Phase I/II Double-blind, Randomized, Placebo-controlled, Descending Age, Dose-escalation Study of the Safety, and Immunogenicity of the Trivalent P2-VP8 Subunit Rotavirus Vaccine in Healthy South African Adults, Toddlers and Infants
Phase: PHASE1
Conditions: Healthy
Sponsor: PATH

 
 
 
 
VAC 041 
 
A Phase I/II double-blind, randomized, placebo-controlled, 
descending-age, dose-escalation study to examine 
the safety, tolerability and immunogenicity of the 
trivalent P2-VP8 subunit rotavirus vaccine in healthy 
South African adults, toddlers and infants 
 
 
 
21 June 2016 
 
 
Sponsored by: 
 
PATH Vaccine Solutions 
 
 
 
 
National Principal Investigator: 
 
Michelle Groome, MBBCh, DCH, MSc Med 
 
 
 
 
Version 4.0 
 

Study Protocol: VAC 041 
 
PATH Vaccine Solutions 
Version 4.0 
 
21 June 2016 
 
i 
TABLE OF CONTENTS 
 
List of ABBREVIATIONS and ACRONYMS ....................................................................... viii 
PROTOCOL SIGNATURE PAGE ............................................................................................. 1 
PARTICIPATING INSTITUTIONS ............................................................................................. 2 
STUDY SYNOPSIS ................................................................................................................... 4 
1. 
BACKGROUND AND INTRODUCTION ..........................................................................16 
1.1. 
Burden of Disease ....................................................................................................16 
1.2. 
Pathogen ...................................................................................................................16 
1.3. 
Non-replicating Rotavirus Vaccine Approach Using VP8 Subunit ........................17 
1.4. 
Potential Safety Risks ..............................................................................................18 
1.5. 
Summary of Nonclinical Studies with Monovalent (P[8]) P2-VP8 .........................18 
1.5.1. Non-GLP Nonclinical Pharmacology/Proof of Concept Studies ........................ 19 
1.5.1.1. Evaluation of the Antibody Response in Guinea Pigs Administered 
Monovalent (P[8]) VP8 or P2-VP8 .......................................................19 
1.5.1.2. Evaluation of the Antibody Response in Guinea Pigs Administered 
Monovalent (P[8]) P2-VP8 with or without Aluminum Phosphate 
Adjuvant ..............................................................................................20 
1.5.1.3. Evaluation of the Rotavirus Neutralizing Antibody Response in 
Guinea Pigs .........................................................................................20 
1.5.1.4. Protection from Rotavirus Disease in Neonatal Piglets ........................21 
1.5.2. GLP Immunogenicity and Toxicology Study in NZW Rabbits ............................ 23 
1.5.2.1. Study Description ................................................................................23 
1.5.2.2. Toxicology Results ..............................................................................24 
1.5.2.3. Immunogenicity Results.......................................................................25 
1.6. 
Summary of Nonclinical Studies with Trivalent (P[4], P[6], P[8]) P2-VP8 Vaccine
 ...................................................................................................................................27 
1.6.1. Evaluation of the Antibody Response in Guinea Pigs Administered Either 
Trivalent or Monovalent P2-VP8 Vaccine ......................................................... 27 
1.6.2. GLP Immunogenicity and Toxicology Study of the Trivalent P2-VP8 Subunit 
Vaccine in NZW Rabbits ................................................................................... 28 
1.6.2.1. Study Description ................................................................................29 
1.6.2.2. Toxicology Results ..............................................................................29 
1.6.2.3. Immunogenicity Results.......................................................................30 
1.7. 
Clinical Studies .........................................................................................................30 
1.7.1. First-in-human testing of monovalent (P[8]) P2-VP8 subunit vaccine in adults 
(VAC 009) ........................................................................................................ 30 
1.7.2. Clinical testing of monovalent (P[8]) P2-VP8 subunit vaccine in toddlers and 
infants in South Africa (VAC 013) ..................................................................... 36 
2. 
HYPOTHESIS, SCIENTIFIC RATIONALE, OBJECTIVES AND STUDY DESIGN ...........42 
2.1. 
Study Hypothesis .....................................................................................................42 
2.2. 
Scientific Rationale ..................................................................................................42 

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2.3. 
Overall Clinical Development Strategy ...................................................................43 
2.4. 
Study Design ............................................................................................................44 
2.4.1. Primary Objectives ........................................................................................... 44 
2.4.2. Secondary Objectives ....................................................................................... 44 
2.4.3. Exploratory Objective ....................................................................................... 45 
2.4.4. Primary Endpoints ............................................................................................ 45 
2.4.5. Secondary Endpoints: ...................................................................................... 45 
2.4.6. Exploratory Endpoint ........................................................................................ 46 
3. 
STUDY PRODUCT ..........................................................................................................46 
3.1. 
Product Description .................................................................................................46 
3.2. 
Products and Manufacturers ...................................................................................46 
3.3. 
Presentation and Formulation .................................................................................46 
3.4. 
Stability and Storage ................................................................................................47 
3.5. 
Preparation and Administration ..............................................................................47 
3.5.1. Dose Preparation and Administration ............................................................... 48 
3.5.2. Accountability and Disposal .............................................................................. 48 
4. 
STUDY POPULATION .....................................................................................................49 
4.1. 
Clinical Trial Sites .....................................................................................................49 
4.2. 
Study Population ......................................................................................................49 
4.3. 
Eligibility ...................................................................................................................49 
4.4. 
Inclusion Criteria ......................................................................................................49 
4.5. 
Exclusion Criteria .....................................................................................................50 
5. 
STUDY PROCEDURES ...................................................................................................51 
5.1. 
Recruitment ..............................................................................................................51 
5.2. 
Screening, Randomization, and Masking Procedures (for all age-groups) ..........51 
5.2.1. Initial and Continuing Informed Consent ........................................................... 51 
5.2.2. Screening – Day -7 (infants) or Day -28 (adults and toddlers) to Day -1 ........... 51 
5.2.3. Randomization ................................................................................................. 52 
5.2.4. Masking procedures ......................................................................................... 52 
5.2.5. Group A (adults) ............................................................................................... 53 
5.2.5.1. Day 0 – First Dose ...............................................................................53 
5.2.5.2. Day 7, 35 and 63 ± 1 day (to be defined by 7 days after most recent 
study injection day) – Clinic visit follow-up ...........................................54 
5.2.5.3. Day 28 (-2 to +7 days) and Day 56 (-2 to +7 days) days (to be defined 
by 28 days after the previous study injection) – Study injection days ...54 
5.2.5.4. Day 84 ± 4 days (to be defined by 28 days post-3rd study injection day) 
– Clinic follow-up visit and immunogenicity sampling ...........................55 
5.2.5.5. Day 224 ± 14 days (to be defined by 168 days post-3rd study injection 
day) – Final participant contact (telephone or clinic visit) .....................56 
5.2.6. Group B (toddlers) ............................................................................................ 56 
5.2.6.1. Day 0 – Study Injection Day ................................................................56 

