Non-specific Effects of Rabies Vaccine on the Incidence of Common Infectious Disease Episodes: a Randomized Controlled Trial
Phase: PHASE4
Conditions: Influenza-like Illness, Upper Respiratory Disease, Diarrhea, Fever
Sponsor: Ross University School of Veterinary Medicine

 
 
Non-specific effects of rabies vaccine on the incidence of common infectious disease 
episodes: study protocol for a randomized controlled trial 
 
Protocol Number: 18-04-FL 
Principal Investigator: Darryn Knobel 
Sponsor: Ross University School of Veterinary Medicine (RUSVM) 
Sponsor’s Protocol Reference Number: 18-04-FL 
Co-Sponsor: Serum Institute of India Pvt. Ltd. (SIIPL) 
 
ClinicalTrials.gov ID: NCT03656198 
 
Funded by: RUSVM Center III 
Version Number:  v.3.1 
29 August 2019 
 
 
 

Protocol 18-04-FL (v.3.1) 29 August 2019 
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Summary of Changes from Previous Version: 
Affected 
Section(s) 
Summary of Revisions Made 
Rationale 
Roles and 
responsibilities 
(p. 3) 
Designation of Donna Barry as 
Manager of RUSVM Student Health 
Services was removed, and 
designation of Marshalette Smith-
Antony as interim Manager of 
RUSVM Health Services was added. 
Change in leadership of RUSVM 
Health Services. Dr. Donna Barry 
left the role when she left 
RUSVM, but remained on the 
study as study nurse practitioner 
Roles and 
responsibilities 
(p. 3) 
Addition of Shianne England as 
study nurse. 
Shianne England brought in as 
nurse in RUSVM Health Services 
after Dr. Donna Barry’s 
departure. 
3.2.4 
Addition of secondary objective to 
safety analysis (sub-group analysis 
for potential effect measure 
modification of safety endpoints by 
sex). 
Evidence in the literature of 
modification of vaccine safety 
outcomes by sex. Alignment with 
secondary objective of sub-group 
analysis for potential effect 
measure modification of Primary 
Endpoint and Secondary 
Endpoints 1-7 by sex (Section 
3.2.2; third bullet point). 
 
3.2.5 
Addition of secondary objective to 
vaccine efficacy ancillary study 
(sub-group analysis for potential 
effect measure modification of 
vaccine efficacy endpoint by sex).  
 
Extension of vaccine efficacy 
ancillary study from two to three 
consecutive cohorts (including Fall 
2019). 
Evidence in the literature of 
modification of vaccine efficacy 
outcomes by sex. Alignment with 
secondary objective of sub-group 
analysis for potential effect 
measure modification of Primary 
Endpoint and Secondary 
Endpoints 1-7 by sex (Section 
3.2.2; third bullet point). 
 
To accommodate this secondary 
objective, the vaccine efficacy 
ancillary study was extended to a 
third cohort (Fall 2019) to 
increase the number of males in 
the ancillary study. 

Protocol 18-04-FL (v.3.1) 29 August 2019 
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Roles and responsibilities 
 
Darryn Knobel1 BVSc PhD [Principal Investigator] 
Anne Conan1 DVM PhD [Study coordinator and Data analyst] 
Donna Barry1 APN DNP [Study nurse practitioner] 
Marshalette Smith-Antony1 RN [Study nurse and interim Manager of RUSVM Health 
Services] 
Juliet Battice1 RN [Study nurse] 
Shianne England1 RN [Study nurse] 
Felix Toka1 DVM PhD DSc [Immunology specialist] 
Bradford D. Gessner2 MD MPH [Medical advisor] 
Christianah Ibironke Odita3 DVM MVPH MSc [PhD Student] 
Kristen Boyle1 BSc MSc [Research Assistant] 
 
1Ross University School of Veterinary Medicine, Basseterre, St Kitts 
2EpiVac Consulting Services, Anchorage, Alaska 
3University of Pretoria, Pretoria, South Africa 
 
 
 
 
 
 

Protocol 18-04-FL (v.3.1) 29 August 2019 
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TABLE OF CONTENTS 
STATEMENT OF COMPLIANCE ........................................................................................................ 6 
1 
STUDY SCHEME ....................................................................................................................... 7 
2 
INTRODUCTION ....................................................................................................................... 8 
2.1 
BACKGROUND AND RATIONALE .................................................................................................. 8 
3 
HYPOTHESIS, OBJECTIVES AND ENDPOINTS ......................................................................... 10 
3.1 
PRIMARY HYPOTHESIS ................................................................................................................ 10 
3.2 
OBJECTIVES AND ENDPOINTS .................................................................................................... 10 
3.2.1 
PRIMARY ............................................................................................................................................... 10 
3.2.2 
SECONDARY .......................................................................................................................................... 11 
3.2.3 
TERTIARY .............................................................................................................................................. 13 
3.2.4 
SAFETY .................................................................................................................................................. 14 
3.2.5 
VACCINE EFFICACY ANCILLARY STUDY ................................................................................................. 15 
3.2.6 
IMMUNOLOGY ANCILLARY STUDY ....................................................................................................... 15 
4 
STUDY DESIGN....................................................................................................................... 16 
5 
STUDY POPULATION ............................................................................................................. 16 
5.1 
STUDY SETTING .......................................................................................................................... 16 
5.2 
ELIGIBILITY CRITERIA .................................................................................................................. 17 
5.2.1 
INCLUSION CRITERIA ............................................................................................................................ 18 
5.2.2 
EXCLUSION CRITERIA ............................................................................................................................ 18 
5.2.3 
SCREEN FAILURES ................................................................................................................................. 18 
6 
STUDY INTERVENTIONS ........................................................................................................ 20 
6.1 
VACCINE GROUP ......................................................................................................................... 20 
6.2 
CONTROL GROUP ....................................................................................................................... 20 
6.3 
DOSING AND ADMINISTRATION ................................................................................................ 20 
6.4 
STUDY INTERVENTION COMPLIANCE ......................................................................................... 21 
6.5 
DISCONTINUATION OF STUDY INTERVENTION .......................................................................... 21 
7 
PARTICIPANT TIMELINE......................................................................................................... 22 
7.1 
PARTICIPANT DISCONTINUATION/WITHDRAWAL FROM THE STUDY ....................................... 23 
7.2 
LOST TO FOLLOW-UP ................................................................................................................. 25 
8 
SAMPLE SIZE .......................................................................................................................... 25 
9 
RECRUITMENT ....................................................................................................................... 26 
10 
CONSENT ........................................................................................................................... 27 
11 
ALLOCATION...................................................................................................................... 28 
11.1 
SEQUENCE GENERATION ............................................................................................................ 28 
11.2 
ALLOCATION CONCEALMENT ..................................................................................................... 28 
11.3 
BLINDING (MASKING) ................................................................................................................. 29 
12 
DATA COLLECTION METHODS........................................................................................... 31 
13 
DATA MANAGEMENT ........................................................................................................ 33 
14 
STATISTICAL METHODS ..................................................................................................... 34 
15 
HARMS .............................................................................................................................. 35 
15.1 
DEFINITIONS OF ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS ........................................ 35 
15.2 
CLASSIFICATION OF ADVERSE EVENTS ....................................................................................... 36 
15.3 
IDENTIFICATION OF ADVERSE EVENTS ....................................................................................... 38 
15.4 
REPORTING OF ADVERSE EVENTS .............................................................................................. 39 
16 
RESEARCH ETHICS APPROVAL ........................................................................................... 39 

Protocol 18-04-FL (v.3.1) 29 August 2019 
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17 
TRIAL MONITORING .......................................................................................................... 40 
18 
PROTOCOL AMENDMENTS ............................................................................................... 40 
19 
CONFIDENTIALITY ............................................................................................................. 41 
20 
DECLARATION OF INTERESTS ............................................................................................ 41 
21 
ACCESS TO DATA ............................................................................................................... 42 
22 
DISSEMINATION POLICY.................................................................................................... 42 
23 
REFERENCES ...................................................................................................................... 42 
 
 
 

Protocol 18-04-FL (v.3.1) 29 August 2019 
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STATEMENT OF COMPLIANCE 
The trial will be carried out in accordance with International Conference on Harmonisation 
Good Clinical Practice (ICH GCP) and the World Medical Association Declaration of Helsinki 
- Ethical Principles for Medical Research Involving Human Subjects   
 
All personnel involved in the conduct of this study have completed Human Subjects 
Protection and ICH GCP Training. 
 
