Phase 1 Randomized, Placebo-Controlled, Dose-Escalation Study of the Safety, Reactogenicity, and Immunogenicity of Trivalent (S. Enteritidis/S. Typhimurium/S. Typhi Vi) Conjugate Vaccine Against Invasive Salmonella Disease Administered Parenterally to Healthy U.S. Adults
Phase: PHASE1
Conditions: Risk Reduction
Sponsor: University of Maryland, Baltimore

 
 
 ____________________________________________________________________________________________ 
 
 ____________________________________________________________________________________________ 
1 
Phase 1 Randomized, Placebo-Controlled, Dose-Escalation 
Study of the Safety, Reactogenicity, and Immunogenicity of 
Trivalent Salmonella (S. Enteritidis/S. Typhimurium/S. Typhi 
Vi) Conjugate Vaccine against Invasive Salmonella Disease 
Administered Parenterally to Healthy U.S. Adults  
 
Protocol Number: Salmonella conjugates CVD 1000 
 
Clinical Principal Investigator: Wilbur H. Chen, MD, MS 
Center for Vaccine Development and Global Health 
University of Maryland School of Medicine 
685 West Baltimore Street, Suite 480 
Baltimore, MD 21201, U.S.A. 
 
IND Sponsor: University of Maryland, Baltimore 
IND Sponsor’s Representative: Myron M. Levine, MD, DTPH 
Center for Vaccine Development and Global Health 
University of Maryland School of Medicine 
University of Maryland, Baltimore 
685 West Baltimore Street, Suite 480 
Baltimore, MD 21201, U.S.A. 
 
In Collaboration With: Bharat Biotech International Ltd. 
Hyderabad, India 
 
Funding Source: The Wellcome Trust 
London, UK 
 
 
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STATEMENT OF COMPLIANCE 
This trial is to be conducted in accordance with Good Clinical Practices (GCP) as required by 
the following: 
• 
United States Code of Federal Regulations (CFR) applicable to clinical studies (45 CFR 
Part 46, 21 CFR Part 50, 21 CFR Part 54, 21 CFR Part 56, and 21 CFR Part 312) 
• 
International Conference on Harmonization (ICH) E6; 62 Federal Register 25691 (1997) 
• 
The Health Insurance Portability and Accountability Act (HIPAA) Privacy Rule-Final 
Modification (45 CFR  Parts 160 and 164) 
• 
The local laws and regulations, which prevail over the University of Maryland, Baltimore 
Compliance with these standards provides public assurance that the rights, safety, and well-
being of study subjects are protected, consistent with the principles that have their origin in the 
Declaration of Helsinki. 
All key personnel (all individuals responsible for the design and conduct of this trial) have 
completed Human Subjects Protection Training. 
 
 
 

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SIGNATURE PAGE 
The signature below constitutes the approval of this protocol and the attachments, and provides 
the necessary assurances that this trial is to be conducted according to all stipulations of the 
protocol, including all statements regarding confidentiality, and according to local legal and 
regulatory requirements and applicable United States of America federal regulations and ICH 
guidelines. 
 
Principal Investigator: 
_________________________________________________ 
 
Name / Title (Print) 
Signature: 
_________________________________________________ 
Date: 
_________________________________________________ 
 
 
 

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Table of Contents 
Statement of Compliance ...................................................................................................................... 2 
Signature Page ..................................................................................................................................... 3 
Abbreviations ........................................................................................................................................ 6 
Protocol Synopsis ................................................................................................................................. 8 
Key Personnel..................................................................................................................................... 11 
1 Background.................................................................................................................................... 12 
1.1 
Epidemiology and Burden of Disease .............................................................................. 12 
1.2 
Vaccine Development ..................................................................................................... 12 
1.3 
Pre-clinical Testing .......................................................................................................... 14 
1.3.1 
Rabbit non-GMP product study ........................................................................ 14 
1.3.2 
GLP Toxicology Study ...................................................................................... 16 
1.4 
Clinical Studies ............................................................................................................... 16 
1.5 
Risks and Potential Benefits ............................................................................................ 17 
1.5.1 
Risks................................................................................................................ 17 
1.5.2 
Potential Benefits ............................................................................................. 18 
2 Study Design ................................................................................................................................. 19 
2.1 
How the Trivalent Conjugate Vaccine is expected to be used in sub-Saharan Africa ........ 20 
2.2 
Work flow of the Phase 1 study in U.S. adults.................................................................. 21 
2.3 
Primary Objectives and Outcome Measures .................................................................... 23 
2.4 
Secondary Objectives and Outcome Measures ............................................................... 23 
2.5 
Exploratory Objectives and Outcome Measures .............................................................. 23 
3 Study Population ............................................................................................................................ 25 
3.1 
Inclusion Criteria ............................................................................................................. 26 
3.2 
Exclusion Criteria ............................................................................................................ 26 
3.3 
Enrollment and Randomization Procedures ..................................................................... 28 
3.3.1 
Re-screening procedure ................................................................................... 28 
3.4 
Withdrawals and Discontinuation from Study ................................................................... 28 
4 Investigational Products ................................................................................................................. 30 
4.1 
Description of Investigational Products ............................................................................ 30 
4.1.1 
Trivalent, with preservative (multi-dose vials).................................................... 30 
4.1.2 
Placebo............................................................................................................ 30 
4.2 
Formulation, Storage, Packaging, and Labeling ............................................................... 30 
4.2.1 
Trivalent, with preservative (multi-dose vials).................................................... 30 
4.2.2 
Placebo............................................................................................................ 30 
4.3 
Preparation and Administration ....................................................................................... 30 
4.4 
Study Product Accountability ........................................................................................... 31 
4.5 
Masking of the Study Product .......................................................................................... 31 
5 Study Procedures........................................................................................................................... 32 
5.1 
Clinical Evaluations ......................................................................................................... 32 
5.2 
Clinical Laboratory Evaluations ....................................................................................... 32 
5.3 
Research Laboratory Assays........................................................................................... 33 
6 Study Schedule .............................................................................................................................. 34 
6.1 
Screening Visit (Day -45 till -2) ........................................................................................ 34 
6.2 
Visit 1 - Enrollment & Vaccination Dose #1 (Day 1).......................................................... 34 
6.3 
Visit 2 Follow Up (Day 2) ................................................................................................. 35 
6.4 
Visit 3 Follow Up (Day 8 ±1) ............................................................................................ 35 
6.5 
Visit 4 Follow Up (for Cohorts A-B) or Vaccination Dose #2 (for Cohorts C and D) (Day 
29 ±3) ............................................................................................................................. 35 
6.6 
Visit 5 Follow Up, only for Cohort C and D (Day 36 ±1) .................................................... 36 
6.7 
Follow Up, Visit 5 for Cohort A-C, Visit 6 for Cohort C and D (Day 57 ±3) ........................ 36 
6.8 
Final Follow Up, Visit 6 for Cohort A-B (Day 180 ±14), Visit 7 for Cohort C and D (Day 
210 ±14) ......................................................................................................................... 36 
6.9 
Unscheduled Visits.......................................................................................................... 37 
6.10 Early Termination ............................................................................................................ 37 
7 Assessment of Safety..................................................................................................................... 38 
7.1 
Specification of Safety Parameters .................................................................................. 38 

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7.2 
Methods and Timing for Assessing, Recording, and Analyzing Safety Parameters........... 38 
7.3 
Halting Rules .................................................................................................................. 40 
7.4 
Safety Oversight ............................................................................................................. 40 
8 Assessment of Immunogenicity ...................................................................................................... 42 
8.1 
Enzyme-linked Immunosorbent Antibody Assay (ELISA) ................................................. 42 
8.2 
Opsonophagocytic Activity (OPA) assay .......................................................................... 42 
8.3 
Serum Bactericidal Activity (SBA) assay .......................................................................... 42 
8.4 
Antibody Secreting Cells (ASC) assay ............................................................................. 42 
8.5 
Circulating B cell memory cells (BM) and Cell-mediated Immunity (CMI) (cytokines, T 
cell effector and memory (including circulating T follicular/helper cells -cTfh-), T cell 
homing............................................................................................................................ 43 
8.6 
Antibodies in Oral Fluid ................................................................................................... 43 
9 Quality and Monitoring ................................................................................................................... 44 
9.1 
Quality Control and Quality Assurance ............................................................................ 44 
9.2 
Clinical Monitoring ........................................................................................................... 44 
9.3 
Site Monitoring Plan ........................................................................................................ 45 
10 Statistical Considerations ............................................................................................................... 46 
10.1 Study Hypothesis and Sample Size Considerations ......................................................... 46 
10.1.1 Sample size in Part 2 of the study .................................................................... 46 
10.2 General Statistical Principles ........................................................................................... 46 
10.3 Go/No-Go Milestones...................................................................................................... 47 
11 Ethics/Protection of Human Subjects .............................................................................................. 48 
11.1 Ethical Standard.............................................................................................................. 48 
11.2 Institutional Review Board ............................................................................................... 48 
11.3 Informed Consent Process .............................................................................................. 48 
11.4 Exclusion of Women, Minorities, and Children (Special Populations) ............................... 49 
11.5 Subject Compensation .................................................................................................... 49 
11.6 Subject Confidentiality ..................................................................................................... 49 
11.7 Future Use of Stored Specimens ..................................................................................... 50 
12 Data Handling and Record Keeping................................................................................................ 51 
13 Publication Policy ........................................................................................................................... 52 
14 References .................................................................................................................................... 53 
Appendix A: Schedule of Events (Cohort A-B) ..................................................................................... 57 
Appendix A: Schedule of Events (Cohort C and D) .............................................................................. 58 
Appendix B: Clinical Laboratory Values, for Study Eligibility ................................................................. 59 
Appendix C: Grading Scales for Laboratory Toxicity and Reactogenicity.............................................. 60 
  
 

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ABBREVIATIONS 
ADH 
Adipic acid dihydrazide 
AE 
Adverse Event 
ALT 
Alanine aminotransferase 
ALS 
Antibodies in lymphocyte supernatant 
ANC 
Absolute neutrophil count 
ASC 
Antibody secreting cells 
AST 
Aspartate Aminotransferase 
BM 
B memory cell 
Bn 
Naïve B cell 
BMI 
Body mass index 
BP 
Blood pressure 
BUN 
Blood urea nitrogen 
CBC 
Complete blood count 
CDAP 
1-cyano-4-dimethylaminopyridinium tetrafluoroborate 
CFR 
Code of Federal Regulations 
CFU 
Colony forming units 
cGMP 
Current Good Manufacturing Practices 
CI 
Confidence interval 
CMI 
Cell-mediated immunity 
COPS 
Core-O polysaccharide 
COVID-19 
Coronavirus disease 2019, caused by the SARS-CoV-2 virus 
CRF 
Case report form 
CRP 
C-reactive protein 
cTfh 
Circulating T follicular/helper cell 
CVD 
Center for Vaccine Development  
ELISA 
Enzyme-linked immunosorbent assay 
ELISPOT 
Enzyme-linked immunospot 
FDA 
Food and Drug Administration 
FliC 
Flagellin subunit protein (phase 1 for S. Typhimurium) 
GCP 
Good Clinical Practice 
GLP 
Good Laboratory Practice 
GMBS 
succinimidyl 4-maleimidylbutyrate  
(a.k.a. N-γ-maleimidobutyryl-oxysuccinimide ester) 
GMT 
Geometric mean titer 
HBsAg 
Hepatitis B surface antigen 
HCV 
Hepatitis C virus 
HEENT 
Head, eyes, ear, nose, throat 
Hg 
Hemoglobin 
HIPAA  
Health Insurance Portability and Accountability Act 
HIV 
Human Immunodeficiency Virus 
HRP 
Horseradish peroxidase 
HSF1 
Health Sciences Facility building 1 
ICF 
Informed consent form 
IDS 
Investigational Drug Service 

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ICH 
International Conference on Harmonization 
IgA 
Immunoglobulin A 
IgG 
Immunoglobulin G 
IM 
Intramuscular 
IND 
Investigational new drug 
iNTS 
Invasive non-typhoidal Salmonella 
IP 
Investigational product (a.k.a. study product) 
IRB 
Institutional Review Board 
ISM 
Independent safety monitor 
IU 
International units 
KDO 
2-keto-3-deoxyoctonate 
LPS 
Lipopolysaccharide 
mcg or µg 
Micrograms  
mL 
Milliliters  
MOP 
Manual of procedures 
NTS 
Non-typhoidal Salmonella 
OPA 
Opsonophagocytic activity 
OPS 
O polysaccharide 
PBS 
Phosphate buffered saline 
pH 
Potential of hydrogen 
PBMC 
Peripheral blood mononuclear cells 
PI 
Principal investigator 
QA 
Quality assurance 
QC 
Quality control 
QMP 
Quality management plan 
S. (italicized S.) 
Salmonella, bacteria of the genus 
SAE 
Serious adverse event 
SBA 
Serum bactericidal activity 
SD 
Standard deviation 
SFC 
Spot-forming cell 
SMC 
Safety monitoring committee 
SOP  
Standard operating procedure 
TBIL 
Total bilirubin 
TLR5 
Toll-like receptor 5 
TT 
Tetanus toxoid 
U.S. or U.S.A. 
United States (of America) 
WBC 
White blood cell 
WHO 
World Health Organization 
 
 
 

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PROTOCOL SYNOPSIS 
Title: 
Phase 1 Randomized, Placebo-Controlled, Dose-Escalation Study of the 
Safety, Reactogenicity, and Immunogenicity of a Trivalent (S. 
Enteritidis/S. Typhimurium/S. Typhi Vi) Conjugate Vaccine against 
Invasive Salmonella Disease Administered Parenterally to Healthy U.S. 
Adults 
Phase: 
1, adaptive design 
Population: 
104 healthy adults, ages 18 through 45 
Site: 
Two site, outpatient trial  
Subject 
Participation 
Duration: 
Each subject will participate for approximately 6-7 months from the time of 
enrollment through completion of the study.   
Investigational 
Products: 
• Study product #1: trivalent S. Enteritidis [25 µg]/S. Typhimurium [25 
µg]/S. Typhi Vi conjugate vaccine, presented in multi-dose vials 
containing 2-phenoxyethanol preservative. 
• Study product #2: placebo, a buffer containing the 2-phenoxyethanol 
preservative. 
 
Primary 
Objectives: 
 
1. Safety and Reactogenicity: To assess the frequency and severity of 
solicited local (i.e., injective site) and systemic (such as fever) AEs during 
the first 7 days following each dose of vaccine. 
2. Safety: To assess the frequency and severity of unsolicited AEs within 28 
days of each dose of vaccine and the occurrence of any SAEs through 6 
months after the last dose of vaccine 
3. Immunogenicity: To measure the proportion of subjects that achieve a 
four-fold increase in titer, as compared to baseline, of specific serum IgG 
anti-COPS (S. Enteritidis or S. Typhimurium), anti-Vi (S. Typhi) 
polysaccharide, and anti-FliC (S. Enteritidis or S. Typhimurium) antibody 
at days 29 and 57, as measured by ELISA. 
Secondary 
Objectives 
1. Safety: To assess the frequency and severity of abnormalities in clinical 
safety laboratory parameters at 7 days following the first dose of vaccine. 
2. Immunogenicity: To measure the proportion of subjects that achieve a 
four-fold increase in titer, as compared to baseline, of specific serum IgG 
anti-COPS (S. Enteritidis or S. Typhimurium), anti-Vi (S. Typhi) 
polysaccharide, and anti-FliC (S. Enteritidis or S. Typhimurium) antibody 
at day 180/210, as measured by ELISA. 
3. Immunogenicity: To calculate the GMT of serum IgG anti-COPS (S. 
Enteritidis or S. Typhimurium), anti-Vi polysaccharide, and anti-FliC (S. 
Enteritidis or S. Typhimurium) antibodies at days 1, 29, 57, and 180/210, 
as measured by ELISA. 
 