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5.2.6.2. Day 3 (to be defined by 3 days after study injection day) – Clinic follow-
up visit .................................................................................................57 
5.2.6.3. Day 7 ± 1 day (to be defined by 7 days after study injection day) – Clinic 
follow-up visit .......................................................................................57 
5.2.6.4. Day 28 ± 4 days (to be defined by 28 days after study injection day) – 
Clinic follow-up ....................................................................................58 
5.2.6.5. Day 168 ± 14 days (to be defined by 168 days post-injection day) – 
Final participant contact (telephone or clinic visit) ................................58 
5.2.7. Groups C and D (infants) .................................................................................. 58 
5.2.7.1. Day 0 – First Dose ...............................................................................58 
5.2.7.2. Day 3, 31 and 59 (to be defined by 3 days after most recent study 
injection day) – Clinic visit follow-up ....................................................59 
5.2.7.3. Day 7, 35 and 63 ± 1 day (to be defined by 7 days after most recent 
study injection day) – Clinic visit follow-up ...........................................60 
5.2.7.4. Day 28 (-2 to +7 days) (to be defined by 28 days after the previous 
study injection) – Study injection day ...................................................61 
5.2.7.5. Day 56 (-2 to +7 days) (to be defined by 28 days after the previous 
study injection) – Study injection day ...................................................62 
5.2.7.6. Day 84 ± 4 days (to be defined by 28 days post-3rd Study injection day) 
– Clinic follow-up visit, immunogenicity sampling and first dose of 
Rotarix .................................................................................................63 
5.2.7.7. Day 89 – Clinic visit for stool sampling (RMPRU only) .........................63 
5.2.7.8. Day 91 – Clinic visit for stool sampling (RMPRU only) .........................63 
5.2.7.9. Day 93 – Clinic visit for stool sampling (RMPRU only) .........................64 
5.2.7.10. Day 112 and 140 + 14 days (to be defined by 28 days after the previous 
Rotarix vaccination) – Clinic visit for second and third dose of Rotarix .64 
5.2.7.11. Day 224 ± 14 days (to be defined by 168 days post-3rd injection day) – 
Final contact (telephone or clinic visit) .................................................64 
5.3. 
Interim Contacts and Unscheduled Visits ..............................................................64 
5.4. 
Stool Samples from Infants with Diarrhea ..............................................................64 
5.5. 
Early Termination .....................................................................................................64 
5.6. 
Procedure Methods ..................................................................................................65 
5.6.1. Vital Signs ........................................................................................................ 65 
5.6.2. Height/length and Weight ................................................................................. 65 
5.6.3. Physical Examination ....................................................................................... 65 
5.6.4. Medical History ................................................................................................. 65 
5.6.5. Injection Site Examination ................................................................................ 65 
5.6.6. Reactogenicity Assessment .............................................................................. 66 
5.6.7. Clinical Laboratory Testing ............................................................................... 66 
5.6.8. Withdrawal from Further Study Injection and Early Termination from Study ..... 66 
5.6.9. Interim Contacts and Visits ............................................................................... 67 
5.6.10. Contraception and Pregnancy .......................................................................... 67 
6. 
LABORATORY EVALUATIONS ......................................................................................67 
6.1. 
Sample collection, distribution and storage ...........................................................67 
6.2. 
Safety clinical laboratory assays.............................................................................67 

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6.3. 
Immunological assays .............................................................................................68 
6.4. 
Assay qualification, standardization and validation ..............................................68 
6.5. 
Stool testing for rotavirus ........................................................................................68 
6.6. 
Rotarix shedding ......................................................................................................68 
6.7. 
Biohazard Containment ...........................................................................................69 
7. 
STATISTICAL DESIGN AND ANALYSIS ........................................................................69 
7.1. 
Overview and General Design .................................................................................69 
7.2. 
Randomization and blinding procedures ................................................................70 
7.3. 
Objectives and Endpoints ........................................................................................70 
7.3.1. Primary Objectives: .......................................................................................... 70 
7.3.2. Secondary Objectives ....................................................................................... 71 
7.3.3. Exploratory Objective ....................................................................................... 72 
7.4. 
Sample Size ..............................................................................................................72 
7.4.1. Primary Safety Endpoints ................................................................................. 72 
7.4.2. Primary Immunogenicity Endpoints .................................................................. 74 
7.5. 
Analytical Methodology ...........................................................................................75 
8. 
SAFETY ASSESSMENT AND REPORTING ...................................................................77 
8.1. 
Safety Events ............................................................................................................77 
8.2. 
Reporting Period ......................................................................................................77 
8.3. 
Definitions .................................................................................................................78 
8.3.1. Adverse Event (AE) or Medical Event ............................................................... 78 
8.3.2. Serious Adverse Events (Defined as Serious Adverse Events, Serious 
Suspected Adverse Reactions or Serious Adverse Reactions) ......................... 78 
8.3.3. Unexpected Adverse Event .............................................................................. 78 
8.4. 
Toxicity Grading .......................................................................................................78 
8.4.1. Guidelines for Determining Causality of an Adverse Event ............................... 79 
8.5. 
Adverse Event Identification, Resolution and Reporting .......................................79 
8.5.1. AE Resolution ................................................................................................... 80 
8.5.2. General Recording and Reporting Procedures ................................................. 80 
8.5.3. SAE Recording and Reporting Procedures ....................................................... 80 
8.5.4. Site Reporting of Events ................................................................................... 81 
8.6. 
Serious Adverse Event Notification ........................................................................81 
8.6.1. Notifications and Review .................................................................................. 81 
8.6.2. Expedited Reporting ......................................................................................... 82 
8.6.3. Notifying the Protocol Safety Review Committee .............................................. 83 
8.6.4. Notifying the Ethics Committee/Institutional Review Board ............................... 83 
9. 
SAFETY MONITORING ...................................................................................................83 
9.1. 
Safety Review Committee ........................................................................................83 
9.2. 
Data and Safety Monitoring Board (DSMB) ............................................................83 

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9.3. 
Withdrawal for Safety ...............................................................................................84 
9.4. 
Study Pause ..............................................................................................................84 
9.4.1. Study Pause Rules ........................................................................................... 84 
9.4.2. Study Pause Procedure .................................................................................... 85 
9.5. 
Dose Escalation ........................................................................................................85 
10. DATA MANAGEMENT ....................................................................................................86 
10.1. Case Report Form Development and Completion ..................................................86 
10.2. Details of Data Management ....................................................................................86 
10.2.1. Data Validation ................................................................................................. 86 
10.2.2. Source Data Verification ................................................................................... 86 
10.2.3. Definition of Source Data .................................................................................. 87 
10.2.4. Definition of Source Document ......................................................................... 87 
10.3. Database Locking Procedures ................................................................................87 
10.4. Record Archival ........................................................................................................87 
10.5. Screen Failures .........................................................................................................87 
10.6. Protocol Deviations ..................................................................................................87 
11. STUDY MONITORING .....................................................................................................88 
11.1. Independent Auditing ...............................................................................................88 
11.2. Regulatory Agency Auditing ....................................................................................89 
12. OBLIGATIONS AND ROLES OF THE SPONSOR, SITE PIS AND STUDY PERSONNEL
 .........................................................................................................................................89 
13. ETHICAL CONSIDERATIONS AND INFORMED CONSENT ..........................................90 
13.1. Informed Consent Process ......................................................................................90 
13.2. Risk/Benefit ...............................................................................................................90 
13.3. Protocol Review Process .........................................................................................90 
13.4. Participant Confidentiality .......................................................................................91 
13.5. Reimbursement ........................................................................................................91 
13.6. Storage of Specimens ..............................................................................................91 
14. APPENDICES ..................................................................................................................91 
14.1. APPENDIX I: Schedule of Events ............................................................................92 
14.1.1. Schedule of Events for Group A (Adults) .......................................................... 92 
14.1.2. Schedule of Events for Group B (Toddlers) ...................................................... 93 
14.1.3. Schedule of Events for Groups C & D (Infants) ................................................. 94 
14.2. APPENDIX II: Toxicity Grading Scales ....................................................................95 
14.3. APPENDIX III: Memory Aid .......................................................................................98 
14.4. APPENDIX IV: Literature Cited ................................................................................99 
 
 
 