The protocol, informed consent form(s), recruitment materials, and all participant 
materials will be submitted to the Institutional Review Board (IRB) for review and 
approval. Approval of both the protocol and the consent form must be obtained before any 
participant is enrolled.  Any amendment to the protocol will require review and approval 
by the IRB before the changes are implemented to the study.  In addition, all changes to the 
consent form will be IRB-approved; a determination will be made regarding whether a new 
consent needs to be obtained from participants who provided consent, using a previously 
approved consent form. 
 
 
 

Protocol 18-04-FL (v.3.1) 29 August 2019 
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1 
STUDY SCHEME 
 
Weekly illness 
survey 3
Excluded: Not eligible
Excluded: No informed 
consent
Excluded: No visit for Dose 1 
by week 12 of semester
Excluded: Permanent CI or 
postponement of Dose 1 
beyond week 12 of semester
Study information shared in 4th
semester/Orientation Week 
List of all VP/1st/5th semester students 
obtained from RUSVM IT Services
Invitation to participate sent via email 
to students
Online assessment of eligibility
Documentation of informed consent
Visit to Student Health Services for 
Dose 1
Screening for contraindications (CIs)
Randomization
Allocated to 
vaccine
Allocated to 
placebo
Received Dose 1 
(day 0)
Received Dose 2 
(day 7)
Received Dose 3 
(day 21)
Received Dose 1 
(day 0)
Received Dose 2 
(day 7)
Received Dose 3 
(day 21)
Baseline health questionnaire
Weekly illness 
survey 1
Weekly illness 
survey 2
Weekly illness 
survey 26
Loss to follow-up or discontinued 
INTERVENTION
INTERVENTION
ASSESSMENT
Excluded: No response

Protocol 18-04-FL (v.3.1) 29 August 2019 
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2 
INTRODUCTION 
2.1 
BACKGROUND AND RATIONALE 
Non-specific effects (NSEs) of vaccines – also known as heterologous effects [1] – are those 
immune-mediated effects of vaccines on morbidity and mortality that are not explained by 
the prevention of the targeted disease [2]. Hypothetically, NSEs may be beneficial or 
detrimental. It has been proposed that this is determined by the type of vaccine [3], with 
NSEs of live vaccines (such as oral polio vaccine (OPV), Bacillus Calmette–Guérin (BCG) and 
measles-containing vaccines (MCV)) reducing all-cause mortality and NSEs of non-live 
vaccines (such as inactivated polio vaccine (IPV) and diphtheria tetanus and whole cell 
pertussis (DTP) combined vaccines) increasing all-cause mortality. It has also been 
reported that these NSEs on all-cause mortality are generally more pronounced in females 
than in males [4]. A systematic review of the evidence for NSEs of BCG, DTP and MCV on all-
cause mortality in children under five years old provides support for these conclusions, 
although much of the evidence came from observational studies with a high risk of bias [5], 
and the importance and implications of these effects remain controversial [6]. There is a 
clear need for new data from randomised controlled trials (RCT), but there are logistic and 
ethical challenges to trials in children with relevant vaccines in high-mortality settings [6]. 
 
Recently, it was proposed that rabies vaccine (a non-live vaccine) has protective NSEs in 
people and in animals [7-9]. The higher number of cases of meningitis and of cerebral 
malaria among children receiving the RTS,S malaria vaccine than in controls who received 
rabies vaccine in a recent large RCT [10] can be explained by a protective NSE of rabies 

Protocol 18-04-FL (v.3.1) 29 August 2019 
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vaccine [7, 8].  In a population-based cohort study, rabies vaccine was shown to be 
associated with decreased all-cause mortality in free-roaming dogs in a high-mortality 
setting [9]. A review of the literature revealed older studies in mice, using live attenuated 
rabies vaccine, that provided protection against Klebsiella pneumonia sepsis [11] and 
mortality following intracerebral injection of a neurotropic strain of herpes virus [12]. 
Studies have shown that part of the rabies virus (the nucleoprotein, which is present in the 
vaccine) acts as a non-specific immunological enhancer [13]. More recently, a controlled 
trial by the PI has shown that rabies vaccine reduces the risk of respiratory disease in 
feedlot cattle (manuscript in preparation). Further randomized controlled trials are needed 
to test for the effect in people.   
 
A non-specific protective effect of rabies vaccine would have implications for prevention of 
rabies in endemic areas. In rabies-endemic areas, recognition of nonspecific effects of 
rabies vaccine in humans could affect public health decisions: Although rabies vaccine is 
known to be a safe and effective vaccine, its routine use as pre-exposure prophylaxis in 
children is not recommended as it is not cost-effective in most situations, in which the 
incidence of exposure to rabies is relatively low [14]. A substantial non-specific protective 
effect against other infections would improve the cost-comparability of routine pre-
exposure prophylaxis vs. a reliance on post-exposure prophylaxis alone in areas where dog 
rabies is endemic. Post-exposure prophylaxis is not always affordable or readily available 
in many developing countries; hence incorporating rabies vaccine into routine childhood 
vaccination schedules could prevent rabies cases.  
 

Protocol 18-04-FL (v.3.1) 29 August 2019 
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The aim of the proposed study is to determine if rabies vaccine reduces the incidence rate 
of episodes of common infectious disease syndromes in a population of veterinary 
students. The primary study endpoint will be a composite outcome of common infectious 
diseases (CID) including upper respiratory, diarrheal, and febrile illnesses. CIDs was chosen 
as the endpoints because of their common occurrence (and thus ability to have adequate 
study power), because of their public health and economic importance, and because no 
studies have yet documented rabies immunization effects on milder disease outcomes.  
 
3 
HYPOTHESIS, OBJECTIVES AND ENDPOINTS 
3.1 
PRIMARY HYPOTHESIS 
Compared to an unvaccinated control group, administration of at least one dose of a three-
dose course of rabies vaccine to previously-unvaccinated subjects leads to at least a 25% 
relative reduction in the rate of self-reported new episodes of common infectious disease 
syndromes (respiratory, diarrheal and febrile illness) over a 26-week period. 
 
3.2 
OBJECTIVES AND ENDPOINTS 
3.2.1 PRIMARY 
 
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
Primary 
To determine if the incidence 
rate of self-reported episodes 
of common infectious disease 
(CID) syndromes (respiratory, 
diarrheal and febrile illness) 
over a 26-week period is 
significantly different between 
previously-unvaccinated 
Number of self-reported new 
episodes of acute common 
infectious disease (CID), 
defined as any of the 
following: upper respiratory 
illness (URI) or influenza-like 
illness (ILI) or diarrhea (DIA) 
The assumption underlying 
the choice of a composite 
primary endpoint is that the 
biological mechanism that 
protects against infections 
that cause URI or ILI will also 
protect against infections that 
cause diarrhea or acute 

Protocol 18-04-FL (v.3.1) 29 August 2019 
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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
subjects that receive at least 
one dose of a three-dose 
course of rabies vaccine and 
those subjects that receive a 
placebo injection. 
 