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Exploratory 
Objectives 
1. Immunogenicity: To measure the titers of functional antibodies by serum 
bactericidal antibody (SBA) activity assay and opsonophagocytic activity 
(OPA) assay against S. Typhimurium, S. Enteritidis, and S. Typhi 
bacterial cells at days 1, 29, 57, and 180/210. 
2. Immunogenicity: To measure the antibody secreting cells (ASC), specific 
for anti-COPS, Vi, FliC, and TT; possibly including homing markers and 
the measurement of antibodies in lymphocyte supernatants (ALS) 
3. Immunogenicity: To measure memory B and T cells, specific for anti-
COPS, anti-Vi, anti-FliC, and anti-TT activity; including the B cells that 
exhibit mucosal homing markers integrin α4β7 and/or the lymph node 
homing marker CD62L 
4. Immunogenicity: To conduct functional genomic and proteomic studies to 
further explore the immune response to vaccination. 
5. Immunogenicity: To measure the anti-Vi polysaccharide, anti-TT, anti-
COPS, and anti-FliC antibody levels in oral fluid at days 1, 29, 57, and 
18/210, as measured by ELISA (Cohorts C and D) 
 
Brief 
Description of 
Study Design 
 
This is intended to be an adaptive design study.1 In Part 1 of the study, we 
will conduct a double-blinded, randomized, placebo-controlled, dose-
escalation study. Participants will be sequentially enrolled into Part 1 of the 
study in three dose-escalating steps (Steps 1-3, Cohorts A-C), consisting of 
fractional vaccine doses containing: 6.25 µg (Step 1), 12.5 µg (Step 2), or 25 
µg (Step 3) of each specific polysaccharide by weight. Within each step, 
participants will be randomly allocated to receive a single dose of trivalent 
conjugate vaccine or placebo. In Step 3, participants may receive one dose 
or two-doses, separated by 28 days, of the vaccine. 
Part 2 of the study is intended to expand the safety and immunogenicity data 
from a selected dose level of the vaccine (anticipated to be the 25 µg per 
conjugate dose level). The target dose for this part of the study is to be 
informed by safety and immunogenicity data from the dose-escalation 
cohorts, with an intent to select the most promising immunogenic dose of the 
vaccine. Part 2 of the study will consist of a cohort of 60 participants who will 
be randomly allocated to receive a blinded two-dose regimen in one of the 
following three possible treatment groups:  
• a single dose of the trivalent conjugate vaccine, followed by a dose of 
placebo 28 days later (n=25); 
• two doses of trivalent conjugate vaccine, separated by 28 days (n=25); 
• or two doses of placebo, separated by 28 days (n=10).  
Special Notation: Due to the limited shelf-life of the vaccine, which is being determined by an 
ongoing stability testing plan, the availability of this pilot production lot of vaccine may be 
limited in time.  Furthermore, since March 2020 the global pandemic due to SARS-CoV-2 virus 
(causing COVID-19 infections) has significantly disrupted the ability to conduct this study.  
Therefore, the Step 3 (Cohort C) has been significantly revised such that both a single and two-
dose regimens of the 25 µg dosage of vaccine will be evaluated, albeit with a very small 
sample size. This was originally intended to be evaluated with a large sample size in Part 2 
(Cohort D), but due to the extraordinary present circumstances, the revised design of the study 
is intended to provide similar information which is crucial for the vaccine development plan. 
Furthermore, in order to provide some bridging immunogenicity data, a small number of 
participants in Cohort C will be randomized to receive either a single or two-dose regimen of 

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the 12.5 µg dosage of the vaccine. It is possible that Part 2 (Cohort D) will not be conducted, 
due to lack of vaccine. 
Furthermore, because of the COVID-19 situation, for Cohort A the follow-up visits at 6 months 
post-vaccination were conducted by phone call contact and thus the collection of specimens 
was unable to be conducted. For Cohort B, the follow-up visits at 2 months and 6 months post-
vaccination were conducted by phone call contact and thus the collection of specimens was 
unable to be conducted. Therefore for Cohort A and B participants, a single clinic visit for the 
collection of blood and oral fluid will be requested to measure the long-term immune responses 
(>6 months post-vaccination). 
Pharmaron CPC, Inc. (Baltimore, MD) is being added as a site for the enrollment of Cohort C. 

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KEY PERSONNEL 
Principal Investigator: 
Wilbur H. Chen, MD, MS, FACP, FIDSA 
Professor of Medicine 
Chief, Adult Clinical Studies section 
Center for Vaccine Development & Global Health 
University of Maryland School of Medicine 
Direct Office Phone: 410-706-1188 
Mobile Phone: 443-414-4462 
Email: wchen@som.umaryland.edu  
 
Clinical Co-Principal Investigator: 
Mohamed Al-Ibrahim, MB, ChB, FACP 
Pharmaron CPC Inc. 
800 W. Baltimore St., 5th Floor 
Baltimore MD 21201 
Office: (410) 706-8772 
Cell: (410) 245-6888 
Email: Mohamed.al-ibrahim@pharmaron-us.com 
 
Immunology Co-Investigators 
Marcela F. Pasetti, PhD 
Professor of Pediatrics and Microbiology & Immunology 
Direct Office Phone: 410-706-2341 
Email: mpasetti@som.umaryland.edu 
 
Marcelo B. Sztein, MD 
Professor of Pediatrics, Medicine, and Microbiology & Immunology 
Direct Office Phone: 410-706-2345 
Email: msztein@som.umaryland.edu  
 
 
 

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1 
BACKGROUND 
1.1 
Epidemiology and Burden of Disease 
Salmonella enterica serovars Typhi and Paratyphi A and B and certain non-typhoidal 
Salmonella enterica (NTS) serovars are important causes of invasive Salmonella disease 
worldwide.  “Typhoid Fever” is caused by Salmonella enterica serovar Typhi (S. Typhi), while 
“Paratyphoid Fever” is caused by Salmonella enterica serovar Paratyphi A (S. Paratyphi A) or 
Salmonella enterica serovar Paratyphi B (S. Paratyphi B). Both Typhoid Fever and Paratyphoid 
Fever manifest as persistent fevers, abdominal discomfort, headache and malaise. Without 
prompt and effective antibiotic treatment, these infections may lead to intestinal hemorrhage, 
intestinal perforation and peritonitis, myocarditis, hepatitis, neurological consequences and 
death. In the pre-antibiotic era the case fatality rate of typhoid fever was circa 15%. 
In industrialized countries in North America, Europe and in Australia and New Zealand, various 
NTS serovars are common causes of gastroenteritis, as the result of sporadic food-borne 
transmission or associated with outbreaks.  In older children and adults, these NTS infections 
typically result in uncomplicated diarrheal illness that does not require antibiotic treatment and 
invasive infections due to NTS serovars in healthy adults are rare. However, among certain sub-
populations (such as infants < 3 months of age, the elderly, and immunocompromised hosts) in 
industrialized countries, invasive NTS (iNTS) infections such as bacteremia, septicemia, and 
meningitis can be observed and are associated with notable case fatality.2,3 The most common 
Salmonella serovars associated with invasive disease in the U.S. include: S. Typhimurium, S. 
Enteritidis, S. Heidelberg, S. Dublin, and S. Schwarzengrund.4  
Whereas invasive disease due to NTS (bacteremia, septicemia, meningitis, etc.) is relatively 
rare in the USA and industrialized countries, a recognized burden of iNTS disease exists in sub-
Saharan Africa, where it was recently estimated that ~2 million cases (~227 cases/100,000 
infants and young children) occur annually.5  The case fatality ratio of iNTS disease in Africa is 
~20%, resulting in an estimated ~681,000 deaths each year.5 In sub-Saharan Africa four 
serovars, S. Typhimurium, S. Enteritidis, I:4,[5],12:i-, and S. Dublin, account for > 85-95% of all 
iNTS cases.6,7 Notably, the strains of S. Typhimurium, I:4,[5],12:i- and S. Enteritidis associated 
with invasive NTS disease in sub-Saharan Africa are genomically distinct from strains of those 
serovars circulating in the USA and Europe.8,9 For example, S. Typhimurium and I:4,[5],12:i- 
strains in the USA and Europe are overwhelmingly of multi-locus sequence type ST19, whereas 
the African strains are ST313, an unusual MLST type. Similarly, the S. Enteritidis strains in 
Africa are genomically distinct. Notably, most cases of iNTS in infants and toddlers in sub-
Saharan Africa do not present with or have a history of gastroenteritis. The vast majority of the 
iNTS isolates from sub-Saharan Africa encode resistance to multiple clinically-relevant 
antibiotics.8,10    
1.2 
Vaccine Development 
To address this important public health problem, iNTS vaccines are being developed.11,12 The 
polysaccharides of encapsulated bacteria (e.g., Haemophilus influenzae type b, Streptococcus 
pneumoniae, Neisseria meningitidis) linked to carrier proteins have resulted in safe, 
immunogenic (in young children) and highly effective parenteral polysaccharide conjugate 

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vaccines.  One typhoid polysaccharide conjugate vaccine, Typbar-TCV®, manufactured by 
Bharat Biotech, Hyderabad, India, that was first licensed in India, was pre-qualified in 2017 by 
the World Health Organization (WHO) for procurement by United Nations Agencies and its use 
in infants as young as six months of age has been recommended by WHO’s Scientific Group of 
Experts (SAGE).13,14 In contrast, currently there are no licensed NTS vaccines. 
CVD investigators have been developing candidate polysaccharide protein conjugate vaccines 
for the prevention of iNTS disease.  The vaccine development strategy involves targeting two 
surface virulence antigens of the key NTS Salmonella serovars including the core 
polysaccharide and O-polysaccharide (COPS) plus FliC protein, the unpolymerized flagellin 
subunits of flagella motility organelles.12,15-19   
The outer membrane lipopolysaccharides of Salmonella can be divided into 3 distinct regions: 
an endotoxin lipid A moiety; a highly conserved, core oligosaccharide region; and a serogroup-
specific, repeating oligosaccharide polymer (O-polysaccharide [OPS]). The majority of human 
invasive isolates fall antigenically within Salmonella group A (includes S. Paratyphi A), Group B 
(includes S. Paratyphi B, S. Typhimurium and I:4,[5],12:i-), Group C (includes S. Paratyphi C, S. 
Choleraesuis and S. Newport), and Group D (includes S. Typhi, S. Enteritidis and S. Dublin).  
Serovars of Salmonella serogroups A, B, and D share a common mannose-rhamnose-galactose 
trisaccharide backbone. Distinct dideoxyhexose sugars linked to the mannose residues of the 
common side chain provide serogroup specificity. Thus, if the dideoxyhexose sugar linked to the 
mannose is a paratose, this results in immunodominant antigen “2”, denoting a serogroup A 
Salmonella isolate. If the sugar linked to the mannose is an abequose, this results in an 
immunodominant antigen “4”, denoting serogroup B Salmonella. Finally, if the sugar linked to 
the mannose is a tyvelose, this results in immunodominant antigen “9”, denoting serogroup D 
Salmonella.12  
Specific antibody responses against the O polysaccharide antigens of the conjugate, if 
protective, should provide some degree of protection against all the different serovar pathogens 
within that O serogroup. For example, both S. Enteritidis and S. Dublin serovars fall within O 
serogroup D. Immune responses against Group D O antigen should contribute to protection 
against both of these Group D serovars. CVD investigators have devised practical methods to 
purify COPS from attenuated “reagent strains” that have been engineered to enhance the yield 
of COPS and to facilitate the large-scale manufacture of these antigens under enhanced 
occupational safety.18  The vaccine candidates to be evaluated in this study are based on the 
COPS of S. Typhimurium (a Group B serovar) and S. Enteritidis (a Group D serovar). 
The S. Enteritidis conjugate component in our trivalent vaccine is generated by 1-cyano-4-
dimethylaminopyridinium tetrafluoroborate (CDAP) chemistry to link S. Enteritidis COPS to the 
FliC Phase 1 flagellin protein subunits of S. Enteritidis that had been derivatized with adipic acid 
dihydrazide (ADH) using carbodiimide. This strategy is similar to the approach used for 
MenAfriVac™, which is comprised of the group A meningococcus capsular polysaccharide 
linked to tetanus toxoid (TT).20 MenAfriVac has been administered to infants, toddlers, children, 
and adults in countries within the African Meningitis belt.21 CDAP linkage and ADH derivitization 
are also used by GlaxoSmithKline (GSK) Vaccines in preparing their multivalent pneumococcal 
conjugate vaccine (Synflorix™) and by Sanofi Pasteur Vaccines in preparing their quadrivalent 
meningococcal conjugate vaccine (Menactra™). Synflorix™ and Menactra™ have extensive 
track records of safety in infants, children and adults, including in sub-Saharan Africa. Similarly, 
experimental S. Paratyphi A conjugates generated at the National Institute of Child Health and 

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Human Development by John B. Robbins and colleagues in the late 1990s consisted of S. 
Paratyphi A COPS linked to tetanus toxoid carrier protein and utilized ADH as a linker with 
CDAP conjugation.22 The S. Paratyphi A conjugates prepared by the Robbins Laboratory at 
NICHD were safe and immunogenic in Phase 1 and Phase 2 clinical trials that involved adults, 
teenagers and children 2-4 years of age.23 Interestingly, among the pre-school age children who 
received either one or two spaced doses of S. Paratyphi A conjugate 6 weeks apart, there was 
no evidence of a booster effect following administration of the second dose of conjugate.23   
The S. Typhimurium conjugate component in our trivalent vaccine is generated by modification 
at the polysaccharide terminal 2-keto-3-deoxyoctonate (KDO) carbonyl with an aminooxythiol 
linker. The aminooxy forms an oxime bond with the KDO ketone that is further reduced with 
sodium cyanoborohydride. S. Typhimurium FliC protein (phase 1 flagellin subunit) is derivatized 
with the amine reactive reagent succinimidyl 4-maleimidylbutyrate (GMBS) to introduce a 
maleimide moiety that is then linked to the reactive sulfhydryl of the derivatized COPS molecule 
via formation of a thioether bond. There is no FDA-licensed conjugate vaccine for human use 
that utilizes the specific method of conjugation employed for our S. Typhimurium 
glycoconjugate. However, a synthetic Hib capsular polysaccharide-based glyconjugate vaccine 
that is licensed and manufactured in Cuba (and that is pre-qualified by the WHO) utilizes a 
maleimide linker.24 We have extensive pre-clinical experience with S. Typhimurium conjugate 
vaccines prepared utilizing this approach, and have published reports that document the 
tolerability and immunogenicity of such conjugates in animal models.15,16,19,25 John Robbins and 
team at the NICHD also successfully used aminooxy linker based approaches for synthesis and 
preclinical evaluation of COPS-based conjugates for a variety of gram-negative pathogens.26,27 
The S. Typhi conjugate component in our trivalent conjugate vaccine is essentially Typbar-
TCV™, a Vi conjugate vaccine generated by carbodiimide-mediated modification of S. Typhi Vi 
polysaccharide with ADH that introduces reactive hydrazide groups that are then used to link to 
tetanus toxoid (TT) via a second carbodiimide step. Typbar-TCV is a licensed typhoid vaccine in 
India and a number of other countries and has been extensively used in pediatric and adult 
populations.28 The World Health Organization conferred pre-qualification status for Typbar-TCV 
on January 3, 2018.14,29 It is now a vaccine which can be procured by United Nations agencies.   
Therefore, CVD investigators have devised novel candidate core-O polysaccharide-flagellin 
protein conjugates as a vaccine strategy for preventing iNTS and we have combined the iNTS 
conjugates with a Vi-TT conjugate (Typbar-TCV) to create a Trivalent Conjugate vaccine for 
preventing invasive iNTS disease and typhoid fever. 
1.3 
Pre-clinical Testing 
Early non-GMP formulations of monovalent S. Enteritidis and S. Typhimurium COPS:FliC 
conjugate vaccines were immunogenic and protected mice against fatal infections with the 
homologous serovar NTS pathogen.15-19  
1.3.1 Rabbit non-GMP product study 
Non-GMP bivalent and trivalent formulations of the Salmonella conjugate vaccines were also 
evaluated in a rabbit model.15 New Zealand White rabbits were immunized with 3 sequential 
doses of a monovalent S. Enteritidis, bivalent S. Enteritidis/Typhimurium, and trivalent S. 
Enteritidis/Typhimurium/Typhi conjugate vaccine formulations, separated 14 days apart, and 