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LIST OF TABLES AND FIGURES 
Figure 1: 
Study overview and timeline ................................................................................... 15 
Figure 2: 
Continental Variation in the Distribution of Human Group A Rotavirus P Types 
Ascertained by Analysis of Strains Collected between 1982-2003.......................... 17 
Table 1: 
Preclinical Studies Supporting P2-VP8 Subunit Vaccine Development .................. 19 
Table 2: 
Geometric Mean Neutralizing Titers to Human Rotavirus Strains: Before and After 
Immunization .......................................................................................................... 21 
Table 3: 
Seroconversion to Rotavirus Strains Following Immunization ................................. 21 
Figure 3: 
Rotavirus Geometric Mean Neutralizing Antibody Titers Following Vaccination and 
Challenge ............................................................................................................... 22 
Figure 4: 
Days to Onset of Diarrhea and Viral Shedding by Treatment ................................. 23 
Table 4: 
Geometric Mean Titers by Treatment Following Four Doses of Monovalent (P[8]) 
P2-VP8 Subunit Vaccine with or without Aluminum Hydroxide [Al(OH)3] ................ 26 
Figure 5: 
Geometric Mean Titers by Interval and Treatment .................................................. 26 
Table 5: 
Preclinical Studies Supporting Trivalent Rotavirus P2-VP8 Subunit Vaccine 
Development .......................................................................................................... 27 
Table 6:  
Serum IgG Binding Antibody Responses in Guinea Pigs following vaccination with 
either Monovalent or Trivalent P-VP8 Vaccines. ..................................................... 28 
Table 7.  
Neutralizing Antibody titers to Strains of Rotavirus Expressing Various P Types .... 28 
Table 8. 
Serum IgG Binding Antibody Responses in NZW Rabbits Following Vaccination with 
180 µg Trivalent P2-VP8 Vaccine (Study Day 45) .................................................. 30 
Table 9:  
Maximum Injection Site Reactogenicity per Participant .......................................... 31 
Table 10:  Maximum Systemic Reactogenicity per Participant ................................................ 32 
Table 11:  Maximum Adverse Event Severity per Participant .................................................. 32 
Figure 6:  
Serum Anti-P2-VP8 IgG Titers by ELISA ................................................................ 33 
Table 12:  Proportion of Participants with ≥4-fold Increase in Anti-P2-VP8 IgG Titers by 
ELISA ..................................................................................................................... 33 
Figure 7:  
Serum Anti-P2-VP8 IgA Titers by ELISA ................................................................ 34 
Table 13:  Proportion of Participants with ≥4-fold Increase in Anti-P2-VP8 IgA Titers by 
ELISA ..................................................................................................................... 34 
Figure 8:  
Serum Neutralizing Antibody Titers against Wa Strain ........................................... 35 
Table 14:  Proportion of Participants with ≥4-fold Increase in Neutralizing Antibody Titers 
against Wa Strain ................................................................................................... 35 
Table 15:  Proportion of Participants with ≥4-fold Increase in Neutralizing Antibody Titers at 
Day 84 against Other Strains ................................................................................. 35 
Table 16:  Participants with Detectable Antibody in Lymphocyte Supernatant (ALS) to P2-VP8 
Antigen 7 Days after the Third Study Injection ........................................................ 36 
Table 17:  Maximum Injection Site Reactogenicity per Participant in VAC 013 ........................ 37 

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Table 18: 
Maximum Systemic Reactogenicity per Participant in VAC 013 ............................. 39 
Table 19: 
Maximum Adverse Event Severity per Participant in VAC 013 ............................... 41 
Table 20: 
Anti-P2-VP8 IgG Responses in Infants ................................................................... 41 
Table 21: 
Anti-P2-VP8 IgA Responses in Infants ................................................................... 41 
Table 22: 
Neutralizing Antibody Responses to Wa Strain in Infants ....................................... 42 
Table 23: 
Rotarix Shedding in Infants .................................................................................... 42 
Table 24: 
Dosing Schedule .................................................................................................... 48 
Table 25:  Probabilities of observing AEs ................................................................................ 74 
Table 26:  Power calculations for Comparing GMT in Two Groups of Infants (Cohorts C and D 
combined) .............................................................................................................. 75 
Figure 9: 
Reporting Decisions for Adverse Events ................................................................ 81 
 
 

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VAC 041 
LIST OF ABBREVIATIONS AND ACRONYMS 
ABBREVIATION/ 
ACRONYM 
DEFINITION 
Ab 
AE 
Al(OH)3 
CCHMC 
CDC 
CFR 
cm 
CRF/eCRF 
CSR 
CRO 
dL 
DSMB 
EC 
ECG 
EDC 
ELISA 
EPI 
FAM-CRU 
FDA 
g 
GCP 
GCLP 
GLP 
GMT 
IB 
ICH 
ICF 
IND 
kg 
L 
MCC 
MedDRA 
MedDRA PT 
MedDRA SOC 
mEq 
mg 
mL 
µg 
Antibody 
Adverse Event 
Aluminum Hydroxide Adjuvant 
Cincinnati Children’s Hospital Medical Center 
(U.S.) Centers for Disease Control 
Code of Federal Regulations 
Centimeter 
Case Report Form/electronic Case Report Form 
Clinical Study Report 
Contract Research Organization 
Deciliter 
Data Safety Monitoring Board 
Ethics Committee 
Electrocardiogram 
Electronic Data Capture 
Enzyme-linked Immunosorbent Assay 
Expanded Program on Immunization 
Family Clinical Research Unit, Tygerberg Hospital 
(U.S.) Food and Drug Administration 
Gram 
Good Clinical Practice 
Good Clinical Laboratory Practice 
Good Laboratory Practice 
Geometric Mean Titer 
Investigators’ Brochure 
International Conference on Harmonisation 
Informed Consent Form 
Investigational New Drug 
Kilogram 
Liter 
Medicines Control Council, South Africa 
Medical Dictionary for Regulatory Activities 
Medical Dictionary for Regulatory Activities Preferred Term 
Medical Dictionary for Regulatory Activities System Organ Class 
Milliequivalent 
Milligram 
Milliliter 
Microgram 

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ABBREVIATION/ 
ACRONYM 
DEFINITION 
NIAID 
NICD 
NIH 
NS 
OHRP 
PBMCs 
PE 
PI 
PVS 
SAE 
SDMC 
SDV 
SRC 
SOP 
TV 
ULN 
VS 
WRAIR 
WBC 
WHO 
WIRB 
(U.S.) National Institute for Allergy and Infectious Diseases 
National Institute for Communicable Diseases 
(U.S.) National Institutes of Health 
Normal Saline 
(U.S.) Office of Human Research Protections 
Peripheral Blood Mononuclear Cells 
Physical Examination 
Principal Investigator 
PATH Vaccine Solutions 
Serious Adverse Event 
Statistical Data Management Center 
Source Data Verification 
Safety Review Committee 
Standard Operating Procedure(s) 
Trivalent 
Upper Limit of Normal 
Vital Signs 
Walter Reed Army Institute of Research 
White Blood Cell 
World Health Organization 
Western Institutional Review Board 
 
 
 

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VAC 041 
PROTOCOL SIGNATURE PAGE 
 
PATH Vaccine Solutions, the study Sponsor, will keep a list of Investigator(s), who must provide 
a Curriculum Vita (CV) and a copy of their medical licenses to the Sponsor or Sponsor 
representatives. The Sponsor will keep a list and qualification records of all relevant Sponsor 
study personnel. 
 
The Emmes Corporation will provide medical monitoring, data management, statistical analysis, 
quality assurance and safety and pharmacovigilance services. Site monitoring will be performed 
through SCT Consulting, a contract research organization (CRO) experienced in clinical site 
monitoring. 
 
Site Contact 
Phone Number 
Email Address 
Respiratory and Meningococcal Pathogens Research Unit (RMPRU) PI 
Michelle Groome, MBBCh, DCH, MSc Med 
+27 011 983 4283 
groomem@rmpru.co.za 
Shandukani Research Centre PI  
Lee Fairlie, MD 
 +27 11 358 5317  
lfairlie@wrhi.ac.za 
Family Clinical Research Unit (FAM-CRU) 
Julie Morrison, MBCHB, MRCPCH, DCH, FCPaed   +27 21 938-4153 
morrison@sun.ac.za 
 
 
 
 
SIGNATURE OF SITE PI:  
 
“I have read the foregoing protocol and agree to conduct the study as outlined herein.”  
 