Primary analysis will be based 
on an intention-to-treat 
analysis.  
or undifferentiated febrile 
illness (UFI). 
 
URI is defined as (two or more 
of the following: runny or 
blocked nose/sneezing/sore 
throat/cough) and (absence of 
itchy or watery eyes). 
ILI is defined as [fever (feeling 
feverish, or an axillary, oral or 
otic temperature of 100°F or 
higher)] and (cough or sore 
throat). 
DIA is defined as three or 
more loose stools within a 24-
hour period. 
UFI is defined as [fever 
(feeling feverish, or an 
axillary, oral or otic 
temperature of 100°F or 
higher)] and (not meeting the 
case definition of URI, ILI or 
DIA). 
To be defined as a new 
episode, illness must be 
preceded by at least one week 
without any CID. 
Time Frame: Weekly self-
reporting of occurrence or 
non-occurrence of episodes of 
CID for a maximum of 26 
weeks, starting 10-14 days 
after allocation. 
 
undifferentiated febrile illness, 
and therefore there is no need 
to separate those outcomes, 
and no biological basis 
currently for doing so. If rabies 
vaccine acts non-specifically to 
prevent some infectious 
diseases, its biological basis is 
unknown. However, the only 
existing data suggest it may 
work as a T-cell enhancer, 
which in theory could prevent 
any infectious disease. 
 
Assessment of occurrence of 
study syndromes will be done 
on a weekly basis, through 
completion of a weekly survey 
of self-reported symptoms of 
illness by participants. For the 
composite outcome of CID, a 
week without any illness must 
occur before an illness is 
considered a new episode; a 
similar requirement will exist 
within individual disease 
syndromes.  
 
 
 
3.2.2 SECONDARY 
 
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
Secondary 

Protocol 18-04-FL (v.3.1) 29 August 2019 
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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
• 
To compare, between the 
same two groups over the 
same time period, 
1) The rate of self-
reported new episodes 
of respiratory illness 
(URI or ILI), DIA and 
UFI. 
2) The rate of self-
reported new episodes 
of  
a)  ILI 
b) URI 
c) DIA 
d) UFI 
3) The rate of clinically-
confirmed episodes of 
the study syndromes 
4) The rate of laboratory-
confirmed episodes of 
the study syndromes 
 
• 
First event, per protocol 
analysis (receipt of all 3 
doses) and extended per 
protocol analysis (receipt 
of at least one dose) of 
primary endpoint and 
secondary endpoints 1-7 
(primary analyses will 
focus on all events and 
intention-to-treat 
analyses). 
 
• 
Sub-group analyses for 
potential effect measure 
modification of Primary 
Endpoint and Secondary 
Endpoints 1-7 by 
1) Sex (males or females) 
2) Season (spring = 
January cohorts; 
summer = May 
cohorts; winter = 
September cohorts) 
1. Number of self-reported 
new episodes of 
respiratory illness (URI or 
ILI), DIA and UFI. 
2. Number of self-reported 
new episodes of URI 
3. Number of self-reported 
new episodes of ILI 
4. Number of self-reported 
new episode of DIA 
5. Number of self-reported 
new episodes of UFI 
6. Number of clinically-
confirmed episodes of the 
study syndromes, defined 
as an episode resulting in a 
visit to the RUSVM Student 
Health Services with a 
recorded ICD10 of J00 
(acute nasopharyngitis); 
J11 (influenza due to 
unidentified influenza 
virus); R19.7 (diarrhea) or 
R50.9 (fever, unspecified). 
7. Number of laboratory-
confirmed episodes of the 
study syndromes, defined 
as clinically-confirmed 
episodes with laboratory 
diagnosis of influenza 
virus, respiratory syncytial 
virus or metapneumovirus 
(URI/ILI episodes) or 
rotavirus or norovirus 
(DIA episodes) . 
 
For Secondary Endpoint 1, in 
any week a single participant 
could report one of the 
following: 
 
• 
No CID episode 
• 
One CID episode (URI or 
ILI or DIA or UFI) 
• 
Two CID episodes (either 
URI or ILI, and DIA) 
 
By this definition, a participant 
cannot experience more than 
two CID episodes within a 
week, as occurrence of URI 
together with ILI is considered 
an episode of ILI only, and 
occurrence of URI, ILI and/or 
DIA precludes the occurrence 
of UFI. 
 
We also will attempt to 
determine impact on 
etiologically confirmed 
outcomes but we expect few 
episodes to meet this criteria 
and thus study power will be 
low. Etiological confirmation 
will be done for a limited 
range of pathogens only 
(influenza virus, respiratory 
syncytial virus, 
metapneumovirus, rotavirus 
and norovirus) through an 
independent research protocol 
(RUSVM IRB protocol #17-11-
FL) and the test results sent 
back to the RUSVM Student 
Health Services. 
 

Protocol 18-04-FL (v.3.1) 29 August 2019 
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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
3) Class (new arrivals [VP 
and 1st semester] vs. 
5th semester) 
 
• 
Subset analyses of Primary 
Endpoint and Secondary 
Endpoints 1-7 in the 
subset of participants 
excluding those 
participants categorized as 
immunosuppressed 
(reporting use of 
immunosuppressive 
medications or treatments 
or occurrence of 
immunosuppressive 
medical conditions in 
baseline questionnaire)  
 
 
3.2.3 TERTIARY 
 
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
Tertiary/Exploratory 
• 
Among the vaccinated 
group only, to compare 
Primary Endpoint and 
Secondary Endpoints 1-7 
between high responders 
and low responders, 
defined by RVNA titers 
measured by rapid 
fluorescent foci inhibition 
test (RFFIT) at day 180 
after first vaccination. 
 
• 
Primary Endpoint and 
Secondary Endpoints 1-7 
 
See above 
• 
Explore latency period and 
duration of effect on 
Primary Endpoint and 
Secondary Endpoints 1-5. 
 
• 
Primary Endpoint and 
Secondary Endpoints 1-5 
 
• 
Explore the effect of dose 
(1 vs. 2 vs. 3) on Primary 
• 
Primary Endpoint and 
Secondary Endpoints 1-5 
 

Protocol 18-04-FL (v.3.1) 29 August 2019 
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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
Endpoint and Secondary 
Endpoints 1-5. 
 
3.2.4 SAFETY 
 
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
Safety 
• 
To compare, between the 
same two groups, 
1) The rate of solicited 
adverse events (AEs) 
through 3 days after 
each injection (dose 1, 
2 and 3) 
2) The rate of unsolicited 
adverse events and 
serious adverse events 
(SAEs) through four 
weeks after first 
injection 
 
• 
As a secondary objective, 
sub-group analysis for 
potential effect measure 
modification of safety 
endpoints by sex (males or 
females) 
1. Number of solicited self-
reported events over three 
days following each 
injection of the following 
adverse events: 
a. Local reactions 
(limited to the site 
of the injection): 
pain, erythema, 
oedema, pruritus 
and induration. 
b. Systemic reactions: 
fever, shivering, 
malaise, asthenia, 
faintness, 
dizziness, 
headache, myalgia, 
arthralgia, nausea 
and abdominal 
pain. 
c. Hypersensitivity or 
allergic reactions: 
anaphylaxis, 
urticaria, rash and 
erythema 
multiforme. 
2. Number of unsolicited 
adverse events and SAEs 
reported to RUSVM 
Student Health Services  
through four weeks after 
first injection 
 
Solicited adverse events are 
those adverse events listed in 
the package insert of the study 
intervention (Rabivax-S rabies 
vaccine) 
 

Protocol 18-04-FL (v.3.1) 29 August 2019 
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3.2.5 VACCINE EFFICACY ANCILLARY STUDY 
 
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
Vaccine efficacy ancillary study 
• 
To compare, between 
participants in each group 
over three consecutive 
cohorts (Spring 2019, 
Summer 2019 and Fall 
2019)(n = ~350), 
concentrations of rabies 
virus neutralizing 
antibody (RVNA) before 
first injection (day -7) and 
six months after first 
injection (day 180). 
 