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serum was collected pre-vaccination and post-final immunization (at day 44). High titers of 
serogroup-specific anti-COPS and anti-Vi (in the trivalent formulation) IgG antibodies were 
elicited by the bivalent and trivalent formulations; robust anti-FliC antibody were also elicited but 
the responses to FliC were lower relative to the anti-COPS titers. (Figure below) 
 
In vitro functional antibody activity was demonstrated with the complement-mediated serum 
bactericidal activity (SBA) assay.  Furthermore, in vivo functional antibody activity was 
measured with outbred CD-1 mice which were passively immunized with pooled pre- or post-
vaccination rabbit sera or PBS; then subsequently challenged intraperitoneally with S. Enteritidis 
R11 or S. Typhimurium D65 (representative virulent Malian blood serovar isolates). Mice which 
received PBS or pre-vaccination sera demonstrated >90% fatality with S. Enteritidis and ~63% 
fatality with S. Typhimurium, whereas mice that received post-vaccination serum were protected 
against lethal challenge (vaccine efficacy ~88% for S. Enteritidis and 100% for S. Typhimurium). 
The Kaplan-Meier survival curves for the respective pre- and post-immune sera were compared 
using log-rank analysis and P values were calculated as <0.0001 for both serovar challenges.15 
(Figure below) 
 

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1.3.2 GLP Toxicology Study 
As part of our submission of an Investigational New Drug Application (IND) to the U.S. Food and 
Drug Administration (FDA), the four products manufactured at Bharat Biotech International, Inc. 
and two of which are to be tested in the Phase 1 trial described herein have been evaluated in a 
Rabbit Toxicology Test performed by Noble Life Sciences, a Contract Research Organization 
located in Sykesville, Carrol County, MD, that specializes in performing animal toxicology tests 
under the tenets of Good Laboratory Practices (GLP). Eighty pathogen-free rabbits, 40 males 
and 40 females, were injected; 20 rabbits with each vaccine test product (3 vaccine test 
products: single-dose bivalent, multi-dose bivalent, and multi-dose trivalent) and 20 with the 
placebo test product (buffer containing 2-phenoxyethanol). The Rabbit Toxicology study was 
initiated July 20, 2018 and the first dose of study vaccines were administered on August 13, 
2018; a total of four sequential doses separated by 28 days was administered. All animals 
survived to the scheduled necropsy. There were no test article-related adverse changes in 
mortality, clinical observations, physical exams, injection site scores, body weights, food 
consumption, organ weights, organ-to-body weight ratios, body temperature, ophthalmology or 
urinalysis.  The antigen-specific ELISA IgG responses following the first dose of vaccine for the 
20 rabbits that received the trivalent multi-dose test article are shown in the table below. 
According to these pre-clinical data, the vaccines appear to be immunogenic after a single dose. 
ELISA IgG response following the first dose of vaccine for rabbits that received the trivalent multi-dose vaccine  
Typhimurium titers (EU/mL) 
 
 
Enteritidis titers (EU/mL) 
 
 
Typhi Vi titers (EU/mL) 
 
   GMT, pre 
3.4 
 
   GMT, pre 
7.2 
 
   GMT, pre 
24.0 
   GMT, post (day 29) 
1454.2 
 
   GMT, post (day 29) 
4381.4 
 
   GMT, post (day 29) 
2409.5 
   Geometric Mean Fold-rise 
426.8 
 
   Geometric Mean Fold-rise 
610.6 
 
   Geometric Mean Fold-rise 
99.7 
   Minimum fold-rise 
41.0 
 
   Minimum fold-rise 
75.6 
 
   Minimum fold-rise 
12.5 
   Maximum fold-rise 
6977.1 
 
   Maximum fold-rise 
3202.8 
 
   Maximum fold-rise 
337.5 
1.4 
Clinical Studies 
No prior human clinical studies have been performed with the trivalent (iNTS conjugates 
combined with Typhoid Vi conjugate) candidate Salmonella vaccines, that will be evaluated in 
the study described in this clinical protocol.  
However, the S. Typhi Vi capsular polysaccharide-TT conjugate vaccine (Typbar-TCV®, Bharat 
Biotech, Hyderabad, India) component is licensed as a monovalent conjugate vaccine in India28 
and has been pre-qualified by the WHO and recommended by the WHO SAGE for use in 
adults, children and infants as young as 6 months of age.  Among the adverse effects 
associated with Typbar-TCV are the following:30 
• 
Common (≥1% and <10%): fever, pain at injection site, and swelling 
• 
Uncommmon (≥0.1% and <1%): injection site tenderness, itching, arthralgia, cold, 
cough, vomiting, and myalgia 
The iNTS conjugates in our trivalent vaccine use Phase 1 flagellin subunits from the 
homologous iNTS serovars as the carrier protein. Flagellin can act as an adjuvant through 
binding to Toll-like receptor 5 (TLR5) and the induction of innate and adaptive immune signaling 
via an MyD88-dependent pathway and an MyD88-independent mechanism.31,32 Genetically-
engineered fusion proteins consisting of influenza virus hemagglutinin or matrix protein fused to 
a TLR5 stimulating portion of the FljB flagellin subunit of S. Typhimurium Phase 2 flagella have 
been evaluated in clinical trials. Some dose levels were associated with reactogenicity and 

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elevated levels of C-reactive protein, suggesting strong stimulation of the innate immune 
system.33-35 Importantly, the method of linkage of the Phase 1 flagellin FliC subunits to COPS in 
our NTS conjugates masks the TLR5 stimulating portion of flagellin. Pre-clinical assays of our 
conjugates have demonstrated >100-fold reduction in TLR5 activity using an NF-κB-luciferase 
reporter assay. Most importantly, rabbits tolerated the vaccine without mounting febrile 
responses or exhibiting elevated C-reactive protein levels following vaccination in the rabbit 
toxicology test. 
There is considerable justification for our expectation that a 25 µg (by weight of polysaccharide 
per dose of each conjugate) dose of our iNTS conjugate vaccine will be safe and immunogenic, 
based on results with various Vi conjugate vaccines containing 25 µg of Vi linked to various 
carrier proteins. As mentioned, Typbar-TCV, which contains 25 µg of Vi polysaccharide linked to 
TT, has an excellent safety record in adults, children and infants.28  
In a field trial in Vietnam involving children 2-5 years of age, a two-dose regimen of another Vi 
conjugate vaccine consisting of 25 µg of S. Typhi Vi polysaccharide linked to recombinant 
exotoxin A of Pseudomonas aeruginosa as the carrier protein was well tolerated, immunogenic 
and significantly protective against confirmed typhoid fever.36  
1.5 
Risks and Potential Benefits 
1.5.1 Risks 
Risks related to Bivalent or Trivalent vaccine: 
Since this is an investigational vaccine and has not been given to humans previously, the risks 
associated with administration are unknown. These vaccines are killed and purified and will not 
cause Salmonella infection. With an injectable vaccine, recipients may have fever, swelling, 
redness and pain at the injection site. An allergic reaction, including anaphylaxis, could possibly 
occur. The Typhoid component of the trivalent vaccine is virtually the same as the licensed 
vaccine called Typbar-TCV™. This licensed vaccine is known to cause fever in 10% of children 
who receive it.  
Blood Drawing risks: 
Blood drawing is sometimes associated with pain at the site from which the blood is drawn, 
bruising, and occasional lightheadedness and rarely, fainting or infection. Bruising at the site of 
blood drawing can be prevented by applying pressure for several minutes. To reduce the risk of 
infection, the area is wiped clean with alcohol and germ-free equipment is used. It is the policy of 
the American Red Cross that recipients of an experimental vaccine may not be able to donate 
blood for one year after receipt. 
Risks of Confidentiality: 
Efforts will be made to keep personal health information confidential. There is a small risk of loss 
of confidentiality by an unauthorized person viewing study participant records. In order to maintain 
confidentiality, the PI and the study staff will store study records in a locked area of a limited 
access locked office. Study records and samples will be coded with a number, not the participant’s 
name. Records will be used by the FDA, the data coordinating center and monitoring group, CVD 
and the IRB only in connection with carrying out their obligations relating to the study, and every 
effort will be made to keep the records as confidential as possible, within the limits of the law. 

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Risks to Pregnancy or Fetus: 
If a woman is pregnant or becomes pregnant, this research may hurt the baby or the pregnancy 
in ways that are unknown. Women of childbearing potential must use birth control to be sure that 
they do not become pregnant. The only birth control methods that work well enough to be sure 
that women of childbearing potential will not become pregnant are oral contraceptives (“the pill”), 
intrauterine devices (IUDs), contraceptive implants under the skin, or contraceptive injections, 
and condoms with foam. 
1.5.2 Potential Benefits  
Participants will not benefit directly from their participation in this study. However, their 
participation may help the investigators better understand the response to these Salmonella 
vaccines and whether these vaccines will help to prevent the disease.  
 

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2 
STUDY DESIGN 
Part 1.  The first part of the study will be conducted as a traditional first-in-human, double-
blinded, randomized, placebo-controlled, dose-escalation study. This part of the study is 
designed to assess the safety, reactogenicity, and preliminary immunogenicity of a single 
parenteral (i.e., intramuscular) dose of a trivalent (S. Enteritidis/S. Typhimurium/S. Typhi) 
conjugate vaccine in comparison to a placebo control consisting of buffer plus preservative (2-
phenoxyethanol). 
In total, approximately 44 healthy eligible participants will be sequentially enrolled into Part 1 of 
the study in three steps based on dose.  
In Step 1 (Cohort A), 10 participants will be randomly allocated to one of two treatment groups 
to receive: 
• 
trivalent vaccine, 6.25 mcg of each COPS and Vi, in 0.125 ml volume (N=8) mixed 
with 0.375 ml of placebo (phosphate buffered saline + 2-phenoxyethanol + Tween 80 
stabilizer) to reach a volume of 0.5 ml; 
• 
or placebo, 0.5 ml volume (N=2). 
In Step 2 (Cohort B), 12 participants will be randomly allocated to one of two treatment groups 
to receive: 
• 
trivalent vaccine, 12.5 mcg of each COPS and Vi, in 0.25 ml volume (N=10) mixed 
with 0.25 ml of placebo to reach a volume of 0.5 ml; 
• 
or placebo, 0.5 ml volume (N=2). 
In Step 3 (Cohort C), 22 participants will be randomly allocated to one of five treatment groups 
to receive: 
• 
one dose of trivalent vaccine, 25 mcg of each COPS and Vi, in 0.5 ml volume (N=6); 
• 
one dose of trivalent vaccine, 12.5 mcg of each COPS and Vi, in 0.5 ml volume 
(N=4); 
• 
two doses of trivalent vaccine, 25 mcg of each COPS and Vi, in 0.5 ml volume (N=6); 
• 
two doses of trivalent vaccine, 12.5 mcg of each COPS and Vi, in 0.5 ml volume 
(N=4); 
• 
or placebo, 0.5 ml volume (N=2). 
Each of the escalated dosage cohorts is designed to be slightly larger in size in order to have a 
higher probability to detect potential reactogenicity with the increasing quantities of flagellin, 
which is a component (the protein carrier of the NTS conjugates), while increasing the number 
of subjects exposed in a step-wise fashion.   
Following collection of the 28 day post-vaccination clinical data and rapid measurement of 
serum IgG ELISA antibody levels, there will be an interim analysis of the safety and 
immunogenicity in preparation for advancement to Part 2 of the study.  
Part 2. The second part of the study is intended to expand the safety and immunogenicity data 
from a selected dose level of the vaccines.  The target dose for this part of the study will be 
informed by the reactogenicity data from Part 1 of the study with particular attention on the dose 
level of vaccine administered in Step 3 (administration of the highest dose) and by the 
immunogenicity data from the dose-escalation cohorts, with an intent to select the most 
immunogenic yet well-tolerated dose of the vaccine formulations. Based on experiences with 
existing licensed and unlicensed Vi conjugate vaccines, we anticipate that the trivalent 

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conjugate vaccine containing 25 mcg of polysaccharide of each conjugate will likely be the most 
immunogenic dose and will hopefully be as well-tolerated as monovalent Vi conjugate vaccines. 
Part 2 of the overall study will intend to enroll 60 eligible participants who will be randomized to 
one of three possible treatment arms:  
• 
Trivalent conjugate vaccine followed 28 days later by a second dose of trivalent 
conjugate vaccine (n=25); 
• 
Trivalent conjugate vaccine followed 28 days later by a dose of placebo (n=25); 
• 
A dose of placebo followed 28 days later by a second dose of placebo (n=10). 
2.1 
How the Trivalent Conjugate Vaccine is expected to be used 
in sub-Saharan Africa  
By administration of a Trivalent Conjugate Vaccine, we will aim to prevent two distinct clinico-
epidemiologic forms of invasive salmonellosis that affect somewhat different age groups in sub-
Saharan Africa, including invasive disease (septicemia, bacteremia, meningitis, etc.) caused by 
certain non-typhoidal serovars (in particular, S. Typhimurium and its variants, and S. Enteritidis) 
and typhoid fever caused by S. Typhi. The S. Typhimurium and S. Enteritidis conjugates in the 
Trivalent Conjugate Vaccine are intended to prevent iNTS disease which exhibits a peak 
incidence from 6-23 months of age.7,37 The iNTS incidence drops somewhat in children 24-35 
months of age and then falls further in those 36-47 months of age.7,37 Thus, to diminish the iNTS 
burden the Trivalent Conjugate Vaccine will have to be administered in early infancy to elicit 
immune responses in time to protect children 6-11 months of age when maternally-transferred 
antibodies will have waned. The Typbar-TCV component of the Trivalent Conjugate is intended 
to prevent typhoid fever, a disease that shows a low incidence in infancy but rises in children 
age 24 months and often peaks in children 5-9 years of age.  
The ultimate preferred regimen to administer the Trivalent Conjugate Vaccine will be determined 
by analysis of the results of future Phase 2 clinical trials that will be carried out in infants and 
toddlers in sub-Saharan Africa with the doses being administered at ages when infants and 
toddlers receive other vaccines within the schedule of the Expanded Program on Immunization 
(EPI). Currently in sub-Saharan Africa, three doses of parenteral pentavalent vaccine 
(diphtheria and tetanus toxoids, whole cell pertussis vaccine, Haemophilus influenzae type b 
conjugate and hepatitis B combination vaccine) are given to young infants at ~6, ~10 and ~14 
weeks of age; another parenteral vaccine administered at these visits is multivalent 
pneumococcal conjugate vaccine. In sub-Saharan Africa the initial measles containing vaccine 
is administered at ~9 months of age (along with yellow fever vaccine in certain countries). A 
second dose of measles containing vaccine is administered at ~15 months of age. Future 
Phase 2 studies in infants and toddlers will compare the safety, immunogenicity, and practicality 
of spaced 2-dose and 3-dose regimens of Trivalent Conjugate Vaccine administered during 
infancy alone or during infancy and toddler age. Moreover, the compatibility of the Trivalent 
Conjugate with concomitantly administered EPI vaccines will have to be established.   
The purpose of the Phase 1 trial in U.S. adults is to gather preliminary safety and 
immunogenicity data to pave the way for initiating future Phase 2 trials in sub-Saharan Africa. 
Part 2 of this study in healthy U.S. adults offers a potential opportunity to determine whether at 
day 56 the anti-COPS and anti-Vi antibodies in participants of the two-dose regimen exhibit 