 
---------------------------------------------------- 
------------------------  
Principal Investigator   
 
 
DD/MM/YY 
 
 
 

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VAC 041 
PARTICIPATING INSTITUTIONS 
Sponsor 
PATH Vaccine Solutions 
2201 Westlake Avenue 
Suite 200 
Seattle, WA 98121 
USA 
Clinical Trial Sites 
Family Clinical Research Unit (FAM-CRU)  
Site 8950 
Faculty of Medicine Health Sciences, 
Stellenbosch University 
Ward J8, Tygerberg Academic Hospital,  
Francie Van Zijl Drive, Parow Valley, 7505 
South Africa 
Telephone: +27 21 938 4302 
 
Respiratory and Meningeal Pathogens Research 
Unit (RMPRU) 
West wing, Nurses Residence 
Chris Hani Baragwanath Academic Hospital 
Soweto 
South Africa 
 
Shandukani Research Centre 
Hillbrow Health Precinct  
22 Esselen Street  
Hillbrow 
Johannesburg, 2001 
South Africa 
Telephone: 011 358 5919 
Research Laboratories 
National Institute for Communicable Diseases 
Centre for Enteric Diseases 
1 Modderfontein Rd. 
Sandringham 
Johannesburg, South Africa 
 
Cincinnati Children’s Hospital Medical Center 
Laboratory for Specialized Clinical Studies 
Division of Infectious Diseases 
333 Burnet Avenue ML6014 
Cincinnati, OH 45229-3039 
USA 
Site Monitoring 
SCT Consulting 
15 Bryanston Homestead Ben Road 
Bryanston, 2021, South Africa 
Mailing address: Postnet Suite 376, P/Bag X51, 
Bryanston, Gauteng 2021, South Africa 
Telephone: +27 82 654 8395 

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Medical Monitoring 
The Emmes Corporation 
401 N. Washington St., Suite 700 
Rockville, MD 20850 
USA 
Data Center and  
Statistical Support 
The Emmes Corporation 
401 N. Washington St., Suite 700 
Rockville, MD 20850 
USA 
Vaccine Developer 
PATH Vaccine Solutions 
2201 Westlake Avenue 
Suite 200 
Seattle, WA 98121 
USA 
Vaccine Manufacturer 
Walter Reed Army Institute of Research (WRAIR) 
Pilot BioProduction Facility 
501 Robert Grant Avenue 
Silver Spring, MD 20910 

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STUDY SYNOPSIS 
TITLE 
A Phase I/II double-blind, randomized, placebo-controlled, descending-age, 
dose-escalation study to examine the safety, tolerability and immunogenicity 
of the trivalent P2-VP8 subunit rotavirus vaccine in healthy South African 
adults, toddlers and infants 
STUDY NUMBER 
VAC 041 
PROJECT PHASE 
Phase 1/2 
SPONSOR 
PATH Vaccine Solutions (PVS) 
2201 Westlake Avenue, Suite 200 
Seattle, WA 98121 
MANUFACTURER 
Walter Reed Army Institute of Research (WRAIR) 
Pilot BioProduction Facility 
501 Robert Grant Avenue 
Silver Spring, MD 20910 
NATIONAL 
PRINCIPAL 
INVESTIGATOR 
Michelle Groome, MBBCh, DCH, MSc Med 
Respiratory and Meningeal Pathogens Research Unit (RMPRU) 
West wing, Nurses Residence 
Chris Hani Baragwanath Hospital 
Soweto 
South Africa 
SITE PRINCIPAL 
INVESTIGATORS 
Julie Morrison, MBCHB, MRCPCH, DCH, FCPaed 
Family Clinical Research Unit (FAM-CRU)  
Site 8950 
Faculty of Medicine Health Sciences, Stellenbosch University 
Ward J8, Tygerberg Academic Hospital,  
Francie Van Zijl Drive, Parow Valley, 7505 
South Africa 
Telephone: +27 21 938 4153 
 
Lee Fairlie, MD 
Shandukani Research Centre 
Hillbrow Health Precinct  
22 Esselen Street  
Hillbrow 
Johannesburg, 2001 
South Africa 
Tel: 011 358 5919 
 
Michelle Groome, MBBCh, DCH, MSc Med 
Respiratory and Meningeal Pathogens Research Unit (RMPRU) 
West wing, Nurses Residence 
Chris Hani Baragwanath Hospital 
Soweto 
South Africa  
CRO MEDICAL 
MONITOR 
Robert Lindblad, M.D. 
The Emmes Corporation 
401 N. Washington St., Suite 700 
Rockville, MD 20850 

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PATH MEDICAL 
OFFICER 
Alan Fix, MD, MS 
455 Massachusetts Avenue, Suite 1000 
Washington, DC 20001 
PATH CLINICAL 
OPERATIONS 
SPECIALIST 
Maureen Power, RN, MPH 
455 Massachusetts Avenue, Suite 1000 
Washington, DC 20001 
PATH CONTACT 
REGARDING 
INVESTIGATIONAL 
PRODUCT 
Stanley Cryz, MD, PhD 
455 Massachusetts Avenue, Suite 1000 
Washington, DC 20001 
CONTRACT 
RESEARCH 
ORGANIZATIONS 
Site Monitoring: 
SCT Consulting 
15 Bryanston Homestead Ben Road 
Bryanston, 2021, South Africa 
Mailing address: Postnet Suite 376, P/Bag X51, Bryanston, Gauteng 2021, 
South Africa 
Phone: 27 82 654 8395 
 
Safety Monitoring & Data Management: 
The Emmes Corporation 
401 N. Washington St., Suite 700 
Rockville, MD 20850 
LABORATORIES 
Research Laboratories:  
 
Nicola Page, PhD 
Head: Virology Division 
National Institute for Communicable Disease 
1 Modderfontein Rd. 
Sandringham 
Johannesburg, South Africa 
 
 Monica McNeal, MS 
Cincinnati Children’s Hospital Medical Center 
Laboratory for Specialized Clinical Studies 
Division of Infectious Diseases 
3333 Burnet Avenue ML6014 
Cincinnati, OH 45229-3039 
 
Clinical Laboratory: 
 
Clinical Laboratory Services, 
Spencer Lister Building, NHLS Central Complex 
Cnr Hospital and De Korte Street 
Braamfontein, Johannesburg 
South Africa 
 
 
Bio Analytical Research Corporation (BARC)  
Waverley Business Park, Unit 7 
Bldg 23B, Wycroft Road 
Mowbray, 7700 
South Africa 

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INVESTIGATIONAL 
PHARMACIES 
Respiratory and Meningeal Pathogens Research Unit (RMPRU) 
West wing, Nurses Residence 
Chris Hani Baragwanath Hospital 
Soweto, South Africa 
 
Family Clinical Research Unit (FAM-CRU) 
Tygerberg Hospital 
Francie Van Zijl Drive 
Parow Valley 7505 
Tygerberg, South Africa 
 
Shandukani Research Pharmacy 
2nd Floor Shandukani Building 
Hillbrow Health Precinct 
C/O Esselen and Klein Street 
Hillbrow  
Gauteng, South Africa 
STUDY 
HYPOTHESES - 
PRIMARY 
Safety 
The trivalent P2-VP8 subunit rotavirus vaccine is safe and well-tolerated in 
healthy South African adults, toddlers and infants. 
 