• 
As a secondary objective, 
sub-group analysis for 
potential effect measure 
modification of vaccine 
efficacy endpoint by sex 
(males or females) 
 
 
 
• 
Geometric mean 
concentration (GMC) of 
RVNA titers measured by 
rapid fluorescent foci 
inhibition test (RFFIT) on 
sera samples collected 7 
days before injection (day 
-7) and 6 months after 
first injection (day 180). 
 
 
3.2.6 IMMUNOLOGY ANCILLARY STUDY 
 
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
Immunology ancillary study 
• 
To compare, between 20 
consecutively enrolled 
participants in each group 
(n = 40), 
1) cytokine response of 
isolated peripheral 
blood mononuclear 
cells (PBMCs) 
stimulated with 
antigens unrelated to 
rabies vaccine 
antigens 
1. Concentration of TNF, IL-
1, IL-6, IFN and IL-17 by 
ELISA assays or Luminex 
system on day 0, 21 and 
90 after first injection 
2. Expression of 84 genes 
involved in innate and 
adaptive immune 
responses, using custom 
RT2 Profiler PCR arrays on 
day 0, 21 and 90 after first 
injection 
 

Protocol 18-04-FL (v.3.1) 29 August 2019 
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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
2) innate and adaptive 
immune gene 
expression profile of 
isolated PBMCs 
stimulated with 
antigens unrelated to 
rabies vaccine 
antigens 
3) changes in the 
proportion of 
monocyte and 
lymphocyte 
subpopulations upon 
stimulation with 
antigens unrelated to 
rabies vaccine 
antigens 
 
3. Proportion of monocyte 
and lymphocyte 
subpopulations of 
peripheral blood 
mononuclear cells 
analyzed in a flow 
cytometer on day 0, 21 
and 90 after first injection 
 
 
4 
STUDY DESIGN 
The trial design is a single-site, two-arm, parallel-group, participant-blinded, randomized, 
placebo-controlled, two-sided comparative study, with an internal pilot study for blinded 
sample size re-estimation. Allocation to study arm will be by block randomization stratified 
by sex within cohort (semester) with a 1:1 allocation ratio. 
 
5 
STUDY POPULATION 
5.1 
STUDY SETTING 
The study will take place at Ross University School of Veterinary Medicine (RUSVM) on the 
island of St Kitts in the Caribbean. The Doctor of Veterinary Medicine (DVM) program at 
RUSVM comprises a preclinical curriculum of seven semesters in St Kitts, and three 
semesters of clinical training at an affiliated school of veterinary medicine in the United 

Protocol 18-04-FL (v.3.1) 29 August 2019 
17
States, Canada, or other international location. There are three semesters per year (starting 
in January, May and September), and one intake (cohort) per semester. Each semester is 15 
weeks, with a break of two or three weeks between semesters. RUSVM also offers a one-
semester Veterinary Preparatory (VP) program. Students who successfully complete the VP 
program are placed into first semester classes. The study will enroll participants from the 
VP program and the 1st and 5th semester classes of the DVM program each semester. 
 
Because St Kitts is free from rabies (https://www.cdc.gov/importation/rabies-free-
countries.html), students are not required to receive pre-exposure rabies prophylaxis 
during their preclinical training (semesters 1-7). All students at RUSVM must have received 
a primary course of rabies vaccine before the end of their preclinical program to provide 
cover during their clinical training (semesters 8-10). This is based on the recommendations 
of the Advisory Committee on Immunization Practices for human rabies prevention [15], 
that veterinarians in the United States are considered a population in the “frequent” risk 
category for rabies exposure, for whom the pre-exposure recommendations are (i) primary 
course of rabies vaccine, followed by (ii) serological testing every two years, and (iii) 
booster vaccination if antibody titer is below acceptable level. Students who have not 
received a primary course of rabies vaccine by their 7th semester are able to do so through 
RUSVM Student Health Services, at a current cost of US$ 200 for the 3-dose course. 
Vaccinations are done by the RUSVM Student Health Services using Rabivax-S, produced by 
the Serum Institute of India Pvt. Ltd (SIIPL). 
 
5.2 
ELIGIBILITY CRITERIA 

Protocol 18-04-FL (v.3.1) 29 August 2019 
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5.2.1 INCLUSION CRITERIA 
A student registered at RUSVM will be eligible for inclusion in the study if s/he is in the VP 
program or the 1st or 5th semester of the DVM program. 
 
5.2.2 EXCLUSION CRITERIA 
A student registered at RUSVM and in the VP program or the 1st or 5th semester of the DVM 
program will be excluded from the study if s/he: 
1) has previously received a dose of rabies vaccine, or 
2) is intending to undertake activities during the course of participation in the study 
that would increase their risk category of rabies exposure above that of the U.S. 
population at large, as defined by the Advisory Committee on Immunization 
Practices (ACIP) for human rabies prevention [15], or 
3) does not provide informed consent for participation, or 
4) enrolls in the study but does not present for the first injection within the first 12 
weeks of the semester (up to and including Week 12), or 
5) has a contraindication to rabies vaccine as described in the Rabivax-S package insert 
(see Section 5.2.3, Screen Failures for details). 
 
5.2.3 SCREEN FAILURES 
Eligible students who consent to participate in the study and who present for the first 
injection within the first 12 weeks of the semester will be screened by study personnel at 
the RUSVM Student Health Services for contraindications to the vaccine, prior to 

Protocol 18-04-FL (v.3.1) 29 August 2019 
19
assignment to study intervention. Contraindications are described in the vaccine package 
insert and are defined as follows: 
1. Contraindications requiring postponement of vaccination: 
a. Fever or an acute illness 
b. Antimalarial medication or other short-term immunosuppressive treatments 
c. Pregnancy 
d. Breastfeeding 
2. Absolute contraindications: 
a. Known systemic hypersensitivity to any of the vaccine components 
(including neomycin) 
b. History of life-threatening reaction to a vaccine containing any of the same 
substances as Rabivax-S 
 
Individuals who fail screening because of a contraindication requiring postponement of 
vaccination may be rescreened once the condition is resolved, so long as they still meet the 
inclusion criteria for the study (a student in the VP program or the 1st or 5th semester of the 
DVM program) and that the intervention regimen (3-dose course on Days 0, 7 and 21) 
could still be completed before the semester break (to comply with this requirement, the 
latest time at which an individual could be rescreened is in Week 12 of the semester). 
 
Individuals who fail screening because of an absolute contraindication will be excluded 
from the study.  
 

Protocol 18-04-FL (v.3.1) 29 August 2019 
20
6 
STUDY INTERVENTIONS 
6.1 
VACCINE GROUP 
The study intervention is at least one dose (1 mL by intramuscular injection) of a three 
dose primary course of Rabivax-S. Rabivax-S is a lyophilized vaccine manufactured by 
Serum Institute of India Pvt. Ltd. containing inactivated purified rabies antigen (Pitman 
Moore, PM3218 as virus strain) produced using Vero ATCC CCL 81 cells. The diluent 
(sterile water for injection) is provided in a separate 1 mL ampoule. After reconstitution, a 
single dose of 1 mL contains an inactivated, purified rabies antigen (not less than 2.5 IU), 
glycine (40 mg), sucrose (40 mg) and human serum albumin (25% 10 mg).  
 
6.2 
CONTROL GROUP 
The intervention (placebo) in the control group is at least one dose (1 mL by intramuscular 
injection) of a three dose primary course (on days 0, 7 and 21) of vaccine diluent (sterile 
water for injection). 
 