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higher avidity and perhaps higher titers than the antibodies at day 56 in recipients of a single 
dose of Trivalent Conjugate Vaccine.  
Serological data demonstrate that a single dose of monovalent Typbar-TCV conjugate 
administered to Indian children 6-23 months of age is highly immunogenic in stimulating Vi 
antibodies and the titers remain elevated for up to 5 years post primary-immunization. There are 
no published or public human data to dictate the serum antibody response to a conjugate 
vaccine consisting of COPS conjugated to a carrier protein. Moreover, adult humans immunized 
with a glycoconjugate vaccine of any sort typically mount a maximal antibody response after the 
first dose that is not boosted following administration of a second dose of glycoconjugate. 
Therefore, the main purpose of administering two-sequential doses of trivalent conjugate 
separated by 4 weeks is to evaluate whether a two-dose regimen presents an increased risk for 
reactogenicity when the second dose is administered. A second dose is not expected to 
increase anti-COPS titers in U.S. adults but it may perhaps increase antibody avidity.  
2.2 
Work flow of the Phase 1 study in U.S. adults 
Eligible subjects will be screened, enrolled, and vaccinated at the CVD Outpatient Vaccine 
Clinic (located on the 4th floor of HSF1) or Pharmaron (located in the UMB BioPark 1 building).  
After the documentation of informed consent, subjects will be screened for eligibility; eligible 
subjects will subsequently be enrolled and randomized to a treatment group.  On Day 1 (and 
Day 29, for participants in Cohorts C and D), study product will be administered, in double-
blinded fashion, as an intramuscular injection into the deltoid muscle. Following vaccination, 
there will be a 20-minute post-vaccination observation period. Following each dose of study 
product, safety and reactogenicity will be measured by a daily oral temperature and solicited AE 
reporting over the subsequent 7 days (via a 7-day memory aid); unsolicited AE reporting will 
occur through 28 days following the last dose of vaccine.  The occurrence of any SAEs will be 
recorded through 6 months after the last dose of vaccine (Day 180 or 210, for participants in 
Cohort C and D). 
Subjects in Steps 1-2 (Cohorts A-B) will complete four additional outpatient visits on Days 8, 29, 
57, and 180 (final visit).  Subjects in Cohorts C and D will complete five additional outpatient 
visits on Days 8, 29 (second dose of masked study product), 36, 57, and 210 (final visit). The 
recording of AEs and concomitant medication change information, the performance of a 
physical exam (if indicated), and the collection of study-specific samples will be performed 
during these outpatient visits.  Clinical safety laboratory tests will be evaluated on Day 8.  The 
final study visit will be for the collection of SAE information (Day 180 or 210). 
After the completion of enrollment of each cohort, the safety data through 7-days post-
vaccination are to be reviewed by an independent Safety Monitoring Committee (SMC). The 
SMC must provide approval prior to the enrollment of the subsequent dose-escalating cohort. 
Immunology data may not be available at the times of these scheduled SMC reviews. 
In addition to the evaluation of safety, immunogenicity assessments are planned to evaluate the 
immune responses induced by the three different vaccine formulations.  The study, however, is 
not statistically powered to determine significance of any pre-specified difference.  Therefore, 
there was no formal sample size calculation, based on immunogenicity, as the basis for the 
designed study.  Nonetheless, the sample size was chosen based on the number of subjects 
appropriate for a phase 1 study in which these vaccines have never been evaluated in humans. 

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Schematic of Study Cohorts 
Cohort 
Vaccine 
Dose of each COPS and of 
Vi  (µg) 
No. 
Subjects 
Dose #1 
Dose #2 
Vaccination 
Days 
A 
(Step 1) 
Trivalent 
Placebo 
6.25 
0 
8 
2 
Vaccine 
Placebo 
- 
- 
1 
SMC review of available safety data through 7-days post-vaccination before proceeding to the next dosage cohort 
B 
(Step 2) 
Trivalent 
Placebo 
12.5 
0 
10 
2 
Vaccine 
Placebo 
- 
- 
1 
SMC review of available safety data through 7-days post-vaccination before proceeding to the next dosage cohort 
C 
(Step 3) 
Trivalent 
Trivalent 
Trivalent 
Trivalent 
Placebo 
25 
25 
12.5 
12.5 
0 
6 
6 
4 
4 
2 
Vaccine 
Vaccine 
Vaccine 
Vaccine 
Placebo 
Vaccine 
Placebo 
Vaccine 
Placebo 
Placebo 
1& 29* 
1 & (29)* 
1& 29* 
1 & (29)* 
1 & (29)* 
SMC review of available safety data through 7-days post-vaccination before proceeding to Part 2 of the study 
D 
(Expanded, confirmatory 
cohort) 
Trivalent 
Trivalent 
Placebo 
(highest, well-tolerated dose 
among cohorts A-C) 
25 
25 
10 
Vaccine 
Vaccine 
Placebo 
Vaccine 
Placebo 
Placebo 
1 & 29* 
1 & (29)* 
1 & (29)* 
Total 
N = 104 
 
* In cohorts C and D, the trivalent vaccine will be administered as a single dose (day 1) or as a two-dose (days 1 and 29) regimen. In order to maintain 
the masking of study products, on day 29 a placebo will be administered when indicated by the parentheses. 
SMC, Safety Monitoring Committee 
 
Schematic of the Flow of the Cohort enrollments and timing of SMC meetings: 
Part 1 
Step 1 (Cohort A)  
single 6.25 µg dose trivalent  
or single dose placebo  
 
[SMC Review] 
  
 
Step 2 (Cohort B)  
single 12.5 µg dose trivalent  
or single dose placebo  
  
 
[SMC Review]  
 
 
 
Step 3 (Cohort C)  
single 25 µg dose trivalent 
or two-dose 25 µg dose trivalent 
or single 12.5 µg dose trivalent 
or two-dose 12.5 µg dose trivalent  
or single dose placebo  
 
 
 
 [Go/No-Go Decision for Part 2 based on interim safety and immunogenicity assessments]  
  
[SMC Review] 
 
Part 2 

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Cohort D  
Trivalent on day 1, (placebo on day 29); 
Trivalent on day 1 and day 29; 
Or Placebo on day 1 and day 29 
 
2.3 
Primary Objectives and Outcome Measures 
The primary objectives and outcome measure of this study are the following: 
1. Safety and Reactogenicity: To assess the frequency and severity of solicited local (i.e., 
injective site) and systemic (such as fever) AEs during the first 7 days following each dose of 
vaccine. 
2. Safety: To assess the frequency and severity of unsolicited AEs within 28 days of each dose 
of vaccine and the occurrence of any SAEs through 6 months after the last dose of vaccine 
3. Immunogenicity: To measure the proportion of subjects that achieve a four-fold increase in 
titer, as compared to baseline, of specific serum IgG anti-COPS (S. Enteritidis or S. 
Typhimurium), anti-Vi (S. Typhi) polysaccharide, and anti-FliC (S. Enteritidis or S. 
Typhimurium) antibody at days 29 and 57, as measured by ELISA. 
2.4 
Secondary Objectives and Outcome Measures 
The secondary objectives and outcome measures of this study are the following: 
1. Safety: To assess the frequency and severity of abnormalities in clinical safety laboratory 
parameters at 7 days following the first dose of vaccine. 
2. Immunogenicity: To measure the proportion of subjects that achieve a four-fold increase in 
titer, as compared to baseline, of specific serum IgG anti-COPS (S. Enteritidis or S. 
Typhimurium), anti-Vi (S. Typhi) polysaccharide, and anti-FliCn (S. Enteritidis or S. 
Typhimurium) antibody at day 180/210, as measured by ELISA. 
3. Immunogenicity: To calculate the GMT of serum IgG anti-COPS (S. Enteritidis or S. 
Typhimurium), anti-Vi polysaccharide, and anti-FliC (S. Enteritidis or S. Typhimurium) 
antibodies at days 1, 29, 57, and 180/210, as measured by ELISA. 
2.5 
Exploratory Objectives and Outcome Measures 
The exploratory objectives and outcome measures of this study include the following: 
1. Immunogenicity: To measure antibodies by serum bactericidal antibody (SBA) activity assay 
and opsonophagocytic activity (OPA) assay against S. Typhimurium, S. Enteritidis, and S. 
Typhi at days 1, 29, 57, and 180/210. 
2. Immunogenicity: To measure the antibody secreting cells (ASC), specific for anti-COPS, Vi, 
FliC, and TT; possibly including homing markers and the measurement of antibodies in 
lymphocyte supernatants (ALS) 
3. Immunogenicity: To measure memory B and T cells, specific for anti-COPS, Vi, FliC, and TT 
activity; including the presence of B cell homing markers as assessed by the expression of 
mucosal homing marker integrin α4β7 and the lymph node homing marker CD62L 

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4. Immunogenicity: To conduct functional genomic and proteomic studies to further explore the 
immune response to vaccination. 
5. Immunogenicity: To measure the anti-Vi polysaccharide, anti-TT, anti-COPS, and anti-FliC 
antibody levels in oral fluid at days 1, 29, 57, and 180/210, as measured by ELISA (Cohorts 
C and D) 
 

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3 
STUDY POPULATION 
The study population will include healthy adult men and women ages 18 to 45 years, inclusive.  
Participants will be recruited from the local Baltimore-Washington DC population through 
established methods, to include: future-contact authorized CVD volunteer database, local 
advertisements, campus flyers, and word-of-mouth.  Potential volunteers who are interested in 
the study are instructed to call the CVD to receive further information about the study.  
The process of informed consent begins when a person contacts the CVD for information about 
the study. CVD research staff receive these inquiries and read through an IRB-approved 
telephone script which describes basic information on the study topic, duration and number of 
visits, procedures, and eligibility criteria. No personal health information is solicited and 
afterwards interested potential participants are given an appointment to attend an Orientation 
Session for formal screening. 
At the Orientation Session, the consent process continues. CVD research staff provide a 
detailed description of all aspects of the study, including the rationale and background, the 
public health significance, the procedures and schedule of visits, and a detailed discussion of 
the risks and the presence or absence of benefit to them, as appropriate. The prospective 
subject is given a copy the study consent form (without the signature page) and is encouraged 
to discuss his/her participation with family members or advisors before agreeing to sign the 
informed consent form and proceeding with any screening procedures. An interested volunteer 
may decide to participate in the study on a separate visit or may continue the consent process 
after the Orientation Session is completed. 
A similar process of recruitment, pre-screening, and consenting process will be performed at 
Phamaron. Pharmaron will use their recruitment process which may include participants outside 
of the local Baltimore-Washington vicinity. Pharmaron will use their established methods of 
recruitment which will include their volunteer database, local advertisements, social media, and 
other methods.  As part of the consenting process, Pharmaron research staff will provide 
detailed description of all aspects of the study, including rationale and background, the public 
health significance, the procedures and schedule of visits, and a detailed discussion of the risks 
and the presence or absence of benefit to them. The recruitment materials will be UMB IRB 
approved. 
Once both the investigator and the volunteer are satisfied that all questions have been 
answered and the volunteer expresses the desire to participate, the volunteer will be allowed to 
sign the consent form. A copy of the signed consent document will be given to the participant. 
After the informed consent form is signed, a one-on-one private meeting with a member of the 
study team is performed, to continue the process of informed consent.  A brief written 
examination is administered to assess the volunteer’s comprehension of the study (i.e., 
Comprehension Assessment Tool). A passing score of ≥70% is required; wrong answers will be 
reviewed with the volunteer and examination may be re-taken once. Upon successful 
completion of the Comprehension Assessment Tool, the research staff can proceed with formal 
screening. 

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3.1 
Inclusion Criteria 
Subjects must meet all inclusion criteria in order to participate in the study: 
1. Ability to provide written informed consent 
2. Age 18 - 45 years, inclusive 
3. Good general health as determined by: vital signs (heart rate <100 bpm; blood pressure 
systolic >90 mm Hg and ≤150 mm Hg; diastolic >45 mm Hg and ≤90 mm Hg; oral 
temperature <100.4ºF), medical history, and a physical examination† within 45 days 
before administration of first dose of vaccine.  
† The intent is to evaluate for acute or ongoing chronic medical conditions which have been 
present for 90 days or more and which could affect the assess of safety or immunogenicity. 
Chronic medical conditions should be stable for at least 60 days; defined as no 
hospitalizations, ER, or urgent care for medical intervention and no change in chronic 
prescription medications for at least 60 days. Changes in medications due to insurance or 
financial reasons and when within the same class of medications or changes for 
improvements in medical conditions are not exclusionary. Medications which are taken prn 
are also no exclusionary. 
4. Expressed interest and availability to fulfill the study requirements 
5. For females of child-bearing potential*, must agree to acceptable birth control&, 4 weeks 
before enrollment and through 4 weeks after last vaccination. 
*  females of child-bearing potential are defined as: not sterilized via tubal ligation, bilateral 
oophorectomy, salpingectomy, hysterectomy, or successful Essure® placement (permanent, 
non-surgical, non-hormonal sterilization) with documented radiological confirmation test at 
least 90 days after the procedure, and still menstruating or <1 year of the last menses if 
menopausal. 
&  acceptable birth control includes: non-male sexual relationships, abstinence from sexual 
intercourse with a male partner, monogamous relationship with vasectomized partner who 
has been vasectomized for 180 days or more prior to the subject receiving the first study 
vaccination, barrier methods such as condoms or diaphragms with spermicide or foam, 
effective intrauterine devices, NuvaRing®, and licensed hormonal methods such as implants, 
injectables, or oral contraceptives. 
6. Agrees not to participate in another clinical trial at any time during the study period. 
7. Agrees to allow for the indefinite storage of blood samples for future research use.  
3.2 
Exclusion Criteria 
Subjects who meet any of the following criteria at baseline will be excluded from participating in 
the study: 
1. History of typhoid vaccination or known history of typhoid infection within 5 years 
2. Unacceptable laboratory abnormality from screening (prior to first vaccination) or upon 
safety laboratory testing (prior to second vaccination) as listed below.  Laboratories with 
abnormalities which are possibly transient in nature may be repeated one time. 
a. Hemoglobin, white blood cell (WBC) count, absolute neutrophil count (ANC), or 
platelet count of an unacceptable value, according to Appendix B 

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b. Creatinine, AST, ALT, total bilirubin, or C-reactive protein of an unacceptable value, 
according to Appendix B  
c. Positive serology for hepatitis C or HIV antibody or hepatitis B surface antigen.  
(Subjects will be informed if their results are positive for hepatitis C, HIV antibody or hepatitis 
B surface antigen and will be referred to a primary care provider for follow up of these 
abnormal laboratory tests.) 
3. For women of child-bearing potential, positive serum pregnancy test (during screening 
within 45 days of enrollment) or positive urine pregnancy test (prior to and within 24 
hours of administering each dose of vaccine). 
4. Nursing mother. 
5. Temperature > 38.0°C (100.4°F) or symptoms of an acute self-limited illness such as an 
upper respiratory infection, including of a COVID-19 infection#, or gastroenteritis within 
10 days prior to each dose of vaccine. 
# Symptoms of a COVID-19 infection include fever, chills, cough, shortness of breath or 
difficulty breathing, fatigue, muscle or body aches, headache, new loss of taste or smell, sore 
throat, congestion or runny nose, nausea or vomiting, and diarrhea. 
https://www.cdc.gov/coronavirus/2019-ncov/symptoms-testing/symptoms.html  
6. Medical, occupational, or family problems as a result of alcohol or illicit drug use during 
the past 12 months. 
7. Diagnosis of schizophrenia or other major psychiatric disease 
8. Failure to pass Comprehension Assessment Tool during screening (70% correct 
answers are required to pass. Subjects will be provided the opportunity to repeat the test 
once).  
9. Receipt of an experimental agent (vaccine, drug, device, etc.) within 28 days before 
enrollment or expects to receive an experimental agent during the study period. 
10. Receipt of any licensed vaccine within 2 weeks (for inactivated vaccines) or 4 weeks (for 
live vaccines) before enrollment in this study. 
11. Known sensitivity to any ingredient in the study vaccine, including a history of severe 
allergic reaction to tetanus vaccine. 
12. Receipt of immunoglobulin or other blood product within the 3 months prior to 
vaccination in this study. 
13. Immunosuppression as a result of an underlying illness or treatment with 
immunosuppressive or cytotoxic drugs, or use of anticancer chemotherapy or radiation 
therapy within the preceding 36 months. 
14. Long-term use (>2 weeks) of oral or parenteral steroids (glucocorticoids), or high-dose 
inhaled steroids (>800 mcg/day of beclomethasone dipropionate or equivalent) within 
the preceding 6 months (nasal and topical steroids are allowed). 
15. Other condition that in the opinion of the investigator would jeopardize the safety or 
rights of a volunteer participating in the trial or would render the subject unable to comply 
with the protocol. 