Immunogenicity 
The trivalent P2-VP8 subunit rotavirus vaccine is immunogenic in healthy 
South African infants and will induce neutralizing immune responses to at 
least 2 of the 3 strains from which vaccine antigens are derived in 60% or 
more of participants in at least one of the study groups. 
STUDY 
OBJECTIVES 
Primary Objectives:  
Safety  
 
To evaluate the safety and tolerability of the trivalent P2-VP8 subunit 
rotavirus vaccine at escalating dose levels in healthy South African adults, 
toddlers and infants 
Immunogenicity 
 
To evaluate the immunogenicity of three doses of the trivalent P2-VP8 
subunit rotavirus vaccine at different dose levels in healthy South African 
infants 
Secondary Objectives: 
Safety  
 
To evaluate the longer term safety (through 6 months after the last 
vaccination) of the trivalent P2-VP8 subunit rotavirus vaccine at escalating 
dose levels in healthy South African adults, toddlers and infants 
 
 
 

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STUDY 
OBJECTIVES 
(cont.) 
Immunogenicity 
 
To evaluate the immunogenicity of two doses of the trivalent P2-VP8 
subunit rotavirus vaccine at different dose levels in healthy South African 
infants 
 
To evaluate the immunogenicity of the trivalent P2-VP8 subunit rotavirus 
vaccine at different dose levels in healthy South African adults and 
toddlers 
Exploratory Objective: 
Efficacy 
 
To evaluate the impact of the trivalent P2-VP8 subunit rotavirus 
vaccination on shedding of Rotarix subsequently administered in healthy 
South African infants as a test of concept 
STUDY 
ENDPOINTS 
Primary Endpoints:   
Safety (all 3 age-groups) 
 
Number of SAEs through 28 days after the last study injection 
 
Number of AEs through 28 days after the last study injection 
 
Number of vaccine-induced local and systemic reactions 
 
Immunogenicity (infants): 
 
Proportion of infants with anti-P2-VP8 IgG & IgA seroresponses by ELISA 
(four-fold increase in antibody titers between baseline and 4-weeks post-
third study injection) in assays using each of the three vaccine antigens 
(P[4], P[6] and P[8]) 
 
Proportion of infants with neutralizing antibody responses (2.7-fold 
increase in antibody titers between baseline and 4-weeks post-third study 
injection) to each of the three rotavirus strains from which the vaccine 
antigens are derived, as well as heterologous strains 
 
Anti-P2-VP8 IgG & IgA geometric mean titers (GMT) (baseline and 4 
weeks third study injection) in ELISA assays using each of the three 
vaccine antigens (P[4], P[6] and P[8]) 
 
Neutralizing antibody GMT (baseline and 4 weeks post-third study 
injection) to each of the three rotavirus strains from which the vaccine 
antigens are derived, as well as heterologous strains 
 
Proportion of infants with neutralizing antibody responses to at least two of 
the three strains from which the vaccine antigens are derived 
 
 
 

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STUDY 
ENDPOINTS 
(cont.) 
Secondary Endpoints: 
Safety (all 3 age-groups) 
 
Number of SAEs at any time during the study 
 
Number of AEs at any time during the study  
Immunogenicity (infants): 
 
Proportion of infants with anti-P2-VP8 IgG & IgA seroresponses by ELISA 
(four-fold increase in antibody titers between baseline and 4 weeks post-
second study injection) in assays using each of the three vaccine antigens 
(P[4], P[6] and P[8]) 
 
Proportion of infants with neutralizing antibody responses (2.7-fold 
increase in antibody titers between baseline and 4 weeks post-second 
study injection) to each of the three rotavirus strains from which the 
vaccine antigens are derived, as well as heterologous strains 
 
Anti-P2-VP8 IgG & IgA geometric mean titers (GMT) (baseline and 4 
weeks post-second study injection) in ELISA assays using each of the 
three vaccine antigens (P[4], P[6] and P[8]) 
 
Neutralizing antibody GMT (baseline and 4 weeks post-second study 
injection) to each of the three rotavirus strains from which the vaccine 
antigens are derived, as well as heterologous strains 
 
Proportion of infants with neutralizing antibody responses to at least two of 
the three strains from which the vaccine antigens are derived (4 weeks 
post-second study injection) 
Immunogenicity (adults and toddlers) 
 
Proportion of adults and toddlers with anti-P2-VP8 IgG & IgA 
seroresponses by ELISA (four-fold increase in antibody titers between 
baseline and 4-weeks post-final study injection) in assays using each of 
the three vaccine antigens (P[4], P[6] and P[8]) 
 
Proportion of adults and toddlers with neutralizing antibody responses 
(2.7-fold increases in antibody titers between baseline and 4-weeks post-
final study injection) to each of the three rotavirus strains from which the 
vaccine antigens are derived, as well as heterologous strains 
 
Anti-P2-VP8 IgG & IgA geometric mean titers (GMT) (baseline and 4 
weeks post final study injection) in ELISA assays using each of the three 
vaccine antigens (P[4], P[6] and P[8]) 
 
Neutralizing antibody GMT (baseline and 4 weeks post-final study 
injection) to each of the three rotavirus strains from which the vaccine 
antigens are derived, as well as heterologous strains 
 
Proportion of adults and toddlers with neutralizing antibody responses to 
at least two of the three strains from which the vaccine antigens are 
derived 
Exploratory Endpoint 
 
Proportion of participants shedding Rotarix after administration of Rotarix 
“challenge” 

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STUDY 
RATIONALE  
Live oral rotavirus vaccines have not performed optimally in developing-country 
populations, similar to the discrepancy in performance observed between 
developed versus developing countries for other oral, live attenuated enteric 
vaccines such as those targeted against cholera and poliomyelitis. Several 
direct and indirect observations support the possibility that parenterally-
administered non-replicating rotavirus vaccines may be successful, including 
observations derived from natural rotavirus infection studies in infants, rotavirus 
challenge studies in adult volunteers, human rotavirus vaccine trials, and 
animal rotavirus vaccine studies. The P2-VP8 vaccine contains a truncated 
rotavirus VP8 subunit fused to the tetanus toxin P2 CD4 epitope. It is 
parenterally administered, which may overcome limitations of oral vaccines in 
developing countries. A monovalent formulation containing the VP8 subunit 
from a P[8] rotavirus strain has been demonstrated to be well-tolerated and 
immunogenic in US adults (VAC 009) and is currently being tested in toddlers 
and infants in South Africa (VAC 013), where it has been generally well-
tolerated. In the study in adults, the monovalent vaccine elicited robust 
neutralizing antibody responses to several homologous P[8] strains of 
rotavirus, but modest response to P[4] strains and meager responses to P[6] 
strains. Thus, the trivalent formulation to be tested in this study, containing VP8 
subunits from P[4], P[6] and P[8] strains was developed, with the goal to 
optimize responses to those strains that account for the vast majority of the 
burden of rotavirus disease globally. Preclinical studies of the trivalent vaccine 
indicate improved responses to P[4] and P[6] strains. Preliminary analysis of 
serologic responses and impact of the monovalent P2-VP8 vaccine on 
shedding of Rotarix, as a proxy model for efficacy, in infants in VAC 013 has 
provided promising results. Those results have also provided the basis for the 
selection of the dose levels of the trivalent vaccine to be tested in this protocol. 
INVESTIGATIONAL 
PRODUCTS 
Vaccine: The TV P2-VP8 subunit rotavirus vaccine produced in E. coli is 
adsorbed onto aluminum hydroxide (0.5625 mg/dose). Three dose levels of 
vaccine will be tested: 15 µg, 30 µg and 90 µg (consisting of equal amounts of 
VP8 antigen derived from a P[4], a P[6] and a P[8] strain of rotavirus), all to be 
administered in 0.5 mL volumes intramuscularly. The three antigens are 
derived from DS1, 1076 and Wa strains, respectively. 
 