6.3 
DOSING AND ADMINISTRATION 
Dosing and administration of the vaccine (Rabivax-S) will be according to the package 
insert, following the schedule for pre-exposure prophylaxis via the intramuscular route; 
that is, 1 mL by intramuscular injection in the deltoid area of the arm on Day 0, Day 7 and 
Day 21. Dosage and administration of the placebo (sterile water for injection) will follow 
the same schedule and route of administration; that is, 1 mL by intramuscular injection in 
the deltoid area of the arm on Day 0, Day 7 and Day 21. In the event of interruption of the 
schedule of administration (of either the vaccine or the placebo), the schedule will be 

Protocol 18-04-FL (v.3.1) 29 August 2019 
21
resumed without repeating a previous dose (as per Table 3 Recommendations for 
Interrupted or Delayed Routine Immunization – Summary of WHO Position Papers, 
updated April 2018; 
http://www.who.int/immunization/policy/immunization_tables/en/).  
 
Vaccine and placebo will be administered to participants at the RUSVM Student Health 
Services by study nurses or nurse practitioner, using a 25G (0.50 x 25mm) needle and 2 mL 
syringe. 
 
6.4 
STUDY INTERVENTION COMPLIANCE 
The number of doses received, and the date received, will be recorded by study personnel 
at RUSVM Student Health Services in Case Report Forms (CRF) for Visits 1-3.  An entry will 
also be made in students’ health records at RUSVM Student Health Services. 
 
6.5 
DISCONTINUATION OF STUDY INTERVENTION 
Prior to administration of each dose of either the vaccine or placebo, participants will be 
assessed by personnel at the RUSVM Student Health Services for contraindications to the 
vaccine (see Section 5.2.3, Screen Failures for listed contraindications). Individuals with 
a contraindication requiring postponement of vaccination may return once the condition is 
resolved to resume the intervention for their allocated group (vaccine or placebo), so long 
as this occurs before study completion (see Section 7, Participant Timeline for definition 
of study completion). In the event of interruption of the schedule of administration (of 

Protocol 18-04-FL (v.3.1) 29 August 2019 
22
either the intervention or the control), the schedule will be resumed without repeating a 
previous dose (see Section 6.3, Dosing and Administration for details). 
 
Participants for whom the study intervention was halted, due to absolute contraindications 
or other clinically-relevant event, or for whom intervention was postponed beyond the 
time of study completion, will remain in the study and complete the weekly illness surveys.  
 
7 
PARTICIPANT TIMELINE 
Students will be informed about the trial prior to the start of the semester for which they 
are eligible (orientation week for VP and 1st semester cohorts, and at the end of 4th 
semester for 5th semester cohorts). Students will be recruited into the study during 
orientation week and/or the first week of the semester, and will receive the first injection 
(day 0) within the first two weeks of the semester. Participants will receive the second 
injection on day 7 and the third injection on day 21. If necessary, participants can begin the 
intervention later, so long as day 0 falls within the first 12 weeks of the semester (see 
Section 5.2.2, Exclusion criteria for details). 
 
Weekly illness surveys will begin in study week 3 (Monday of study week 3, day 10-14 after 
first injection) for all participants, and will continue for 26 weeks (through week 11 of the 
following semester in the case of a two-week semester break, or week 10 of the following 
semester in the case of a three-week semester break). One week after receipt of the final 
(26th) weekly survey, participants will be unmasked and will exit the study. Following exit, 
participants who were allocated to the control group will begin a primary course of rabies 

Protocol 18-04-FL (v.3.1) 29 August 2019 
23
vaccine in weeks 11 or 12 (depending on 3- or 2-week semester break), to be completed by 
the end of the semester (weeks 14 or 15). 
 
A participant is considered to have completed the study one week after receipt of the final 
(26th) weekly survey. 
 
7.1 
PARTICIPANT DISCONTINUATION/WITHDRAWAL FROM THE STUDY 
Participants are free to withdraw from participation in the study at any time upon request. 
Participants who fail to complete surveys for three consecutive weeks will be followed up 
and contacted to determine if they have withdrawn or wish to withdraw from the study. If 
so, they will be informed of their rabies vaccination status and withdrawn from the study. 
Participants will also be withdrawn from the study if for any reason they become aware of 
their study arm allocation (unblinding). The reason for participant withdrawal will be 
captured in the End of Study CRF. 
 

Protocol 18-04-FL (v.3.1) 29 August 2019 
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Schedule of activities for participants in a single cohort (semester) 
*Some semester breaks are three weeks long, in which case Weeks 27 and 28 since allocation will fall in Weeks 9 and 10 of 6th semester 
**Schedule shown here assumes administration of the first dose in week 2 and administration of subsequent doses per schedule (day 7 and day 21). If necessary, 
participants can begin the intervention at any time in the first 12 weeks of the semester.
 
 
1st semester 
Semester break* 
2nd semester 
Weeks since allocation (Study 
week) 
Wk-1 
Wk1 
Wk2 
Wk3 
Wk4 
Etc. 
Wk15 
Wk16 
Wk17 
Etc. 
Wk25 
Wk26 
Wk27 
Wk28 
Weeks of semester/break 
Wk1 
Wk2 
Wk3 
Wk4 
Wk5 
Etc. 
Wk1 
Wk2 
Wk1 
Etc. 
Wk8 
Wk9 
Wk110 
Wk11 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
ENROLLMENT 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Information 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Eligibility screen 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Informed consent 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Contraindication screening 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Allocation 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
INTERVENTION** 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Dose 1 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Dose 2 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
Dose 3 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
ASSESSMENTS 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Baseline 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Weekly illness survey (1-26) 
 
 
 
 
X 
(S1) 
X 
(S2) 
X 
( Etc.) 
X 
(S13) 
X 
(S14) 
X 
(S15) 
X 
(Etc.) 
X 
(S23) 
X 
(S24) 
X 
(S25) 
X 
(S26) 
Solicited adverse events 
 
 
X 
X 
 
X 
 
 
 
 
 
 
 
 
 
Unsolicited adverse events 
 
 
X 
X 
X 
X 
 
 
 
 
 
 
 
 
 
Blood collection (efficacy 
ancillary study) 
 
X 
(D-7) 
 
 
 
 
 
 
 
 
 
X 
(D180) 
 
 
 
Blood collection (immunology 
ancillary study) 
 
 
X 
(D0) 
 
 
X 
(D21) 
 
X 
(D90) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Study exit 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 

Protocol 18-04-FL (v.3.1) 29 August 2019 
25
7.2 
LOST TO FOLLOW-UP 
A participant will be considered lost to follow-up if s/he fails to complete a weekly survey 
for more than three consecutive weeks and is unable to be contacted by the study 
personnel. Contact will be by email to the participant’s registered RUSVM email address. 
The participant will be considered unreachable if s/he does not respond after 3 emails. At 
this point, s/he will be considered to have withdrawn from the study with a primary reason 
of lost to follow-up. 
 
8 
SAMPLE SIZE 
The estimated number of participants needed to achieve the study’s primary objective is 
430 (215 in each group). The sample size was calculated using the approach for comparing 
two negative binomial rates based on true rates (Approach 2 in [17]), implemented using 
the function power.nb.test in package ‘MKmisc’ in R, and assuming the following parameter 
values: 
 
• Alpha level = 0.05 
• Targeted power = 0.8 
• Event rate for control group = 2 (expected mean number of new CID episodes 
[composite primary endpoint] over 26 weeks) 
• Rate ratio under alternative hypothesis = 0.75 
• Average length of participation (accounting for drop-out and non-response) = 21 weeks 
(0.8 of 26-week observation period) 
• Negative binomial dispersion parameter (k in [17]) = 0.4 

Protocol 18-04-FL (v.3.1) 29 August 2019 
26
 
The three nuisance parameters (average length of participation, event rate for control 
group and negative binomial dispersion parameter) were estimated from data collected 
over 7 weeks of a pilot study of rates of CID episodes in 90 RUSVM students (40 in 1st 
semester and 50 in 5th semester) from 21 May to 8 July, 2018 (weeks 3 through 9 of 
summer semester). 
 