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3.3 
Enrollment and Randomization Procedures 
Per International Conference on Harmonisation (ICH) guideline E6: Good Clinical Practice 
(GCP), screening records will be kept at the clinical site to document the reason why an 
individual was screened, but failed trial entry criteria. The reasons why individuals failed 
screening will be recorded in the data system. 
3.3.1 Re-screening procedure 
There may be situations in which a subject is not able to be randomized within 45 days of 
his/her Screening Visit.  In these instances, the subject may need to be re-screened including 
re-consent.  This will involve undergoing all screening procedures again.  
Study participants that meet all inclusion and exclusion criteria and have signed the informed 
consent form will be assigned a subject identification number.  A randomization code will be 
prepared by a CVD statistician and will determine the blinded assignment of each subject to a 
treatment arm. A designated independent individual (e.g., Independent Safety Monitor) will be 
provided with a back-up randomization code list for emergency unblinding purposes and which 
will be maintained in a secure location. 
Subjects, investigators, and study personnel performing any study-related assessments 
following study vaccine administration will be blinded to subject treatment assignment. 
Laboratory personnel performing the immunogenicity assessments will also remain blinded to 
subject treatment assignment. 
3.4 
Withdrawals and Discontinuation from Study 
Participation in the study is strictly voluntary.  Participants have the right to withdraw from the 
study at any time and for any reason, without penalty or prejudice to further treatment.  The 
Principal Investigator and/or designee may, at their own discretion, terminate a subject from 
continuing in the study if it is considered to be in the participant’s best welfare, or if the 
participant is not willing or able to comply with the study requirements.  The reason for 
withdrawal or termination will be documented. Study participants may voluntarily withdraw their 
consent for participation at any time and for any reason, without penalty.  A study participant 
may be withdrawn from the study for the following reasons: 
• 
Medical illness or a new clinical finding which in the opinion of the investigator could 
compromise the safety of the subject or interfere with the evaluation of the safety, 
tolerability, or immunogenicity of the study products. 
• 
The subject no longer meets eligibility criteria. 
• 
As deemed necessary by the investigator for non-compliance or other reasons. 
• 
Subject lost to follow-up. 
• 
Termination of the study. 
• 
New information becomes available that makes further participation unsafe. 
Handling of Participant Withdrawal or Termination. Every effort will be made to undertake 
protocol-specified safety follow-up procedures to capture AEs, serious adverse events (SAEs), 
and unanticipated problems (UPs). In the event of withdrawal or termination from the study, 
reasonable efforts should be made to conduct the following procedures: 

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• 
Review diary card/ memory aid, if still in use prior to withdrawal 
• 
Updating any ongoing AE/SAEs that remain ongoing at time of subject’s last visit prior to 
withdrawal  
• 
Query about AEs, SAEs and concomitant medications if the interval between the 
subject’s last visit and the time of withdrawal is within the protocol defined reporting 
period 
• 
Targeted physical examination, if there are any ongoing AEs at the time of withdrawl 
• 
Blood for safety laboratory testing, if withdrawal occurs before the day 8 visit 
• 
Update contact information 

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4 
INVESTIGATIONAL PRODUCTS 
4.1 
Description of Investigational Products 
4.1.1 Trivalent, with preservative (multi-dose vials) 
This study product consists of a cGMP lot of a trivalent S. Enteritidis/S. Typhimurium/S. 
Typhi Vi conjugate vaccine consisting of a 1:1:1 mix of S. Enteritidis COPS linked to 
Phase 1 Enteritidis flagellin (FliC) subunits and S. Typhimurium COPS linked to Phase 1 
Typhimurium flagellin (FliC) subunits, plus Typbar TCV® (S. Typhi Vi capsular 
polysaccharide linked to tetanus toxoid protein).  The study product is presented in multi-
dose vials containing 2-phenoxyethanol preservative. 
4.1.2 Placebo 
The placebo consists of the buffer vehicle in which the conjugate vaccines are 
suspended, with 2-phenoxyethanol preservative. 
4.2 
Formulation, Storage, Packaging, and Labeling 
4.2.1 Trivalent, with preservative (multi-dose vials) 
Each vial holds 2.5 mL (plus overfill) of material for which a 0.5 mL volume contains S. 
Enteritidis 25 µg (by polysaccharide weight), S. Typhimurium 25 µg (by polysaccharide 
weight), and S. Typhi Vi 25 µg (by polysaccharide weight) conjugate vaccine, plus 
Tween-80 and PBS, and 2-phenoxyethanol preservative. Each multi-dose vial is to be 
stored between 2-8°C, not to be frozen. 
4.2.2 Placebo 
Each vial holds 2.5 mL (plus overfill) of material for which a 0.5 mL volume contains 
PBS, Tween-80, plus 2-phenoxyethanol preservative. Each multi-dose vial of placebo is 
to be stored between 2-8°C, not to be frozen. 
4.3 
Preparation and Administration 
Step 1 (Cohort A). For each dose of vaccine, a 0.25 mL volume from a vial containing study 
product is to be mixed with 0.75 mL of placebo (PBS, Tween-80, and 2-phenoxyethanol).  After 
mixing, 0.5 mL should be withdrawn into a 1 mL syringe -- this volume should contain ~6.25 µg 
of product (by polysaccharide weight) as a dose in 0.5 mL. The 0.5 mL of placebo will contain 
only buffer and preservative. 
Step 2 (Cohort B) and Cohort C.  For each dose of vaccine, a 0.5 mL volume from a vial 
containing study product is to be mixed with 0.5 mL of placebo (PBS, Tween-80, and 2-
phenoxyethanol).  After mixing, 0.5 mL should be withdrawn into a 1 mL syringe-- this volume 
should be equivalent to ~12.5 µg of test product (by polysaccharide weight) dose in 0.5 mL. The 
0.5 mL of placebo will contain only buffer and preservative. 

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Step 3 (Cohort C). For each dose of vaccine, a 0.5 mL volume from a vial containing study 
product is to be directly withdrawn into a 1 mL syringe-- this should be equivalent to ~25 µg (by 
polysaccharide weight) dose in a volume of 0.5 mL. The 0.5 mL of placebo will contain only 
buffer and preservative. 
The diluted study product vial can be used up to 4 hours, if stored at 2-8 °C, or up to a 
maximum of 30 minutes if kept at room temperature.  Prepared diluted vaccine must be 
manually shaken gently before using. Once the mixed vaccine has been drawn into a syringe, it 
should be administered within 30 minutes.  Any unused vaccine or waste material should be 
disposed of in accordance with local requirements. 
Each vial (holding 2.5 mL of material plus overfill) shall only be used to withdraw a maximum of 
5 doses (5 punctures per vial) for each day of scheduled administration. Each multidose vial will 
only be used for one day. At the end of each vaccination day, unused vaccine in the multidose 
vial will be marked as un-usable. The Pharmacy will be responsible for keeping track of the 
number of doses drawn per vial and maintaining proper investigational product accountability on 
the accountability log.  
4.4 
Study Product Accountability 
The study product will be stored in and dispensed by the University of Maryland Medical Center 
(UMMC) Investigational Drug Service (IDS) pharmacy or Pharmaron. The FDA requires 
accounting for the disposition of all investigational products. The Investigator is responsible for 
ensuring that an accurate record of product disposition is maintained and product is dispensed 
only by authorized personnel as required by applicable regulations and guidelines. Records of 
product disposition as required by federal law consist of the date received, date administered, 
quantity administered, and the subject number to whom the study product was administered. 
The investigational pharmacist will be responsible for maintaining accurate records of the 
shipment and accountability of the study product.  
For participants enrolled at CVD, the preparation and dispensing of the study product is 
performed by the IDS Pharmacy and the dosing of subjects is performed by CVD research staff; 
the process and procedures are documented and maintained in a secure location that is 
segregated from the blinded research staff. Participants enrolled at Pharmaron will have the 
study product preparation and administration according to their internal standards.  The 
pharmacy records (IDS pharmacy, CVD, and Pharmaron) will be made available for inspection 
by external monitors and by the relevant regulatory agencies (e.g., FDA) at any time. 
4.5 
Masking of the Study Product 
A prospectively assigned vaccinator and checker will be designated as unblinded personnel and 
will not have any role in the assessment of adverse events in study participants.  Unblinded 
personnel will be responsible for the preparation of the study products and for the blinded 
labeling of the study products. The unblinded staff will be responsible for maintaining the 
randomization key and will secure these study documents in a locked cabinet. 
The PI or the Data and Safety Monitoring Board must submit a written request, to inform each 
other, for emergency unblinding, if this were to be performed. The written request must clearly 
state the justification for the emergency unblinding and describe which specific participants are 
to be unblinded. This document will be filed in the study regulatory file. 

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5 
STUDY PROCEDURES 
5.1 
Clinical Evaluations 
Medical History: Will be obtained by interview of the participants. Participants will be queried 
regarding a history of significant medical disorders of the head, eyes, ears, nose, throat 
(HEENT), mouth, cardiovascular system, lungs, gastrointestinal tract, liver, pancreas, kidney, 
urologic, nervous system, blood, lymph glands, endocrine system, musculoskeletal system, 
skin, and genital/reproductive tract. A history of any allergies, cancer, immunodeficiency, 
psychiatric illness, substance abuse, and autoimmune disease will be solicited. 
Medication History: All current medications and medications taken in the 28 days prior to 
enrollment (prescription and over-the-counter drugs will be included as well as vitamins and 
supplements) through 28 days after the last vaccination or early termination (if prior to 28 days 
from last vaccination), whichever occurs first. Assessment of eligibility also will include a review 
of permitted and prohibited medications (per the exclusion criteria).  
Physical Examination: At screening appropriate study personnel will record oral temperature, 
pulse and blood pressure and perform a focused physical examination to assess general 
wellness which will include the following areas/systems:  HEENT; lymph nodes; skin; 
pulmonary; cardiovascular; abdominal; neurological; and musculoskeletal systems. The physical 
examination should specifically address issues identified by the medical history of the 
participant. At following visits including enrollment, oral temperature, pulse, and blood pressure 
will be recorded and a targeted physical examination may be conducted if indicated based on 
review of medical history.  
Reactogenicity Assessments: Will include brief history for assessment of AE/SAEs just prior to 
and following vaccination (Day 1 for Cohorts A-B and Days 1 and 29 for Cohorts C and D), 
which includes an assessment of systemic and local reactions.  Participants will be observed in-
clinic for 20 minutes following each vaccination. 
Memory Aids: All participants will complete a subject memory aid distributed after each 
vaccination. Subject memory aids will be reviewed with the participant for AE/SAEs during visits 
occurring 7 days after each dose of vaccine. Following review, the appropriate case report form 
(CRF) will be completed by the study staff. Memory aids will not be retained as a source 
document. 
5.2 
Clinical Laboratory Evaluations 
A total of 16 mL blood will be collected for screening clinical labs for the evaluation of eligibility.  
If screening laboratory results are out of range value, they may be repeated once provided there 
is an alternative explanation for the out of range value. Participants must have acceptable 
screening laboratory findings prior to enrollment.  The acceptable ranges for study eligibility are 
listed in Appendix B. 
For the assessment of safety, a total of 12.5 mL of blood will be collected for clinical safety labs 
on Day 1 (pre-vaccination) and the Day 8 (7-days post dose #1) visit. This evaluation is to 
include a CBC with differential for total white blood cells (WBC) count, hemoglobin (Hg), 
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alanine aminotransferase (ALT), total bilirubin (TBIL), and C-reactive protein (CRP).  On Day 2 
(one day after receipt of the first dose of vaccine), a 3.5 mL volume of blood will be collected for 
the assessment of C-reactive protein only. The grading of toxicity for the clinical safety lab 
values that are not in the normal range is provided in Appendix C. 
Due to the U.S. Public Health Emergency with COVID-19, SARS-CoV-2 testing will be 
performed on the days of scheduled vaccination. These test results are not intended to inform 
the eligibility of participants for vaccination, as the results are likely to be available after 2-3 
days. However, for possible pre-symptomatic participants (i.e., eligible due to the absence of 
symptoms at the time of vaccination) the positive test result could be used to provide an 
alternate etiology for the development of symptoms due to COVID-19 rather than due to the 
study product.  The test results of acute infection (positive PCR) or prior infection (positive 
antibody) will also be used in planned stratified analysis of the immunology outcomes, as 
COVID-19 infection may alter the immune response to vaccination. The results of SARS-CoV-2 
testing will be provided to participants at their Day 7 and Day 36 visits. If SARS-CoV-2 testing 
results are received prior to these visits, the research team may call participants to report these 
results. 
All women of child-bearing potential will have a serum pregnancy test performed during 
screening and a urine pregnancy test on the vaccination days. The results of the urine 
pregnancy test must be negative prior to enrollment in the study, and prior to each vaccination. 
When a result is abnormal and satisfies the toxicity grading scale, the result must be recorded 
as a laboratory AE and followed until resolution or it becomes stable. If a laboratory value is 
abnormal but does not satisfy the toxicity grading scale, then the abnormality must be assessed 
by the site PI or designee for clinical significance. For all clinically significant abnormalities or 
grade 1 or higher abnormalities, the value should be recorded as a laboratory AE. Any grade 2 
or higher abnormality must be followed until resolution, it becomes stable, or it becomes grade 1 
or lower.  
5.3 
Research Laboratory Assays 
Blood volumes have been summarized in Appendix A (Schedule of Events).  The Immunological 
Assays to be performed with these blood specimens have been described in Section 8 
(Assessment of Immunogenicity). 
The collection of oral crevicular fluid will be performed with a porous foam swab device that is 
gently rubbed along the gumline until the foam sponge is saturated. The foam swab is then 
inserted into an extraction tube that is prefilled with a buffer solution that elutes the antibodies 
from the foam swab. A dropper tip, attached to the tube and swab, effectively acts as a syringe-
like device which simultaneously squeezes the oral fluid-buffer liquid out of the foam and into 
the collection tube. Antibodies in oral fluid will be measured by the method described in Section 
8. 