Placebo: Normal Saline (NS) 
STUDY 
POPULATION 
 
30 healthy South African adults (18-45 years old, inclusive, at time of first 
vaccination)  
 
30 healthy South African toddlers (≥2 & < 3 years old at time of first 
vaccination) 
 
600 healthy South African infants (≥6 & <8 weeks old at the time of first 
vaccination) 
STUDY DURATION 
Adult and infant participants will be followed for approximately 8 months after 
randomization (6 months following last [3rd] injection). Toddler participants will 
be followed for approximately 6 months after randomization (6 months 
following the single injection). 

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STUDY DESIGN 
The trial will be a double-blind, randomized, placebo-controlled, descending-
age, dose-escalation study in which two dose-levels of vaccine will be tested 
in South African adults and toddlers, and three dose-levels will be tested in 
infants, with progression from one dose-level to the next and one age-group to 
the next based on assessment of safety and tolerability.  
The adult cohorts (Cohort A1 and A2) will consist of 15 participants (12 vaccine 
recipients and 3 placebo recipients) per dose level, who will receive three 
intramuscular injections four weeks apart. The two dose levels of vaccine to be 
tested will be 30 µg (consisting of 10 µg each of the P[4], P[6] and P[8] P2-VP8 
subunit proteins) and 90 µg (consisting of 30 µg each of the P[4], P[6] and P[8] 
P2-VP8 subunit proteins). Progression from low- to high-dose in adults, and to 
low-dose in toddlers, will be based on assessment of safety and tolerability 
during the week after the first injection in the adult low-dose cohort. The adult 
cohorts will receive a series of 3 study injections to allow optimal comparison 
with the results of the VAC 009 study, in which US adults received a series of 
3 study injections of the monovalent vaccine (containing P[8] only, up to 60 µg). 
Similarly, the toddler cohorts (Cohorts B1 and B2) will consist of 15 participants 
(12 vaccine recipients and 3 placebo recipients) per dose level. Participants 
will receive a single intramuscular injection. If found to be safe in the adult 
cohorts, the two dose levels of vaccine to be tested will be 30 µg (consisting of 
10 µg each of the P[4], P[6] and P[8] P2-VP8 subunit proteins) and 90 µg 
(consisting of 30 µg each of the P[4], P[6] and P[8] P2-VP8 subunit proteins). 
Initiation of the toddler low-dose cohort will be based on assessment of safety 
and tolerability of that dose during the week after the first injection in the adult 
low-dose cohort. Progression from low- to high-dose in the toddlers, and to 
low-dose in infants, will be based on assessment of safety and tolerability 
during the week after the first injection in the toddler low-dose cohort. Further, 
progression to the high-dose in toddlers will also be based on assessment of 
safety and tolerability during the week after first injection in the adult high-dose 
cohort. The toddler cohorts will receive only one study injection, as was the 
case for toddlers in VAC 013, as it is the safety data following this single dose 
that will be assessed to determine whether to proceed to the infant cohorts. 
Initial assessment in infants will consist of 16 participants (12 vaccine recipients 
and 4 placebo recipients) per dose level (Cohorts C1, C2 and C3). Participants 
will receive three intramuscular injections four weeks apart. If found to be safe 
in the toddler cohorts, the three dose levels of vaccine to be tested will be 15 µg 
(consisting of 5 µg each of the P[4], P[6] and P[8] P2-VP8 subunit proteins), 
30 µg (consisting of 10 µg each of the P[4], P[6] and P[8] P2-VP8 subunit 
proteins) and 90 µg (consisting of 30 µg each of the P[4], P[6] and P[8] P2-VP8 
subunit proteins). Initiation of the infant low-dose (15 µg) cohort will be based 
on assessment of safety and tolerability during the week after first injection in 
the toddler low-dose (30 µg) cohort. Progression from the 15 µg to the 30 µg 
dose cohort in the infants will be based on assessment of safety and tolerability 

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during the week after the first injection both in the infant 15 µg -dose cohort and 
the toddler 30 µg dose cohort. Progression from the 30 µg to the 90 µg dose 
cohort in the infants will be based on assessment of safety and tolerability 
during the week after the first injection in the infant 30 µg dose cohort. 
Should all three doses be tolerated in the initial assessment in infants, testing 
in infants will be expanded (Group D) to obtain both (a) greater safety 
experience and (b) more robust immunogenicity data for comparison of the 
three doses to support planning of subsequent, advanced phase testing. Group 
D will consist of 4 groups of 138 participants each: 15 µg, 30 µg and 90 µg of 
vaccine and placebo. 
All infants will receive 3 doses of Rotarix, at monthly intervals, starting a month 
after the final study injection (on study Day 84, at the time the post-vaccination 
blood sample is obtained), and fecal shedding of Rotarix will be assessed 
during the week after the first dose on a subset of infants. 
 
 
 

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STUDY SCHEMA (see Appendix I for full schedule of events) 
Group 
 
TV  
P2-VP8 
Dose 
N 
-7a 
to 
-1 
0 
7 
28 
56 
84 
168 
224 
A 
Adult 
A1 
30 µg 
12 
B(S,I) 
X 
B(S) 
X, B(I) 
X, B(I) 
B(I) 
 
F 
Placebo 
3 
B(S,I) 
P 
B(S) 
P, B(I) 
P, B(I) 
B(I) 
 
F 
A2 
90 µg 
12 
B(S,I) 
X 
B(S) 
X, B(I) 
X, B(I) 
B(I) 
 
F 
Placebo 
3 
B(S,I) 
P 
B(S) 
P, B(I) 
P, B(I) 
B(I) 
 
F 
A Total 
30 
B 
Toddler 
B1 
30 µg 
12 
B(S,I) 
X 
B(S) 
B(I) 
 
 
F 
 
Placebo 
3 
B(S,I) 
P 
B(S) 
B(I) 
 
 
F 
 
B2 
90 µg 
12 
B(S,I) 
X 
B(S) 
B(I) 
 
 
F 
 
Placebo 
3 
B(S,I) 
P 
B(S) 
B(I) 
 
 
F 
 
B Total 
30 
C 
Infant 
C1 
15 µg 
12 
B(S,I) 
X 
B(S) 
X 
X, B(I) 
B(I) 
 
F 
Placebo 
4 
B(S,I) 
P 
B(S) 
P 
P, B(I) 
B(I) 
 
F 
C2 
30 µg 
12 
B(S,I) 
X 
B(S) 
X 
X, B(I) 
B(I) 
 
F 
Placebo 
4 
B(S,I) 
P 
B(S) 
P 
P, B(I) 
B(I) 
 
F 
C3 
90 µg 
12 
B(S,I) 
X 
B(S) 
X 
X, B(I) 
B(I) 
 
F 
Placebo 
4 
B(S,I) 
P 
B(S) 
P 
P, B(I) 
B(I) 
 
F 
C Total 
48 
D 
Infant 
 
15 µg 
138 
B(S,I) 
X 
 
X 
X, B(I) 
B(I) 
 
F 
 
30 µg 
138 
B(S,I) 
X 
 
X 
X, B(I) 
B(I) 
 
F 
 
90 µg 
138 
B(S,I) 
X 
 
X 
X, B(I) 
B(I) 
 
F 
 
Placebo 
138 
B(S,I) 
P 
 
P 
 P, B(I) 
B(I) 
 
F 
D Total 
552 
 
 
All Cohorts Total 
660 
 
X = TV P2-VP8 vaccine 
P = placebo 
B = blood sample for (S) safety and/or (I) immunogenicity testing 
F = final study f/u visit/call 
aFor adult and toddler cohorts, -28 to -1 
For each age group, progression from one dose level to the next will require review of clinical and safety lab 
data through 7 days after the first dose at the lower dose level. Safety laboratory blood specimens will be drawn 
at baseline for all participants (screening) and 7 days after first study injection in Groups A, B and C. 
Blood samples for immunogenicity analysis will be obtained at baseline for all participants, Days 28, 56 and 
84 for Group A (adults), Day 28 for Group B (toddlers), and Days 56 and 84 for Groups C and D (infants). 
For each dose level, progression from adults in Group A to toddlers in Group B and then to infants in Group C 
will require review of clinical and safety laboratory data through 7 days after study injection in the older group. 
(see Figure 1) 
These determinations will be made by the Safety Review Committee (SRC). 