An internal pilot study will be done using data from the first 300 participants to complete 
the study. As the expected enrollment rate is <100 participants per semester, this will allow 
capture of seasonal variation in event rates (≥3 semesters in internal pilot study). The 
three nuisance parameters will be re-estimated from the internal pilot study data and the 
sample size recalculated, using a blinded sample size re-estimation method for count data 
[18, 19], which maintains required power without an increase in the type I error. The 
sample size will only be increased and not decreased on the basis of the internal pilot study 
(that is, if the recalculated sample size is smaller than the original sample size, the original 
sample size will still be used).  
 
9 
RECRUITMENT 
The PI or Study Coordinator will present information on the study to VP and 1st semester 
students during their orientation week prior to the start of their program, and to 5th 
semester students during their 4th semester.  They will be provided with a link to a website 
that contains more information about the project, including the informed consent 
documents and contact details of the study personnel. A question-and-answer session with 

Protocol 18-04-FL (v.3.1) 29 August 2019 
27
the PI will be scheduled. Prospective participants will have the opportunity to ask 
questions via email or in person with the study investigators, either during scheduled 
information sessions or by appointment.  
 
To compensate study participants for their time and inconvenience, the study will provide 
the primary course of vaccine at no cost to all participants (participants allocated to the 
control group [including participants who withdraw or are withdrawn] will receive the 
vaccine after exit from the study). Current cost of rabies vaccination for RUSVM students is 
US$ 200 for a 3-dose primary course. 
 
10 CONSENT 
A list of all VP/1st/5th semester student names and email addresses will be obtained from 
the RUSVM IT Department, and an invitation to join the study will be sent by email to these 
students.  The email will contain a unique link to a survey (Appendix A), which will be used 
to establish eligibility and seek agreement to participate from eligible students. Initial 
agreement to participate in the study will be documented electronically as selection of the 
option, “I agree to participate in the study” for the unique survey linked to the participants 
identifiers (name, surname and email address). Participants who agree in the online survey 
will still need to sign a paper copy of the informed consent document (Appendix B) in 
person with a designated member of the study team (Principal Investigator, Study 
Coordinator or Nurse Practitioner). Only participants who sign a paper copy of the 
informed consent document with a member of the study team will be considered enrolled 
and will be allocated to a study group. A signed and dated copy of the consent form will be 

Protocol 18-04-FL (v.3.1) 29 August 2019 
28
given to the participant.  Paper copies of signed consent forms will be collected and stored 
by the Principal Investigator in a locked file cabinet in an area with limited access (PI’s 
office).  
 
Following enrollment, participants will be given a baseline health questionnaire to 
complete (Baseline CRF; Appendix C) and will be given instructions to visit the RUSVM 
Student Health Services Office within the first two weeks of the semester for screening and 
allocation into one of the two study arms. The completed baseline health questionnaire will 
be checked by study personnel at RUSVM Student Health Services at Visit 1, collected and 
stored by the Principal Investigator in a locked file cabinet in an area with limited access 
(PI’s office). 
 
11 ALLOCATION 
11.1 SEQUENCE GENERATION 
Allocation of participants to study arms will be done by restricted randomization 
(permuted block design with stratification). Stratification will be by cohort (three cohorts 
per year: January, May and September) and sex (within cohort). Within strata, 
randomization will be done by computer-generated randomly permuted blocks of varying 
size (using the function blockrand in the package ‘blockrand’ in R). Enrolled students will 
be allocated in a 1:1 ratio to one of the two study arms. 
 
11.2 ALLOCATION CONCEALMENT 

Protocol 18-04-FL (v.3.1) 29 August 2019 
29
Randomization cards will be created by the Principal Investigator or Study Coordinator 
(using the function plotblockrand in the package ‘blockrand’ in R), placed in opaque 
envelopes with a window, and sealed, with the sequential subject number (starting from 1 
for each sex in each cohort) visible in the envelope window. On the first day of the 
semester, envelopes will be placed in the RUSVM Student Health Services Office. Envelopes 
will be drawn sequentially by study personnel at Student Health Services as participants 
come in for the first injection (day 0), and opened to determine allocation. After the 
injection is administered, the RUSVM Student Health Services member will complete the 
Case Report Form for this visit (Visit 1 CRF; Appendix D), including student name, surname, 
email address, allocation group, intervention actually received, date received, and the batch 
number and expiry date of the vaccine or diluent. An entry will be also be made in students’ 
health records. Data from the Visit 1 CRF will be entered into a password-protected file 
(‘Participant allocation file’) in a restricted-access folder on the RUSVM network. Access to 
the folder will be restricted to study personnel only. When participants return for the 
second (day 7) and third (day 21) injections in the course, study staff at RUSVM Student 
Health Services will refer to the file to determine the participant’s allocation. After 
injection, study personnel will complete CRF Visit 2 (Appendix E) or Visit 3 (Appendix F). 
  
11.3 BLINDING (MASKING) 
Participants will be blinded to their study arm allocation. The intervention procedure will 
be identical for both arms (intramuscular injections at Student Health Services on days 0, 7 
and 21). Participants allocated to the control group will receive an intramuscular injection 

Protocol 18-04-FL (v.3.1) 29 August 2019 
30
of sterile water for injection using identical syringes and needles as for the vaccine group. 
The injection will be prepared in a separate room to maintain participant blinding.  
 
Unblinding of participants will occur in circumstances in which there is a need to 
determine their rabies pre-exposure vaccination status; for example, in the event of a 
possible rabies exposure or if participants wish to undertake activities during the course of 
their participation in the study that would increase their risk category of rabies exposure. 
Rabies post-exposure management differs between patients who have not been previously 
vaccinated and those who have been previously vaccinated. The ACIP-recommended 
treatments for patients who have not been previously vaccinated [16] are rabies immune 
globulin (RIG) infiltrated around the wound (if present), and four injections of rabies 
vaccine (one each on days 0, 3, 7 and 14). For previously-vaccinated patients, ACIP 
recommends that no RIG should be administered, and only two injections of rabies vaccine 
(one each of days 0 and 3). Thus, in the event of a suspected rabies exposure that requires 
post-exposure prophylaxis, participants should establish their vaccination status as soon as 
possible. 
 
Participants can determine their vaccination status by contacting the Student Health 
Services to request access to their health records, or by contacting study personnel with 
access to the ‘Participant allocation file’ and/or Visit 1 CRF (Principal Investigator, Study 
Coordinator, study personnel at Student Health Services). Steps for determining 
vaccination status and emergency contact details of all relevant personnel will be provided 

Protocol 18-04-FL (v.3.1) 29 August 2019 
31
in the informed consent document and in the weekly survey sent to participants. 
Participants who are unblinded will exit the study at that point.   
 
12 DATA COLLECTION METHODS 
After enrollment, participants will complete a questionnaire that will capture data on 
relevant baseline characteristics including sex, age, health and vaccination status. (Baseline 
CRF; Appendix C). Participants will complete a short survey each week, starting in study 
week 3, to capture self-reported episodes of illness (respiratory, diarrheal and febrile 
illness; Primary Endpoint and Secondary Endpoints 1-5) in the preceding week (Appendix 
G). Surveys will be sent by email to all participants each Monday. Participants who have not 
completed the survey by Wednesday afternoon will be sent a request by email to complete 
the survey for the preceding week. Surveys will be sent weekly, for 26 weeks. A participant 
is considered to have completed the study one week after receipt of the final (26th) weekly 
survey for that participant’s cohort.  
 