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6 
STUDY SCHEDULE  
6.1 
Screening Visit (Day -45 till -2) 
After a participant has signed informed consent and has successfully completed the 
Comprehension Assessment Tool, the following will be performed: 
• 
Assignment of study subject identification number 
• 
Complete medical history will be obtained by interview 
• 
Record all concomitant medications taken within 28 days prior to signing the informed 
consent form 
• 
Measure oral temperature, blood pressure, and pulse. 
• 
Collect height and weight 
• 
A targeted physical examination is to be performed by a licensed study clinician 
• 
A blood volume of 16 mL is to be obtained for screening laboratories, consisting of: 
o Hematology (4 mL, lavender, EDTA tube): total WBC, ANC, Hg, platelets 
o Chemistries (8.5 mL, red, serum separator tube): creatinine, AST, ALT, TBIL, 
and CRP 
o Serologies (from same 8.5 mL SST tube for chemistries): hepatitis B surface 
antigen (HBsAg) and hepatitis C virus (HCV) antibody 
o Human immunodefienciency virus (HIV) antibody (3.5 mL, serum separator tube) 
o For women of child-bearing potential: serum β-HCG (to be analyzed from the 
specimen obtained for chemistries) 
6.2 
Visit 1 - Enrollment & Vaccination Dose #1 (Day 1) 
A participant that satisfies eligibility will be given an appointment for enrollment. The following 
activities will be completed prior to vaccination: 
• 
interim medical history or concomitant medications 
• 
A targeted physical examination, if indicated based on review of interim medical history 
• 
Measure oral temperature, blood pressure, and pulse 
• 
Final review of all inclusion and exclusion criteria 
• 
A blood volume of 15 mL for the collection of serum, 80 mL for the collection of 
peripheral blood mononuclear cells (PBMC), and 12.5 mL for safety laboratory 
assessment, consisting of: 
o Hematology (4 mL, lavender, EDTA tube): total WBC, ANC, Hg, platelets 
o Chemistries (8.5 mL, red, serum separator tube): creatinine, AST, ALT, TBIL, 
CRP 
o SARS-CoV-2 antibody test (5 mL), to be performed at University of Maryland 
Pathology Associates (UMPA) 
• 
Collect nasopharyngeal swab for SARS-CoV-2 PCR test, to be performed at University 
of Maryland Pathology Associates (UMPA) 
• 
For women of child-bearing potential, a urine pregnancy will be performed 
• 
Collect oral fluid (crevicular fluid) 
• 
Confirmation of ongoing informed consent 

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Enrollment and randomization may be performed after eligibility has been confirmed.  Once 
randomization has been completed, a prospectively assigned unblinded vaccinator and 
unblinded checker will check the randomization treatment assignment and prepare the masked 
study product.  The injection site will be cleaned with an alcohol swab. The assigned study 
product will be administered to the deltoid muscle by intramuscular route. The study product 
administered, the location, and time of administration will be documented on the appropriate 
source document. 
Following receipt of dose #1 of study product, the participant will be directly observed for at least 
20 minutes.  The PI (or designee) may determine that a participant requires longer on-site 
observation or clinical assessment, as needed for significant adverse events.  Prior to 
discharge, the participant will be provided with training on the completion of a Memory Aid, 
given a digital thermometer, and provided follow-up visit information and contact phone 
numbers for study staff. 
6.3 
Visit 2 Follow Up (Day 2) 
The participant will return to clinic to complete the following: 
• 
Review of the Memory Aid and retraining on the use of the Memory Aid, if necessary 
• 
Any other unsolicited adverse events (AEs) will be recorded 
• 
A blood volume of 3.5 mL for safety laboratory assessment of CRP 
• 
Prior to discharge, a reminder of the date and time for the next visit  will be given to the 
participant 
6.4 
Visit 3 Follow Up (Day 8 ±1) 
The participant will return to clinic to complete the following: 
• 
Interim medical history or concomitant medications 
• 
A targeted physical examination, if indicated based on review of interim medical history 
• 
Collection and review of the Memory Aid for reactogenicity information and completion of 
the corresponding source document.  
• 
Any other unsolicited adverse events (AEs) will be recorded 
• 
A blood volume 12.5 mL for safety laboratory assessment, consisting of: 
o Hematology (4 mL, lavender, EDTA tube): total WBC, ANC, Hg, platelets 
o Chemistries (8.5 mL, red, serum separator tube): creatinine, AST, ALT, TBIL, 
CRP 
• 
A blood volume of 40 mL for PBMC (for Cohorts B, C, and D only) 
• 
If available, the result from the PCR testing performed on Day 1 will be provided to the 
participant 
• 
Prior to discharge, a reminder of the date and time for the next visit will be given to the 
participant 
6.5 
Visit 4 Follow Up (for Cohorts A-B) or Vaccination Dose #2 
(for Cohorts C and D) (Day 29 ±3) 
The participant will return to clinic to complete the following, prior to vaccination: 
• 
Interim medical history or concomitant medications 

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• 
A targeted physical examination, if indicated based on review of interim medical history 
• 
Collection of any AEs will be recorded 
• 
A blood volume of 20 mL for serum and 80 mL for PBMC 
o 5 mL for a SARS-CoV-2 antibody test, to be performed at University of Maryland 
Pathology Associates (UMPA) 
• 
Collect nasopharyngeal swab for SARS-CoV-2 PCR test, to be performed at University 
of Maryland Pathology Associates (UMPA) 
• 
Collect oral fluid (crevicular fluid) 
• 
For women of child-bearing potential, a urine pregnancy will be performed (for Cohort C 
and D only) 
• 
Confirmation of eligibility for dose #2, if Cohort C and D 
For Cohort C and D, the unblinded vaccinator and unblinded checker will administer the 
appropriate study product and record the procedure on the respective source document.  
Following receipt of vaccine, the participant will be directly observed for at least 20 minutes and 
given instructions on the completion of a Memory Aid.  Prior to discharge, follow-up visit 
information and contact numbers will be provided. 
6.6 
Visit 5 Follow Up, only for Cohort C and D (Day 36 ±1) 
The participant will return to clinic to complete the following: 
• 
Interim medical history or concomitant medications 
• 
A targeted physical examination, if indicated based on review of interim medical history 
• 
Collection and review of the Memory Aid for reactogenicity information and completion of 
the corresponding source document.  
• 
Any other unsolicited AEs will be recorded 
• 
A blood volume of 40 mL for PBMC 
• 
The result from the PCR testing performed on Vaccination Dose #2 (Day 29) will be 
provided to the participant 
• 
Prior to discharge, a reminder of the date and time for the next visit will be given to the 
participant 
6.7 
Follow Up, Visit 5 for Cohort A-C, Visit 6 for Cohort C and D 
(Day 57 ±3) 
The participant will return to clinic to complete the following: 
• 
Interim medical history or concomitant medications 
• 
A targeted physical examination, if indicated based on review of interim medical history 
• 
Collection of any AEs (for Cohort C and D only) or SAEs will be recorded 
• 
A blood volume of 15 mL for serum and 80 mL for PBMC 
• 
Collect oral fluid (crevicular fluid) 
6.8 
Final Follow Up, Visit 6 for Cohort A-B (Day 180 ±14), Visit 7 
for Cohort C and D (Day 210 ±14) 
The participant will return to clinic to complete the following: 
• 
Interim medical history  

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• 
A targeted physical examination, if indicated based on review of interim medical history 
• 
Collection of SAEs will be recorded 
• 
A blood volume of 15 mL for serum and up to 80 mL for PBMC 
• 
Collect oral fluid (crevicular fluid) 
6.9 
Unscheduled Visits 
Unscheduled visits may be performed at the participant’s request or by judgement of the PI (or 
designee) when necessary for the diagnosis and/or management of a finding or AEs.  All 
unscheduled visits are to be documented. 
6.10 Early Termination 
A participant may withdraw from the study for any reason or the PI (or designee) may withdraw 
the subject for the welfare of the participant. At this visit, vital signs (oral temp, BP, and pulse) 
and AE recording will be completed. 
 

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7 
ASSESSMENT OF SAFETY 
7.1 
Specification of Safety Parameters 
Safety will be assessed by the frequency and severity of: 
1. Solicited adverse events, reactogenicity events occurring from the time of each 
vaccination through 7 days after vaccination, to include: 
a. Local injection site reactions: pain, erythema (redness), induration (swelling), and 
ecchymosis (bruising). 
b. Systemic reactions: fever, chills, fatigue (tiredness), malaise (general unwell 
feeling), myalgia (body aches), (arthralgia (joint aches), nausea, and headache. 
2. Study product-related unsolicited adverse events, non-serious adverse events occurring 
from the time of each vaccination through 28 days after vaccination. 
3. Study product-related serious adverse events occurring from the time of the first 
vaccination and through the final visit (or time of early withdrawal, when applicable) for 
each participant. 
4. New-onset chronic medical conditions occurring from the time of the first vaccination and 
through the final visit (or time of early withdrawal, when applicable) for each participant. 
 
Safety assessment will also include laboratory parameters which will be evaluated at baseline 
(pre-vaccination) and 7 days after the first dose of vaccine and are also to include: WBC, ANC, 
Hg, platelet counts, creatinine, AST, ALT, TBIL, and CRP. CRP will also be evaluated at one 
day after receiving the first dose of vaccine (Day 2).  
7.2 
Methods and Timing for Assessing, Recording, and 
Analyzing Safety Parameters 
Adverse Event (AE):  ICH E6 defines an AE as any untoward medical occurrence in a patient 
or clinical investigation subject administered a pharmaceutical product regardless of its causal 
relationship to the study treatment. An AE can therefore be any unfavorable and unintended 
sign (including an abnormal laboratory finding), symptom, or disease temporally associated with 
the use of medicinal (investigational) product. The occurrence of an AE may come to the 
attention of study personnel during study visits and interviews of a study recipient presenting for 
medical care, or upon review by a study monitor.  
All AEs including local and systemic reactions not meeting the criteria for “serious adverse 
events” should be captured on the appropriate CRF. Information to be collected includes event 
description, time of onset, clinician’s assessment of severity, relationship to study product 
(assessed only by those with the training and authority to make a diagnosis, which would 
include MD, PA, and Nurse Practitioner, listed on the FDA Form 1572), and time of 
resolution/stabilization of the event. All AEs occurring while on study must be documented 
appropriately regardless of relationship. All AEs will be followed to adequate resolution.  
Any medical condition that is present at the time that the patient is screened should be 
considered as baseline and not reported as an AE. However, if it deteriorates at any time during 
the study, it should be recorded as an AE. 

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Special Circumstance of COVID-19 Pandemic. While there is a U.S. Public Health 
Emergency for COVID-19, there is the risk of community transmission of infections. The testing 
of both anti-SARS-CoV-2 antibody and presence of virus by PCR on the days of vaccination 
(both dose #1 and #2) are intended to provide the investigators with information on potential 
asymptomatic or presymptomatic infections at vaccination, but the results will not be available in 
time prior to vaccination.  Therefore, the results are intended to potentially provide alternate 
etiology for reactogenicity that might be observed post-vaccination which could be due to a 
COVID-19 infection rather than to the study product. The results of SARS-CoV-2 testing will be 
provided to participants at their Day 7 and Day 36 visits. If SARS-CoV-2 testing results are 
received prior to these visits, the research team may call participants to report these results. 
Severity of Event: All AEs will be assessed by the clinician using a protocol defined grading 
system. For events not included in the protocol defined grading system, than the following 
guidelines will be used to quantify intensity.  
• 
Mild – Grade 1: events require minimal or no treatment and do not interfere with the 
patient’s daily activities. 
• 
Moderate – Grade 2: events result in a low level of inconvenience or concern with the 
therapeutic measures. Moderate events may cause some interference with functioning. 
• 
Severe – Grade 3: events interrupt a patient’s usual daily activity and may require 
systemic drug therapy or other treatment. Severe events are usually incapacitating. 
Changes in the severity of an AE should be documented to allow an assessment of the duration 
of the event at each level of intensity to be performed. Adverse events characterized as 
intermittent require documentation of onset and duration of each episode. 
Relationship to Study Product:  The clinician’s assessment of an AE's relationship to Vaccine 
is part of the documentation process, but it is not a factor in determining what is or is not 
reported in the study. If there is any doubt as to whether a clinical observation is an AE, the 
event should be reported. All AEs must have their relationship to study product assessed using 
the terms: related or not related. In a clinical trial, the study product must always be suspect. To 
help assess, the following guidelines are used. 
• 
Related – The event is temporally related to the administration of the study product and 
no other etiology explains the event. 
• 
Not Related – The event is temporally independent of study product and/or the event 
appears to be explained by another etiology. 
Serious Adverse Event (SAE): An adverse event or suspected adverse reaction is considered 
“serious” if, in the view of either the investigator or sponsor, it results in any of the following 
outcomes:  
• 
Death, 
• 
a life-threatening adverse event*,  
• 
inpatient hospitalization or prolongation of existing hospitalization,  
• 
a persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions, or  

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• 
a congenital anomaly/birth defect.   
• 
Important medical events that may not result in death, be life-threatening, or require 
hospitalizations may be considered serious when, based upon appropriate medical 
judgment they may jeopardize the patient or subject and may require medical or surgical 
intervention to prevent one of the outcomes listed in this definition.  Examples of such 
medical events include allergic bronchospasm requiring intensive treatment in an 
emergency room or at home, blood dyscrasias or convulsions that do not result in 
inpatient hospitalization, or the development of drug dependency or drug abuse. 
* Life-threatening adverse event. An adverse event is considered “life-threatening” if, in the view 
of either the investigator or sponsor, its occurrence places the patient or subject at immediate 
risk of death.  It does not include an adverse event, had it occurred in a more severe form, might 
have caused death. 
All SAEs will be: 
• 
recorded on the appropriate SAE Form 
• 
followed through resolution by a study physician 
• 
reviewed and evaluated for intensity and causality by a physician listed on the Form FDA 
1572 as the Principal Investigator or Sub-Investigator. 
• 
reviewed by the Independent Safety Monitoring Committee and the IRB. 
7.3 
Halting Rules 
If any of the following halting rules are met, the study will not proceed with the remaining 
enrollment or dose escalation without a review by and recommendation from an Independent 
Safety Monitoring Committee: 
• 
Two or more subjects in a dosage cohort experiences the same systemic toxicity, except 
for fever, of grade 3 or above and judged to be associated with vaccine. 
• 
Two or more subjects in a dosage cohort experiences a fever at or greater than grade 3 
within 7 days of vaccination. 
• 
Two or more subjects in a dosage cohort experiences the same grade 2 or greater 
abnormal laboratory value and judged to be associated with vaccine.  
• 
Three or more subjects in a dosage cohort experience the same localized toxicity (local 
pain or induration) at grade 3 or above judged to be associated with vaccine. 
• 
Any death occurring within 7 days following administration of vaccine that was not the 
result of trauma or accident. 
• 
Any subject experiences an SAE determined to be related to vaccine. 
The study may also be halted due to PI or Sponsor discretion. 
7.4 
Safety Oversight 
Safety oversight will be under the direction an Independent Safety Monitoring Committee 
(SMC). There will be an Independent Safety Monitor (ISM) who will be independent of the study 
team, local to the study site and will be provided access to study records, if required. The SMC 
will be comprised of the ISM plus at least two independent members with expertise in infectious 

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diseases and/or clinical trials who will advise the Sponsor and the PI. SMC quorum is defined as 
a simple majority. The SMC will be responsible for three scheduled meetings to review the 
available data up to 7-days post-first dose of vaccination for each Cohort; for Cohorts A-C, the 
SMC will determine whether the trial may proceed to the next dosage cohort.  The ISM will 
review all SAEs in real time and provide a written ISM assessment regarding the event.  The 
SMC may receive a final study report, but a recommendation will not be necessary. 
 