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STUDY 
PROCEDURES & 
VISIT SCHEDULE 
Participants at each dose level in Group A will receive three intramuscular 
injections in the deltoid of P2-VP8+ Al(OH)3 or placebo at Days 0, 28 and 56. 
Participants at each dose level in Group B will receive a single intramuscular 
injections in the anterolateral thigh of P2-VP8+ Al(OH)3 or Placebo at Day 0. 
Participants at each dose level in Groups C and D will receive three 
intramuscular injections in the anterolateral thigh of P2-VP8+ Al(OH)3 or 
placebo at Days 0, 28 and 56. EPI vaccines will be co-administered with the 
three study vaccines in all infants (at 6, 10 and 14 weeks of age), with the 
exception of rotavirus. All infants will receive 3 doses of Rotarix, at monthly 
intervals, starting at approximately 28 days after the final study injection (on 
study Day 84, at the time the post-vaccination blood sample is obtained).  
Safety will be assessed according to the following schedule: 
1. Blood draw obtained for eligibility screening and as baseline measure for 
all participants 
2. Blood draw for safety laboratory assessment 7 days after the first study 
injection in Groups A, B and C 
3. Clinic visit for reactogenicity and safety assessment (including monitoring 
safety laboratory tests) 7 days following each injection for all participants 
4. Final clinic visit for safety assessment 6 months following last injection for 
all cohorts 
In addition to the blood draws above, adults in Group A will have blood drawn 
for assessment of immune response assessment at baseline and one month 
following each vaccination (Days 28, 56 and 84), toddlers in Group B will have 
blood drawn for immune response assessment at baseline and one month 
following vaccination (Day 28), and infants in Groups C and D will have blood 
drawn for assessment of immune response at baseline and at one month after 
the second and third study injection (Days 56 and 84). When possible, stool 
samples will be obtained from infants experiencing diarrhea to test for rotavirus. 
Infants will receive Rotarix vaccination 28, 56 and 84 days following completion 
of the 3 study injections (Study Days 84, 112 and 140). Stool samples will be 
obtained to assess Rotarix shedding 5, 7, and 9 days following the first dose of 
Rotarix (Study Days 89, 91, and 93) on a subset of infants. 
SAFETY 
MONITORING 
For progression from lower to higher dose-level and to successively lower age-
groups, a Safety Review Committee (SRC) will evaluate clinical and laboratory 
safety data through Day 7 after the first injection for all participants before 
recommending whether to proceed. 
Throughout the study, the site PIs will provide continuous monitoring for safety, 
and the SRC will review safety data regularly. A DSMB will oversee the study, 
with scheduled periodic meetings, and, as needed, ad hoc meetings.  

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IMMUNOLOGY 
ASSESSMENTS 
 
Anti-P2-VP8 IgG & IgA by ELISA (assessed separately with antigens from 
P[4], [6] and [8] strains) 
 
Homologous and heterologous rotavirus neutralization using strains 
expressing the P[4], P[6] and P[8] serotypes 
 
Anti-rotavirus IgA (to whole viral lysate) by ELISA 
STATISTICAL 
CONSIDERATIONS 
It is assumed that almost all participants enrolled will provide data for safety 
analysis and at least 90% of enrolled participants will be evaluable for 
immunogenicity assessments.  
Safety 
For Cohorts A and B, 12 vaccine recipients per dose group and 24 vaccine 
recipients for the two dose groups combined will provide a greater than 90% 
chance of observing an AE that has a 17.5% and 9.2% rate of occurrence, 
respectively. The two infant cohorts combined (C and D), with 150 vaccine 
recipients per dose group and 450 vaccine recipients for the three dose groups 
combined, will provide a greater than 90% chance of observing an AE that has 
a 1.6% and 0.5% rate of occurrence, respectively. 
Conversely, if no SAEs are observed in 24 and 450 vaccine recipients, the 
study will be able to rule out SAEs occurring at a rate of approximately 11.7% 
and 0.7%, respectively based on the upper bounds of the one-sided 95% 
confidence interval.  
Immunogenicity (Infant Cohorts) 
Based on the results in South African infants who received monovalent P2-VP8 
vaccine or placebo (VAC-013), the strain specific seroresponse rates are 
expected to be ≥80% for at least one of the three P2-VP8 vaccine doses and 
<20% for the placebo group. For the two infant cohorts combined with 135 
evaluable vaccine recipients per dose level (assuming 10% loss), this study is 
designed to provide at least 74% and 95% power (Fisher’s exact test) to detect 
15 and 20 percentage points difference (e.g. 65% vs. 80% and 60% vs. 80%), 
respectively, in seroresponse rates between any two P2-VP8 dose groups. For 
comparisons between a P2-VP8 dose group and the combined placebo 
groups, this study is designed to provide ≥99% power to detect ≥30 percentage 
point difference (e.g. 50% vs. 20%). 
For the sample size calculations of the Geometric Mean Titer (GMT) endpoint 
based on the immunogenicity results in VAC 013, the LOG10 standard 
deviations were estimated to be < 0.6 (range: 0.24 – 0.56) for the P2-VP8 
doses and the placebo group. Using a conservative LOG10 standard deviation 
of 0.6, this study with 135 evaluable infants per group (assuming 10% loss) is 
designed to provide at least 98% power to detect as low as a 2.0-fold difference 
between any two P2-VP8 dose groups or between any P2-VP8 vaccine dose 
(15 µg, 30 µg or 90 µg dose) and the placebo group. 

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Figure 1: 
Study overview and timeline  
 
Note: Time line does not take into account delay of enrollment of infant groups until after peak of rotavirus season. 
 
 

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1. BACKGROUND AND INTRODUCTION 
1.1. Burden of Disease 
Diarrhea is the second-leading cause of death worldwide among children under the age of five, 
killing an estimated 1.3 million annually and hospitalizing millions more, mostly in developing 
countries [1]. The most common cause of severe infantile diarrhea is rotavirus; almost all children 
are infected with rotavirus before their third birthday regardless of geographic location or 
economic status. Global surveillance indicates that rotavirus is associated with up to 40 percent 
of severe, dehydrating diarrheal episodes in young children, resulting in hospitalization and more 
than 450,000 deaths each year. More than 85 percent of these deaths occur in the world’s poorest 
countries [1]. Children in low-income countries are at greater risk of severe rotavirus disease 
because they are more likely to be infected earlier in life, access to urgent medical care can be 
limited or unavailable, and there is a greater prevalence of malnutrition and other co-morbidities. 
In addition to causing loss of life, rotavirus places a burden on health-care systems and families. 
Vaccination offers the best hope for protecting all infants, regardless of economic status, against 
rotavirus disease [2]. 
1.2. Pathogen 
Rotavirus infection is primarily acquired through the fecal oral route. Universal infection in both 
developing and developed countries indicate that improved sanitation alone will not control 
rotavirus infection [3]. Treatment is symptomatic, with an emphasis on maintaining hydration 
either by oral administration of fluids or, in severe disease, via the intravenous route, with a 
favorable prognosis [4]. Unfortunately, provision of such rudimentary supportive care in many 
impoverished areas of the world is limited, contributing to the high mortality rate observed.  
 