As an incentive to enhance participant retention and response rates in weekly illness 
surveys, all participants who complete a weekly survey will be entered into a draw each 
week for a chance to win a EC$25 gift voucher to a food/beverage vendor on the RUSVM 
campus. Selection of a winner each week will be done by computer-generated random 
sampling. The weekly winner will be notified by email in the week following the draw. 
 
Clinically- and laboratory-confirmed episodes of study syndromes (Secondary Endpoints 6 
& 7) will be collected by study personnel at RUSVM Student Health Services, as part of the 

Protocol 18-04-FL (v.3.1) 29 August 2019 
32
routine clinic visits and diagnostics. At the end of each 26-week observation period for each 
study cohort, data from that cohort will compiled by the director of the RUSVM Student 
Health Services, anonymized to protect privacy and aggregated by study arm, before being 
sent to the Data Analyst who will conduct the analysis. 
 
Occurrence of expected Adverse Events (Safety Endpoint 1) will be solicited in an online 
survey (Appendix H) sent to participants 3 days after receiving an injection (for dose 1, 2 
and 3). Occurrence of unsolicited adverse events and serious adverse events (Safety 
Endpoint 2) will be captured by study personnel at RUSVM Student Health Services 
through completion of an Adverse Event form (Appendix I; See Section 15.3, 
Identification of Adverse Events for details). 
 
Participants in the vaccine efficacy ancillary study will have a maximum volume of 8 mL of 
blood collected by venipuncture prior to injection on day -7 (acceptable range -3 through -
11), and again on day 180 (acceptable range 173 through 187). Serum will be separated 
and stored at -20 °C at RUSVM until transport to the laboratory for measurement of RVNA 
by RFFIT. Serum specimens will be labelled with participant study identification numbers 
only. No other participant information will be sent to the laboratory. 
 
Participants in the immunology ancillary study will have a maximum volume of 10 mL of 
blood collected by venipuncture prior to injection on day 0, and again on days 21 and 90. 
Blood specimens will be immediately taken to the RUSVM Research Laboratory for 

Protocol 18-04-FL (v.3.1) 29 August 2019 
33
isolation of PBMCs. Blood specimens will be labelled with participant study identification 
numbers only. No other participant information will be sent to the laboratory. 
 
13 DATA MANAGEMENT 
Weekly survey data on illness episodes, will be collected through the Qualtrics® platform. 
Qualtrics safeguards all customer data, and uses secure data centers to ensure the highest 
protection as per the requirements of the Health Information Technology for Economic and 
Clinical Health Act (HITECH) (www.qualtrics.com/security-statement/). Survey data will 
be accessible only by study investigators. Weekly survey data will be downloaded by the 
data analyst from Qualtrics servers each week. Downloaded data will be stored on a 
password-protected computer and backed up to an encrypted external hard drive. Study 
participant research data for purposes of statistical analysis and scientific reporting, will 
not include the participant’s contact or identifying information. Rather, individual 
participants and their research data will be identified by a unique study identification 
number. The code for re-identification of study data will be held by the data analyst in a 
separate password-protected database. 
 
For the vaccine efficacy ancillary study, RFFIT RVNA titers will be sent by the laboratory to 
the PI or Study Coordinator, who will enter the data in the CRF. 
 
For the immunology ancillary study, laboratory results will be sent by the Immunology 
Specialist to the PI or Study Coordinator, who will enter the data in the CRF. The Data 

Protocol 18-04-FL (v.3.1) 29 August 2019 
34
Analyst will return a deidentified dataset containing the immunology laboratory results to 
the Immunology Specialist for further analysis. 
 
14 STATISTICAL METHODS 
The analysis of the primary endpoint and secondary endpoints 1-7 will be conducted after 
all randomized participants have completed the study (one week after receipt of the final 
weekly survey) or have withdrawn from the study, whichever occurs first. Primary analysis 
will be based on intention-to-treat analysis of all events (that is, not just occurrence of first 
events). The comparison of incidence rates of CID episodes (primary endpoint and 
secondary endpoints 1-7) between the treatment arms will be performed using a negative 
binomial regression model, which accounts for different lengths of observation period. 
Participants’ number of CID episodes will be modelled as a function of treatment group, 
with the number of weeks of observation included as an offset in the model. This analytic 
model estimates the rate ratio, 𝜆t/ 𝜆c , which quantifies the risk of CID episodes associated 
with rabies vaccination (𝜆t) in comparison to placebo injection (𝜆c). Statistical significance 
will be controlled at the 2-sided, 0.05 alpha level, and the estimated rate ratio compared 
with 1, assuming the following statistical hypothesis: 
 
H0 (null hypothesis): Rate ratio = 1 
H1 (alternative hypothesis) Rate ratio ≠ 1 
 
Treatment effect will be based on the coefficient in the model. Statistical significance at the 
pre-specified alpha level will be based on a Wald testing procedure. CID rates (primary 

Protocol 18-04-FL (v.3.1) 29 August 2019 
35
endpoint and secondary endpoints 1-7) for rabies vaccine and placebo, and the rate ratio, 
will be presented and will include 95% confidence intervals. 
 
A detailed description of the statistical methods that will be used for the primary and 
secondary endpoints will be provided in a Statistical Analysis Plan (SAP). 
 
15 HARMS 
15.1 DEFINITIONS OF ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS 
Definitions of adverse events (AEs) and serious adverse events (SAEs) are taken from the 
Office of Human Research Protections Guidance on Reviewing and Reporting Unanticipated 
Problems Involving Risks to Subjects or Others and Adverse Events.  
 
Adverse event means any untoward or unfavorable medical occurrence in a participant, 
including any abnormal sign (for example, abnormal physical exam or laboratory finding), 
symptom, or disease, temporally associated with the subject’s participation in the research, 
whether or not considered related to the subject’s participation in the research.  
 
A serious adverse event is any adverse event temporally associated with the subject’s 
participation in research that meets any of the following criteria: 
1. results in death; 
2. is life-threatening (places the subject at immediate risk of death from the event as it 
occurred); 
3. requires inpatient hospitalization or prolongation of existing hospitalization; 

Protocol 18-04-FL (v.3.1) 29 August 2019 
36
4. results in a persistent or significant disability/incapacity; 
5. results in a congenital anomaly/birth defect; or 
6. any other adverse event that, based upon appropriate medical judgment, may 
jeopardize the subject’s health and may require medical or surgical intervention to 
prevent one of the other outcomes listed in this definition (examples of such events 
include allergic bronchospasm requiring intensive treatment in the 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). 
 
15.2 CLASSIFICATION OF ADVERSE EVENTS 
All adverse events (AEs) will have their relationship to study intervention assessed by the 
Manager of the RUSVM Student Health Services (study nurse practitioner), in consultation 
with the study Medical Advisor, based on temporal relationship and clinical judgment.  
 
The following guidelines will be used to describe severity:  
• Mild – Events require minimal or no treatment and do not interfere with the 
participant’s daily activities.  
• Moderate – Events result in a low level of inconvenience or concern with the 
therapeutic measures. Moderate events may cause some interference with 
functioning. 
• Severe – Events interrupt a participant’s usual daily activity and may require 
systemic drug therapy or other treatment. Severe events are usually potentially life-

Protocol 18-04-FL (v.3.1) 29 August 2019 
37
threatening or incapacitating.  Of note, the term “severe” does not necessarily 
equate to “serious”. 
 