 

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8 
ASSESSMENT OF IMMUNOGENICITY 
8.1 
Enzyme-linked Immunosorbent Antibody Assay (ELISA) 
The serum IgG levels against the respective antigens will be measured, as previously 
described.15,17,18  Briefly, 96-well plates are coated with the respective COPS, Vi, or FliC antigen 
(5 µg/mL). After overnight incubation, plates are washed and sera are tested in serial dilutions. 
Specific antibodies will be detected using peroxidase-labeled anti-human IgG and 3,3,5,5-
Tetramethylbenzidine (TMB) substrate. Test and control sera are run in duplicate. Titers are 
calculated by interpolation of absorbance values of test samples into the linear regression curve 
of a calibrated control (reference serum). The endpoint titers will be reported as ELISA units 
(EU), which represent the inverse of the serum dilution that produces an absorbance value of 
0.2 above the blank. Seroconversion will be defined as a 4-fold increase in the antibody titer 
after immunization. 
8.2 
Opsonophagocytic Activity (OPA) assay 
The OPA assay is to be performed as previously described38,39 with some modifications to the 
originally described Lindow  et al. method.40  Heat-inactivated serum samples will be added to 
bacteria and incubated for 30 min at 37°C to allow for antibody binding (opsonization).  THP-1 
macrophage monolayers will be incubated with opsonized bacteria in antibiotic-free medium for 
30 min. (OPA) and 24 hr (survival analysis).  Following incubation, extracellular bacteria will be 
killed (using gentamicin) and for the opsonophagocytosis assays, macrophages will lysed and 
the lysate will be plated onto LB plates, for enumeration of the colony forming units (CFU). For 
the survival assays, plates will be incubated overnight and the surviving bacteria will be counted 
by lysing and plating the cell lysates onto LB plates as described above. 
8.3 
Serum Bactericidal Activity (SBA) assay 
The SBA assay is planned to be performed as previously described.41 Bacteria, from log-phase 
cultures, are added to a plate for incubation with guinea pig complement and/or baby rabbit 
complement. Serially diluted heat-inactivated serum samples will be added to the plate. The 
SBA antibody titer will be defined as the reciprocal of the highest serum dilution that 
produces>50% killing in relation to the killing observed for the control wells containing bacteria 
and complement only (i.e., no serum). The titers will be determined from the mean bacterial 
count from triplicate wells. 
8.4 
Antibody Secreting Cells (ASC) assay 
Plasmablasts secreting antibodies that recognize the respective COPS, Vi, FliC, or TT antigen 
will be measured using ELISPOT, using previously described methods.42 The frequency of spot-
forming cells (SFC) from replicate wells will be determined and reported as ASC/106 PBMC. A 
positive response will be defined as ≥8 ASC/106 PBMC. Antibodies in lymphocyte supernatants 
(ALS) from these plasmablasts may also be collected and tested using the ELISA assay. From 
some PBMCs there may be the measurement of the mucosal homing potential of plasmablasts. 
PBMC will be enumerated by flow cytometry based on molecules expressed in naïve (Bn; 

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CD19+ IgD+ CD27-) and memory B (BM; CD19+ IgD- CD27+) cells which direct them to home 
to distinct sites (e.g., integrin α4β7, CD62L, CXCR3). 
8.5 
Circulating B cell memory cells (BM) and Cell-mediated 
Immunity (CMI) (cytokines, T cell effector and memory 
(including circulating T follicular/helper cells -cTfh-), T cell 
homing  
Blood will be drawn before each vaccination (Days 1, 29, and 57) and on Days 85 and 209  
post-vaccination and isolated PBMC cryopreserved for studies of specific BM 43-45 and CMI46-50 
responses, including cytokine production (e.g., interferon-γ) and expression of homing 
molecules in effector and memory T cells which might direct them to either mucosal or systemic 
effector sites.   Antigen specific cytokine-production and induction of effector and memory T 
cells, as well as cTfh, will be measured in cryopreserved PBMC stimulated with the appropriate 
antigens (e.g., FliC, tetanus toxoid).  Antigen-stimulated PBMC will be stained for conventional 
flow cytometry (using fluorochrome-conjugated monoclonal antibodies) or mass cytometry 
(using metal-conjugated monoclonal antibodies), run in the corresponding instruments, and the 
data analyzed using supervised and non-supervised flow cytometry analytical packages.  The 
frequency of BM specific for COPS, Vi, FliC, and tetanus toxoid antigens will be measured by a 
previously described ELISPOT-based assay.   
8.6 
Antibodies in Oral Fluid 
As opposed to IgA-rich saliva, oral crevicular fluid (the fluid found in the crevicle between the 
tooth and the gum) is a transudate of serum that is ~1% of the IgG concentration of the blood. 
Using the same method of measure serum ELISA antibody, the antibodies in oral fluid will be 
measured against Vi polysaccharide, TT, FliC, and COPS. (reference: Tapia MD, asetti MF, 
Cuberos, L, et al. Pediatr Infect Dis J Sept 2006; 25(9): 819-825) 

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9 
QUALITY AND MONITORING 
9.1 
Quality Control and Quality Assurance 
All clinical trials conducted by the University of Maryland Center for Vaccine Development (CVD) 
are internally audited for quality assurance (QA) quality control (QC) under the CVD-Wide Quality 
Management Plan (QMP).  The purpose of the QMP is to describe QA/QC procedures designed 
to ensure site and investigator compliance with applicable regulations, adherence to the IRB-
approved protocol, generation of credible, quality data, protect data integrity, and safeguard the 
safety and well-being of study participants. The plan has the authority to enforce and correct 
clinical and laboratory deficiencies observed during the conduct of a trial/research study.   
Quality Management (QM) activities will proceed in compliance with E6(R2) Good Clinical 
Practice:  Integrated Addendum to ICH E6(R1), March, 2018.  Consistent with the University of 
Maryland Center for Vaccine Development site-wide QMP, a Protocol-specific Quality 
Management Plan (QMP) (Appendix B of the site-wide QMP) of the will be developed for this 
protocol.  The Protocol-specific plan applies a risk-based approach to Quality Management (QM) 
focused on the essential elements of clinical trial conduct, i.e., recruitment strategies, the informed 
consent process, eligibility, 
study-related 
procedures, clinical and safety assessments, 
AEs/SAEs, study product management, specimen management, and data management.  The 
plan will also outline site delegation of QC/QA responsibilities, frequency of regulatory file reviews, 
the audit schedule (e.g., sample size, audit frequency, critical data that will be reviewed, 
communication, etc.), documentation and reporting of QA findings/queries, and the development 
of corrective action preventive action (CAPA) plans (as indicated). 
Pharmaron will implement a site-specific QMP, similar to the CVD’s QMP and following the CVD’s 
standards. 
9.2 
Clinical Monitoring 
Site monitoring is conducted to ensure that the rights and well-being of human subjects are 
protected, that the reported trial data are accurate, complete, and verifiable, and that the conduct 
of the trial is in compliance with the currently IRB-approved protocol/amendment(s), with GCP, 
and with applicable regulatory requirement(s). Monitoring refers to the methods used by sponsors 
of investigational studies, or Contract Research Organizations (CROs) delegated site monitoring 
responsibilities, to oversee the conduct of, and reporting of data from, clinical investigations. Site 
monitoring includes ensuring appropriate clinical investigator supervision of study site staff and 
third party contractors. 
In order to ensure protocol compliance, monitoring visits by a sponsor-designated professional 
or monitor will occur at scheduled intervals. The visit frequency will be defined in a monitoring 
plan (refer to Section 8.1 below) and communicated before study start to the principal 
investigator and all other appropriate study personnel. 

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9.3 
Site Monitoring Plan 
Independent site monitoring will be conducted to assess the progress of the clinical trial and 
ensure that the rights, safety, and well-being of study participants are protected, study 
procedures, laboratory procedures, study intervention administration, and data collection 
processes are of high quality that comply with ICH/GCP, federal, state, and local regulatory 
authorities and that the study is conducted in accordance with the IRB-protocol and sponsor 
standard operating procedures (SOPs). Clinical monitoring of this protocol will be performed by 
qualified individuals selected by the sponsor in accordance with ICH/GCP (Section 5.18.2) and 
as detailed in the monitoring plan.   
Site visits will be made at standard intervals as defined by the sponsor. Monitoring visits will 
performed in accordance with ICH/GCP (Section 5.18) and will include, but are not limited to, 
review of regulatory files, participant study charts inclusive of DCFs and source documents 
contained therein, informed consent forms, study product accountability records, medical and 
laboratory reports, etc. Study monitors will meet with investigators to discuss visit findings, any 
problems and/or issues and actions to be taken, if any. The principal investigator will be 
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10 
STATISTICAL CONSIDERATIONS 
10.1 Study Hypothesis and Sample Size Considerations 
The overarching hypothesis of this phase 1 study is that the trivalent S. Enteritidis/S. 
Typhimurium/S. Typhi Vi conjugate vaccine will be well-tolerated at all three dosage levels 
tested and that the highest dosage level will elicit the strongest serum anti-COPS, anti-FliC and 
anti-Vi antibody responses in humans.  The primary hypothesis of safety will be fulfilled if no 
participants develop a vaccine related SAE and each vaccine is well-tolerated.  The secondary 
hypothesis of immunogenicity will be fulfilled if each vaccine elicits an immune response by 
ELISA.   
The trial sample size is intended to minimize the number of subjects exposed to each vaccine 
until sufficient safety data have accumulated to proceed to larger trials. The phase 1 trial sample 
size was not derived to achieve a pre-specified statistical power. Instead, the sample size was 
selected to descriptively evaluate the safety and immunogenicity of these vaccines in a first-in-
man trial. As a result, there will be sufficient power to detect only very large differences in 
immunological responses among groups, and confidence limits will be fairly wide.   
The endpoint measures for the primary, secondary and exploratory objectives are listed in 
Sections 6.1, 6.2 and 6.3. 
10.1.1 Sample size in Part 2 of the study 
The adaptive design for the Part 2 portion of this study will involve the evaluation of single dose 
vs. two-dose regimens of the trivalent vaccine, in comparison to placebo. There is a theoretical 
possibility of immunological “interference” with the addition of antigens in multivalent 
glycoconjugate vaccines.51 Under such circumstances, the responses to some antigens may be 
reduced. There is no expectation for there to be differences in the seroconversion rates 
between single dose and two-dose regimens of glycoconjugate vaccines in healthy U.S. 
adults.52  On the other hand, the design of Part 2 of the study will substantiate moving forward 
with two-dose regimens in sub-Saharan Africa where we do expect that more than a single dose 
will be required in the target pediatric population53,54—so the purpose is the demonstration of 
safety with two sequential doses and not for showing differences in immune responses.   
10.2 General Statistical Principles 
Descriptive Statistics. To summarize safety, the number and percentage of participants in each 
cohort that experiences each AE, categorized by body system and preferred term, will be 
tabulated with by vaccine received along with a corresponding two-sided exact 95% confidence 
interval (95%CI). For seroresponse rate endpoints, two-sided 95%CI for each proportion will be 
calculated.  When determining GMT antibody titer, the log values will be used to construct the 
95%CI using the t-distribution for the mean difference between three pair-wise vaccine groups. 
The mean difference and the corresponding CI limits will then be exponentiated to obtain the 
GMT ratio and corresponding CI.  If the values deviate from a log normal distribution, then a 
non-parametric method may be used for the analysis of GMT. 

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Continuous Measures of Immune Response.  Continuous variables of interest that are not 
normally distributed will be transformed if needed. To compare each vaccine dose group with 
the placebo group, t test or Mann-Whitney U test will be used for continuous variables as 
appropriate   
Multiple Comparisons.  No formal statistical adjustment for multiple comparisons is planned. 
The investigators recognize that the study involves multiple comparisons (among vaccine 
doses, between vaccines, etc.) of safety and immunogenicity parameters, and that statistically 
significant results could be obtained in some cases when there is no difference between the 
groups being compared. However, such instances (type I errors) should be infrequent; e.g., if 
we use p <0.05 as the criterion for statistical significance, only 5% of all comparisons that are 
actually equivalent will be significant. Moreover, it is difficult to know exactly which comparisons 
should be included in an adjustment. (We would not want to include safety assessments, 
because the error of assessing a safe vaccine as unsafe is preferable to thinking that a truly 
unsafe vaccine is safe.) The main purpose of this trial is to provide data for making decisions 
about further study of this vaccine, and statistical significance per se is not the only criterion for 
decision-making in this process.  Nevertheless, the investigators will keep the issue of multiple 
comparisons in mind when interpreting the data and making decisions, even though no formal 
adjustment is made.    
Analyzable Subjects. All vaccinated subjects will be included in the reactogenicity analysis even 
if they have missing data points.  All vaccinated subjects will be included in the immunogenicity 
analysis if they donated at least one pre-vaccination and one post-vaccination specimen.   
COVID-19. For participants in Cohort C, COVID-19 testing (both PCR and antibody) will be 
performed. Because of the potential for COVID-19 infection (either acute infection or recent prior 
infection) to alter immune responses, there will be the intent to stratify the analysis according to 
positive or negative COVID-19 infection.   
10.3 Go/No-Go Milestones 
A set of criteria are to be defined for the safety and tolerability of each of the dose-escalation 
cohorts (Cohorts A-C or Steps 1-3), in order to proceed to the next higher dosage cohort.   
Any dose of vaccine which results in more than one episode of a grade 2 or higher solicited 
local or systemic adverse event of the same category, may be considered too reactogenic for 
dose-escalation, unless the SMC recommends otherwise. 
A set of a priori safety and immunologic benchmarks for the dose-selection of the vaccines in 
Part 2 of the study (Cohort D) is defined. The highest well tolerated dosage (no more than one 
grade 2 or higher solicited symptom of the same category) will be selected for further evaluation 
in Part 2 of the study.  Furthermore, the serum antigen-specific ELISA IgG response rates will 
also be evaluated for dosage selection.  Optimally, the final targeted dosage will result in at 
least 70% response rates with the target dosage of vaccine.  
 
 

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11 
ETHICS/PROTECTION OF HUMAN SUBJECTS 
11.1 Ethical Standard 
The principal investigator will ensure that this trial is conducted in full conformity with the 
principles of the Bellmont Report: Ethical Principles and Guidelines for the Protection of Human 
Subjects of Research of the National Commission for the Protection of Human Subjects of 
Biomedical and Behavioral Research (April 18, 1979) and codified in 45 CFR 46, 21 CFR 50 
and 56, and ICH E6; 62 Federal Regulations 25691 (1997), if applicable.  The principle 
investigator’s institution (University of Maryland, Baltimore, UMB) will hold a current Federal 
Wide Assurance (FWA) issued by the Office of Human Research Protection (HRPO). 
11.2 Institutional Review Board 
The local institution must provide for the review and approval of this protocol and the associated 
informed consent documents by an appropriate ethics review committee or Institutional Review 
Board (IRB). Any amendments to the protocol or consent materials must also be approved before 
they are placed into use unless it is in the best interest of the subjects’ safety to implement 
changes prior to approval. In both the United States and in other countries, only institutions 
holding a current U. S. Federal-Wide Assurance issued by the Office for Human Research 
Protections (OHRP) may participate.  
Refer to: http://www.hhs.gov/ohrp/assurances/    
Prior to enrollment of subjects into this clinical study, the protocol and the informed consent 
form(s) will be reviewed and approved by the appropriate IRB. Any amendments to the protocol 
or consent materials will also be reviewed and approved by the appropriate IRB. The responsible 
official for the IRB will sign the IRB letter of approval of the protocol prior to the start of this clinical 
study. Should amendments to the protocol be required, the amendments will be submitted to the 
IRB; an IRB letter of approval of the amendment must be obtained prior to acting upon the 
amendment in the protocol. 
The contact information for the local IRB and the Human Research Protections Office (HRPO) is: 
University of Maryland, Baltimore 
Human Research Protections Office 
620 W. Lexington Street, Second Floor 
Baltimore, MD 21201, U.S.A. 
Phone 1-410-706-5037 
11.3 Informed Consent Process 
Informed consent is a process that is initiated prior to the individual’s agreeing to participate in 
the study and continues throughout the individual’s study participation.  Extensive discussion of 
risks and possible benefits of this therapy will be provided to the participant.  Consent forms 
describing in detail the study, study procedures and risks are given to the potential participant 
and written documentation of informed consent is required prior to starting any study procedure.  
Consent forms will be IRB-approved and the participant will be asked to read and review the 
document. Upon reviewing the document, the investigator will explain the research study to the 

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participant and answer any questions that may arise.  The participants will sign the informed 
consent document prior to any procedures being done specifically for the study.  The participant 
should have the opportunity to discuss the study with their surrogates or think about it prior to 
agreeing to participate.  The participant may withdraw consent at any time throughout the 
course of the trial.  A copy of the informed consent document will be given to the participant for 
their records.  The rights and welfare of the participant will be protected by emphasizing to them 
that the quality of their medical care will not be adversely affected if they decline to participate in 
this study 
11.4 Exclusion of Women, Minorities, and Children (Special 
Populations) 
This clinical study will include women, children of 18 years of age and older, and all minorities 
who meet the inclusion/exclusion criteria, regardless of religion, sex, or ethnic background. 
11.5 Subject Compensation 
Thoughtful consideration has been made toward subject compensation for healthy volunteer 
participants enrolled into clinical studies involving non-licensed vaccines and experimental 
infections. The overarching ethical issues which the CVD’s compensation scheme attempts to 
address are: (1) avoidance of “undue inducement”, (2) payments which may result in economically 
disadvantaged populations bearing an overwhelming share of the risks and burdens of research, 
and (3) payments which may violate the ethical norms of the investigator-subject relationship by 
turning it into a commercial relationship. The compensation scheme for this study is adopted from 
the Wage-Payment Model.55 Under the Wage-Payment Model, a participant is assumed to require 
little skill, but does require time, effort, and an expectation of experiencing undesirable or 
uncomfortable procedures.  Therefore, the CVD’s compensation rates are calculated to 
approximate the hourly wages of an unskilled high-risk laborer. The compensation rate used in 
this study is an standard rate that is used across all of CVD’s clinical research. This compensation 
rate is clearly described in the informed consent form and is unlikely to be unduly coercive. 
Subjects at the Pharmaron site will be compensated for their time and participation. 
Subject Confidentiality 
Subject (participant) confidentiality is strictly held in trust by the investigators, their staff, and the 
Sponsor.  This confidentiality is extended to cover testing of biological samples and other testing 
in addition to the clinical information relating to participant. 
The study protocol, documentation, data, and all other information generated will be held in strict 
confidence.  No information concerning the study or the data will be released to any unauthorized 
third party without prior written approval of the sponsor. 
The study monitor or other authorized representatives of the sponsor may inspect all documents 
and records required to be maintained by the investigator, including but not limited to, medical 
records (office, clinic, or hospital) and pharmacy records for the participants in this study.  The 
clinical study site will permit access to such records. 

