Biosafety Level 2 (BSL2) facility 
with internal and external quality assurance carried out for tests. All SOPs for studies are updated frequently to meet 
study requirements. The laboratory is equipped with ten biohazard safety hoods for the processing of biological samples 
and for maintaining sterile conditions for specimen processing. For the fractionation of samples one bead beater is 
available, and for the separation of samples temperature-controlled tabletop centrifuges, high-speed centrifuges 
(Beckman) and ultracentrifuge (Beckman Coulter High Speed Centrifuge) are also available. Three Incubators with 
carbon-dioxide gas attachment for the study of B-cell and T-cell responses, four incubators and two shaker incubators 
for bacterial culture and Enzyme Linked Immunosorbent Assay (ELISA) are present. There is a cryostat for sectioning 
of frozen sections and a microtome for paraffin sections. Five ELISA readers (EON) linked to computers for 
determining antibody responses in study samples are present. Facilities for Enzyme Linked Immunosorbent Spot 
(ELISPOT) and Antibody in Lymphocyte Supernatant (ALS) assays are available. The Cellular Technology Limited 
(CTL) automatic immunospots counters as well as stereomicroscopes for enumeration of Antibody Secreting Cells 
(ASCs) are also available. There are facilities for carrying out the extraction of lymphocytes from gut biopsies. Two 
conventional polymerase chain reaction (PCR) machines, two real time PCR (RT-PCR) machines, and one TaqMan 
PCR machine are available for any preferable gene amplification. Two Luminex machine of xMAP company (Magpix 
and Intelliflex) and four Fluorescence-Activated Cell Sorting (FACS) machines (two Acuri, Fusion, and Aria III) are 
present for high throughput analysis. We have liquid nitrogen sample storage facility. In addition, facilities for carrying 
out intestinal lavages and fecal extracts are used for assessing mucosal antibody responses. There are several 
refrigerators and freezers maintained at 4°C, -20°C, and -80°C used for storing specimens from vaccine trials and other 
infectious disease studies. 
 
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. 
 
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