The degree of certainty about causality will be graded using the categories below.  
• Related – The AE is known to occur with the study intervention, there is a 
reasonable possibility that the study intervention caused the AE, or there is a 
temporal relationship between the study intervention and event. Reasonable 
possibility means that there is evidence to suggest a causal relationship between the 
study intervention and the AE. 
• Not Related – There is not a reasonable possibility that the administration of the 
study intervention caused the event, there is no temporal relationship between the 
study intervention and event onset, or an alternate etiology has been established. 
 
Adverse events will also be classified as expected or unexpected. An AE will be 
considered unexpected if the nature, severity, or frequency of the event is not consistent 
with the risk information previously described for the study intervention. Based on the 
information contained in the package insert for Rabivax-S, the following adverse events are 
considered expected: 
• Local reactions (limited to the site of the injection): pain, erythema, oedema, pruritus 
and induration. 
• Systemic reactions: fever, shivering, malaise, asthenia, faintness, dizziness, headache, 
myalgia, arthralgia, nausea and abdominal pain. 

Protocol 18-04-FL (v.3.1) 29 August 2019 
38
• Hypersensitivity or allergic reactions: anaphylaxis, urticaria, rash and erythema 
multiforme.   
 
15.3 IDENTIFICATION OF ADVERSE EVENTS 
The Study Coordinator will review the Visit 1-3 CRFs daily for participants receiving 
injections (dose 1, 2 or 3). The study coordinator will send a link to an online Qualtrics 
survey (Appendix H) by email on day 3 after the date of the injection (dose 1, 2 or 3) to all 
participants. Information on occurrence of expected AEs will be specifically solicited in the 
survey, by asking the question, “Since receiving the study injection 3 days ago, did you 
experience any of the following…?” and providing participants with a list of expected AEs 
(see Section 15.2, Classification of adverse events for details) from which to select. 
Participants will also be asked to grade the severity of any AEs (mild, moderate or severe) 
and the outcome (recovered/resolved without sequelae, recovered/resolved with sequelae 
or ongoing).  Information on occurrence of other AEs (unsolicited) will be sought by asking 
the question, “Besides the signs listed above, have you noticed anything different since 
starting the study?”, and providing participants the opportunity to describe what they 
noticed or experienced, and grade the severity and outcome as above.    
 
Participants will also be encouraged to report any adverse events to RUSVM Student Health 
Services (unsolicited adverse events). An adverse event form (Appendix I) will be 
completed for all adverse events reported to RUSVM Student Health Services, using the 
Vaccine Adverse Event Reporting System (VAERS) writable PDF form 
(https://vaers.hhs.gov/pdf/vaers_form.pdf). The Principal Investigator, together with the 

Protocol 18-04-FL (v.3.1) 29 August 2019 
39
Director of the RUSVM Student Health Services, will review and compare the reports of 
unsolicited adverse events (adverse event forms) against reports of solicited adverse 
events in the AE surveys, to avoid double capture. 
 
15.4 REPORTING OF ADVERSE EVENTS 
The Principal Investigator will report all serious adverse events whether or not considered 
study intervention related, and all nonserious unexpected adverse events considered 
related to the study intervention (See Section 15.2, Classification of adverse events for 
details), to the RUSVM IRB within 48 hours of his becoming aware of the event (through 
weekly review, or through notification from study personnel at the RUSVM Student Health 
Services).  
 
The Principal Investigator will report safety data (adverse events and serious adverse 
events) to the vaccine manufacturer for the first two cohorts within 30 days of 80% of the 
cohort completing four weeks follow-up after first dose. For remaining cohorts, RUSVM will 
report safety data to the vaccine manufacturer for each cohort within 30 days of exit of 
participants from that cohort. 
 
16 RESEARCH ETHICS APPROVAL 
Approval for the study will be sought from the RUSVM Institutional Review Board (IRB). 
The protocol, informed consent form, recruitment materials, and all participant materials 
will be submitted to the IRB for review and approval. Approval of both the protocol and the 
consent form must be obtained before any participant is enrolled.  Any amendment to the 

Protocol 18-04-FL (v.3.1) 29 August 2019 
40
protocol will require review and approval by the IRB before the changes are implemented 
to the study. Subsequent to initial review and approval, the study will be subject to annual 
continuing review by the RUSVM IRB.  
 
17 TRIAL MONITORING 
Trial monitoring will be conducted to ensure that the rights and well-being of trial 
participants 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 approved 
protocol/amendment(s), with International Conference on Harmonisation Good Clinical 
Practice (ICH GCP), and with applicable regulatory requirement(s). Monitors will be 
appointed by the RUSVM Institutional Officer. Details of trial monitoring are documented in 
a Trial Monitoring Plan (TMP). The TMP describes in detail who will conduct the 
monitoring, at what frequency monitoring will be done, at what level of detail monitoring 
will be performed, and the distribution of monitoring reports. 
 
18 PROTOCOL AMENDMENTS 
Any modifications to the protocol that may impact on the conduct of the study, potential 
benefit of the patient or may affect patient safety, including changes of study objectives, 
study design, patient population, sample sizes, study procedures, or significant 
administrative aspects will require a formal amendment to the protocol. Such amendments 
will be submitted to the RUSVM IRB for review and approval prior to implementation. 
 

Protocol 18-04-FL (v.3.1) 29 August 2019 
41
Administrative changes of the protocol are minor corrections and/or clarifications that 
have no effect on the way the study is to be conducted. These administrative changes will 
be documented in a memorandum. During annual continuing review of the study, the 
RUSVM IRB will be notified of administrative changes made to the protocol since the 
previous review. 
 
Protocol version will be in the form x.x. Formal amendments to the protocol will change the 
first number (e.g. version 1.1 to 2.1). Minor administrative changes will change the second 
number (e.g. version 1.1 to 1.2). All minor changes made after a formal amendment will be 
subsumed in any subsequent formal amendment, so that the second number reverts to 1 
for each formal amendment (e.g. version 1.2 to 2.1). 
 
19 CONFIDENTIALITY 
All study-related information will be stored electronically in password-protected databases 
in file folders with restricted access on the RUSVM network or on password-protected 
laptops with back-up to encrypted external hard drives. Any paper copies with participant 
information will be stored in locked file cabinets in areas with limited access. Biological 
specimens will be identified by a coded identification number. All records that contain 
names or other personal identifiers will be stored separately from the code list.  
 
20 DECLARATION OF INTERESTS 
The protocol was shared with Serum Institute of India Pvt Ltd (SIIPL) as co-sponsor 
but  input was limited to the vaccine safety and efficacy components of the protocol. 

Protocol 18-04-FL (v.3.1) 29 August 2019 
42
Further, role of SIIPL in this study is restricted to supply the products (Rabivax-S and 
Sterile Water for Injections) for this study and to be used as per study protocol. The cost for 
immunogenicity testing of sera samples by RFFIT assay will be borne by SIIPL. 
 
Dr. Gessner worked for AMP through 2017 and currently advises the company; AMP 
receives grant support from Sanofi Pasteur, a manufacturer of rabies vaccine. Dr. Gessner 
currently serves as the Global Medical Lead for Pneumococcal Vaccines at Pfizer. 
 
21 ACCESS TO DATA 
The Principal Investigator and Study Coordinator will have access to the full dataset. 
Deidentified data may be shared with the vaccine manufacturers (SIIPL) and other 
institutions upon request to the PI. 
 
22 DISSEMINATION POLICY 
Results of the internal pilot study (sample size recalculation), vaccine efficacy ancillary 
study and related safety data for participants up to this point, and immunology ancillary 
study may be released prior to release of the main study results (Primary Endpoint and 
Secondary Endpoints 1-7). 
 
23 REFERENCES 
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2. Aaby P, Kollmann TR, Benn CS. Nonspecific effects of neonatal and infant vaccination: 
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