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Participant identity data will be contained in paper study records which will be kept in a locked file 
cabinet and in a secure electronic database, accessible only to authorized users at each clinical 
site. The study database will be user-restricted and password-protected. The study database will 
identify participants by a coded study Volunteer ID number assigned by clinical site personnel, 
thus participants will not be identified by name. 
Subject confidentiality and the limits for access to records is described in the informed consent 
form. 
11.6 Future Use of Stored Specimens 
It is intended that any remaining specimens at the closure of the study will be stored at the CVD 
indefinitely and may be used for further immunological analyses. Participants may choose to have 
their specimens destroyed and must do this in a written request. This is described in the consent 
form.  
 
 
 

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12 
DATA HANDLING AND RECORD KEEPING 
The site principal investigator is responsible for ensuring the accuracy, completeness, legibility, 
and timeliness of the data reported. 
Source data will be generated by the site and collected on data collection forms.  Source data 
will be entered into a REDCap data management system, managed by the Clinical and 
Translational Research Informatics Center (CTRIC), University of Maryland School of Medicine. 
REDCap is a secure web-based application for building an electronic database that provides a 
21 CFR Part 11, FISMA, and HIPAA-compliant environment. Quality control audits of all key 
safety, laboratory, and clinical data in the database will be made after data entry has been 
completed.  Coexistent medical conditions, adverse events and other medical events will be 
coded using MedDRA dictionary. Concomitant medications will be coded using WHO-DD 
dictionary. When the database has been declared to be complete and accurate, the database 
will be locked.  Any changes to the database after that time will only be by joint written 
agreement of the study team.  
Data reported in REDCap are derived from the data collection forms and should be consistent 
with the data collection forms or the discrepancies should be explained. 
The sponsor and/or its designee will provide guidance to the site principal investigators and 
other study personnel on making corrections to the data collection forms. 
 
 

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13 
PUBLICATION POLICY 
All investigators funded by the Wellcome Trust must agree to their Open Access Policy. 
https://wellcome.ac.uk/funding/guidance/open-access-policy 
The Open Access Policy contains the following elements: 
1. Expect authors of research papers, monographs and book chapters to maximize the 
opportunities to make their results available for free 
2. Require electronic copies of any research papers that have been accepted for 
publication in a peer-reviewed journal, and are supported in whole or in part by 
Wellcome Trust funding, to be made available through PubMed Central (PMC) and 
Europe PMC as soon as possible and in any event within six months of the journal 
publisher's official date of final publication (similarly, monographs and book chapters 
must be made available through PMC Bookshelf and Europe PMC with a maximum 
embargo of six months) 
3. Expect Wellcome-funded researchers to select publishing routes that ensure the work is 
available immediately on publication in its final published form, wherever such options 
exist for their publisher of choice and are compliant with our policy  
4. Will provide grantholders with additional funding to cover open access charges, where 
appropriate, in order to meet our requirements 
5. Encourage – and where it pays an open access fee, require – authors and publishers to 
license research papers using the Creative Commons Attribution license (CC-BY) so 
they may be freely copied and re-used (for example, for text- and data-mining purposes 
or creating a translation), provided that such uses are fully attributed (CC-BY is also the 
preferred license for monographs and book chapters) 
6. Affirm the principle that it is the intrinsic merit of the work, and not the title of the journal 
or the publisher with which an author's work is published, that should be considered in 
making funding decisions. 
Therefore, following completion of this clinical trial, the investigators intend to publish the results 
of this research in a peer-reviewed scientific journal, within 12 months of locking the database. 
The International Committee of Medical Journal Editors (ICMJE) member journals have adopted 
a trials-registration policy as a condition for publication. This policy requires that all clinical trials 
be registered in a public trials registry, such as ClinicalTrials.gov* (http://clinicaltrials.gov/), 
which is sponsored by the National Library of Medicine. 

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APPENDIX A: SCHEDULE OF EVENTS (COHORT A-B) 
Type of Visit  
Screen 
V 
F 
F 
F 
F 
F 
 Study Day 
-45 to -2 
1 
2 
8 
29 
57 
180 
Window 
 
 
 
±1 
±3 
±3 
±14 
Informed Consent 
X 
X 
 
 
 
 
 
Review Eligibility 
X 
X 
 
 
 
 
 
Medical History 
X 
X 
 
 
 
 
 
Targeted Exam, if indicated 
X 
X 
 
 
 
 
 
Vital Signs (temp, HR and BP) 
X 
X 
 
 
 
 
 
Pregnancy Test 
XS 
XU 
 
 
 
 
 
Concomitant Meds 
X 
X 
 
 
 
 
 
Randomization 
 
X 
 
 
 
 
 
Vaccination  
 
#1 
 
 
 
 
 
7-day Memory Aid (disperse & collect) 
 
d 
x 
c 
 
 
 
AE assessment 
 
X 
X 
X 
X 
 
 
SAE assessment 
 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 
 
Screening Labs1 
16 mL 
 
 
 
 
 
 
Safety Labs2 
 
12.5 mL 3.5 mL 12.5 mL 
 
 
 
Research Blood Draw – serum 
 
15 mL 
 
 
15 mL 
15 mL 
15 mL 
Research Blood Draw – PBMC  
 
80 mL 
 
40 mL* 80 mL 
80 mL 
80 mL 
 
 
 
 
 
 
 
 
Total cumulative volume of blood, mL 
16 
123.5 
127 
139.5 
179.5* 
234.5 
274.5* 
329.5 
469.5* 
424.5 
464.5* 
Outpatient clinic visit: V= Vaccination; F= Follow-up visit;  
Pregnancy testing, by S= Serum pregnancy test; U= Urine pregnancy test 
1 - Screening Laboratory testing will include: 4 mL for Hematology (WBC, ANC, Hemoglobin, Platelet count); 10 mL for Chemistry (creatinine, AST, ALT, 
total bilirubin, and C-reactive protein; inclusive of β-HCG, when indicated); 5 mL for Serology (Hepatitis B surface antigen and Hepatitis C antibody); 
and 3.5 mL for HIV antibody.  
2 - Clinical Safety Laboratory testing on Day 1 and 8 will include: 4 mL for Hematology (WBC, ANC, Hemoglobin, Platelet count) and 10 mL for Chemistry 
(creatinine, AST, ALT, total bilirubin, and C-reactive protein). On Day 2, 3.5 mL for C-reactive protein only. 
* for Cohort B, on the day 8 visit, a 40 mL volume of blood will be drawn for PBMC to allow for ASC and homing studies  
 
 

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APPENDIX A: SCHEDULE OF EVENTS (COHORT C AND D) 
Type of Visit  
Screen 
V 
F 
F 
V 
F 
F 
F 
 Study Day 
-45 to -2 
1 
2 
8 
29 
36 
57 
210 
Window 
 
 
 
±1 
±3 
±1 
±3 
±14 
Informed Consent 
X 
X 
 
 
X 
 
 
 
Review Eligibility 
X 
X 
 
 
X 
 
 
 
Medical History 
X 
X 
 
 
X 
 
 
 
Targeted Exam, if indicated 
X 
X 
 
 
X 
 
 
 
Vital Signs (temp, HR and BP) 
X 
X 
 
 
X 
 
 
 
Pregnancy Test 
XS 
XU 
 
 
XU 
 
 
 
Concomitant Meds 
X 
X 
 
 
X 
 
 
 
Randomization 
 
X 
 
 
 
 
 
 
Vaccination  
 
#1 
 
 
#2 
 
 
 
7-day Memory Aid (disperse & collect) 
 
d 
x 
c 
d 
c 
 
 
AE assessment 
 
X 
X 
X 
X 
X 
X 
 
SAE assessment 
 
X 
X 
X 
X 
X 
X 
X 
Collect oral fluid 
 
X 
 
 
X 
 
X 
X 
Collect NP swab, for SARS-CoV-2 PCR test 
 
X 
 
 
X 
 
 
 
 
Screening Labs1 
16 mL 
 
 
 
 
 
 
 
Safety Labs2 
 
12.5 mL 3.5 mL 12.5 mL 
 
 
 
 
SARS-CoV-2 antibody test 
 
5 mL 
 
 
5 mL 
 
 
 
Research Blood Draw – serum 
 
15 mL 
 
 
15 mL 
 
15 mL 
15 mL 
Research Blood Draw – PBMC  
 
80 mL 
 
40 mL 
80 mL 
40 mL 
80 mL 
80 mL 
 
 
 
 
 
 
 
 
 
Total cumulative volume of blood, mL 
16 
128.5 
132 
184.5 
284.5 
324.5 
419.5 
514.5 
Outpatient clinic visit: V= Vaccination; F= Follow-up visit;  
Pregnancy testing, by S= Serum pregnancy test; U= Urine pregnancy test 
1 - Screening Laboratory testing will include: 4 mL for Hematology (WBC, ANC, Hemoglobin, Platelet count); 10 mL for Chemistry (creatinine, AST, ALT, 
total bilirubin, and C-reactive protein; inclusive of β-HCG, when indicated); 5 mL for Serology (Hepatitis B surface antigen and Hepatitis C antibody); 
and 3.5 mL for HIV antibody.  
2 - Clinical Safety Laboratory testing on Day 1 and 8 will include: 4 mL for Hematology (WBC, ANC, Hemoglobin, Platelet count) and 8.5 mL for 
Chemistry (creatinine, AST, ALT, total bilirubin, and C-reactive protein). On Day 2, 3.5 mL for C-reactive protein only. 
NP= nasopharyngeal swab 

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APPENDIX B: CLINICAL LABORATORY VALUES, FOR STUDY ELIGIBILITY 
Laboratory Parameter 
Units of measure 
Reference Range* 
Study Inclusion 
Total white blood cell count, WBC 
cells x103 / mm3 
4.0 – 10.0 
3.0 – 11.5 
Absolute Neutrophil Count, ANC 
cells / mm3 
1560 – 8100 
1100 – 8910 
Hemoglobin, Hg (male) 
gram / dL 
13.5 – 17.5 
11.0 – 17.6 
Hemoglobin, Hg (female) 
gram / dL 
12.0 – 16.0 
10.3 – 17.6 
Platelet count 
platelet x 103 / mm3 
130 - 400 
125 - 480 
Creatinine (male) 
mg / dL 
0.7 – 1.3 
<1.4 
Creatinine (female) 
mg / dL 
0.6 – 1.1 
<1.2 
Alanine aminotransferase, ALT 
U / L 
7-45 
<68 
Aspartate aminotransferase, AST (male) 
U / L 
15 – 40 
<60 
Aspartate aminotransferase, AST (female) 
U / L 
13 - 30 
<45 
Total bilirubin 
mg/dL 
0.2 – 1.2 
<1.5 
C-reactive protein 
mg/dL 
<0.8 
<1.1 
Hepatitis B surface antigen, HBsAg 
n/a 
Negative 
Negative 
Hepatitis C Virus antibody 
n/a 
Non-reactive 
Non-reactive 
Human Immunodeficiency Virus antibody 
n/a 
Non-reactive 
Non-reactive 
 
* The reference ranges provided are from Garcia Labs, published from 28 July 2016. 
 
 
 
 

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APPENDIX C: GRADING SCALES FOR LABORATORY TOXICITY AND 
REACTOGENICITY 
Parameter 
Mild, Grade 1 
Moderate, Grade 2 
Severe, Grade 3 
WBC, high  (cells x103/mm3) 
11.5 – 13.0 
13.1 – 15.0 
> 15.0 
WBC, low  (cells x103/mm3) 
2.5 – 3.0 
1.5 – 2.4 
< 1.5 
ANC, high  (cells/mm3) 
8900 – 10,000 
10,010 – 12,000  
> 12,000 
ANC, low  (cells/mm3) 
800 - 1000 
600 - 799 
< 600 
Hemogloblin, high  (gram/dL) 
< 1.2 x ULN 
1.2-1.5 x ULN 
> 1.5 x ULN 
Hemogloblin, low  (gram/dL) 
> 0.8 x LLN 
0.8 – 0.7 x LLN 
< 0.7 x LLN 
Platelets, high  (x 103/mm3) 
< 1.3 x ULN 
1.3-1.6 x ULN 
> 1.6 x ULN 
Platelets, low  (x 103/mm3) 
> 0.9 x LLN 
0.7-0.9 x LLN 
< 0.7 x LLN 
Creatinine, high  (mg/dL) 
< 1.3 x ULN 
1.3-1.8 x ULN 
>1.8 x ULN 
ALT, high  (U/L) 
< 2.5 x ULN 
2.5-5.0 x ULN 
> 5.0 x ULN 
AST, high  (U/L) 
< 2.5 x ULN 
2.5-5.0 x ULN 
> 5.0 x ULN 
Total bilirubin 
< 2.0 x ULN 
2.0-5.0 x ULN 
> 5.0 x ULN 
C-reactive protein 
< 2.0 x ULN 
2.0-5.0 x ULN 
> 5.0 x ULN 
 
Pain, Injection site 
Minimal limitation of limb use 
Greater than minimal 
limitation of limb use 
Inability to use limb or 
perform daily activities 
Erythema, Injection site* 
< 5 cm 
5 - 10 cm 
> 10 cm 
Ecchymosis, Injection site 
< 5 cm 
5 - 10 cm 
> 10 cm 
Induration, Injection site 
< 5 cm 
5 - 10 cm 
> 10 cm 
 
Chills 
No interference with daily 
activity 
Some interference with daily 
activity 
Significant interference, 
prevents daily activity 
Fatigue 
Headache 
Malaise 
Myalgia 
Arthralgia 
Nausea 
Oral temperature (fever) 
100.0 – 101.1°F 
101.2 – 102.0 °F 
> 102 °F 
ULN – upper limit of normal; LLN – lower limit of normal 
* Injection site erythema will not count toward a halting rule.  Injection site erythema of any size with ulceration, drainage, sterile abscess, or evidence 
of secondary infection should be recorded as an AE