The vast majority of human disease (~90%) is caused by group A rotavirus. Two surface proteins, 
VP4 and VP7, are able to induce neutralizing antibody during infection, and both are very 
divergent across different rotavirus strains: the VP7 glycoprotein represents the G serotype, while 
the VP4 protein represents the P serotype. The seroepidemiology of rotavirus strains causing 
human disease has been extensively studied [5]. The findings have potentially important 
implications for vaccine development. While geographical differences have been noted in the 
absolute frequency of the various P serotypes, the P[4] and P[8] serotypes predominate (Figure 
2, below), accounting for between 90-99% of strains isolated from Asia, North America, Europe, 
South America and Australia/Oceania. However, in Africa they represent approximately 70% of 
clinical isolates with P[6] accounting for nearly all of the remaining strains. There is no clear 
evidence that protection against rotavirus infection is exclusively homotypic, but if that were the 
case, a bivalent vaccine that contains P[4] and P[8] would provide 90% coverage in all areas 
except Africa. For Africa, a P[6] component would be required for a similar rate of coverage. 
However, more recent data indicate the prevalence of P[6] currently may be about half of previous 
estimates [6, 7]. In contrast, the diversity among G serotypes is greater, with 4-5 serotypes 
representing ~90% of clinical isolates worldwide. 
 
Like the influenza hemagglutinin, the rotavirus VP4 must be cleaved by proteases before the virus 
can be activated [8]. Such cleavage results in the generation of two proteins, VP5 and VP8, which 
remain attached to the virion. Most of the antigenic epitopes in VP4 that induce neutralizing activity 
have been mapped to VP8, including those responsible for the serotype differences among 
different strains. 

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Figure 2: Continental Variation in the Distribution of Human Group A Rotavirus P Types 
Ascertained by Analysis of Strains Collected between 1982-2003  
[Santos 2005, Rev Med Virol (5)] 
 
1.3. Non-replicating Rotavirus Vaccine Approach Using VP8 Subunit 
The VP8 subunit of VP4 protein has been expressed in various systems and demonstrated to 
induce rotavirus-specific neutralizing antibodies and/or protection in various animal models [9-
18]. Monoclonal antibodies to VP8 administered orally are able to confer passive protection in 
mice [19] and natural rotavirus infection, as well as vaccination with live vaccines, induces 
protective anti-VP8 neutralizing antibodies [16]. Mapping of neutralizing monoclonal antibodies 
against VP8 reveals that they recognize linear epitopes in the region between amino acids 64 and 
223 of VP8 [13, 20]. All epitopes that induce neutralizing antibody responses to VP8 serotypes 
P[4], P[6] and P[8] have been mapped to that region [14, 21]), which was selected for inclusion in 
the proposed P2-VP8 subunit rotavirus vaccine. Recombinant VP8 protein vaccine candidates 
have been produced in several systems, including E. coli [13, 18, 22, 23]), lactobacillus [15], yeast 
[24] and plants [25]. A norovirus particle recombinant fusion protein utilizing a truncated VP8 
sequence has also been developed and shown to confer protection against rotavirus in a murine 
challenge model [18]. VP8 produced in E. coli induces high-titered homotypic neutralizing 
antibodies in mice, cattle, rabbits, chickens and guinea pigs [12, 13, 23]), as well heterotypic 
neutralizing antibodies [23]. Passively transmitted antibodies derived from VP8 immunization 
protected offspring of vaccinated dams [12, 25, 26]. The magnitude and breadth of the heterotypic 
neutralizing antibodies induced has been variable and strain dependent. To improve the vaccine 
potential of the recombinant VP8 subunit protein, we synthesized a fusion protein comprised of 
the P2 universal CD4+ T cell epitope (aa 830-844 of tetanus toxin) and a truncated VP8 moiety, 
which contains all known neutralizing epitopes. The P2 epitope has been employed in the 
development of various viral and bacterial experimental vaccines including malaria, H. influenza 
type b and others [27, 28]. 

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The P2-VP8 vaccine to be tested in this study was developed by Dr. Taka Hoshino at NIAID [29]. 
The monovalent (P[8]) vaccine previously tested consists of bacteria-expressed VP8 subunit from 
the Wa strain of human rotaviruses (G1[P8]). A DNA segment encoding the sequence of P2 
epitope from tetanus toxin was fused to the VP8 sequence, resulting in this chimeric protein 
vaccine (P2-VP8). Dr. Hoshino’s laboratory has demonstrated in preclinical testing that the 
immunogenicity of these VP8 proteins could be significantly enhanced when fused with the P2 
epitope of tetanus toxin, which exerts a strong T cell helper function. Further, immunization of 
neonatal piglets with a P2-VP8-P[8] chimeric protein conferred significant protection against 
experimental rotavirus gastroenteritis [30]. Based on results of the initial first-in-human testing of 
the monovalent (P[8]) vaccine (see section 1.7), a trivalent vaccine that includes antigens from 
P[4], P[6] and P[8] strains (DS1, 1076 and Wa, respectively) has been developed to broaden 
responses for these 3 P-types, which together are responsible for the vast majority of global 
disease burden. The trivalent vaccine will be assessed in this study. 
1.4. Potential Safety Risks 
Based on pre-clinical studies, the most commonly expected adverse events are reactions at the 
injection site, including redness, swelling and pain; these findings are consistent with reactions 
seen with licensed vaccines. In addition, as with any vaccine, allergic reactions, including the 
extremely rare occurrence of life-threatening allergic reactions, cannot be excluded. 
The vaccine is to be adsorbed on aluminum hydroxide [Al(OH)3]. There is a wealth of experience 
demonstrating the safety of aluminum salts as an adjuvant, with potential local reactions of 
erythema, subcutaneous nodules and contact hypersensitivity [31]. 
Safety data from first-in-human phase 1 testing of the monovalent (P[8]) P2-VP8 vaccine in adults, 
toddlers and infants have been reassuring and consistent with anticipated predominantly mild 
local reactions. (see Section 1.7 for details). 
1.5. Summary of Nonclinical Studies with Monovalent (P[8]) P2-VP8 
The safety and immunogenicity of the P2-VP8 subunit vaccine was evaluated in three animal 
models (guinea pigs, Gnotobiotic piglets, and New Zealand White rabbits). An overview of these 
preclinical studies supporting the development of the P2-VP8 subunit vaccine is provided below 
in Table 1.  
 
 

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Table 1: 
Preclinical Studies Supporting P2-VP8 Subunit Vaccine Development 
Study Title 
Study Site 
Study Type & 
Compliance 
Animal 
Model 
Adjuvant 
Location of 
Summary 
Evaluation of the 
Antibody Response in 
Guinea Pigs 
Administered VP8 or 
P2-VP8 
National 
Institutes of 
Health, 
Bethesda, MD 
Discovery 
Non-GLP 
Guinea Pig 
None 
Section 
1.5.1.1 
Evaluation of the 
Antibody Response in 
Guinea Pigs 
Administered P2-VP8 
with or without 
Aluminum Phosphate 
Adjuvant 
National 
Institutes of 
Health, 
Bethesda, MD 
Discovery  
Non-GLP 
Guinea Pig 
Aluminum 
Phosphate 
Section 
1.5.1.2 
Evaluation of the 
Rotavirus Neutralizing 
Antibody Response in 
Guinea Pigs 
Covance, 
Denver, PA 
Immunogenicity 
Non-GLP 
Guinea Pig 
Aluminum 
Phosphate 
Section 
1.5.1.3 
Protection from 
Rotavirus Infection in 
Neonatal Piglets 
Virginia Tech, 
Blacksburg, VA 
Immunogenicity 
Challenge 
Non-GLP 
Gnotobiotic 
Piglets 
Aluminum 
Phosphate 
Section 
1.5.1.4 
Repeat Dose 
Intramuscular Safety 
and Immunogenicity 
Study of P2-VP8 
subunit vaccine (with 
and without Aluminum 
Hydroxide Adjuvant) in 
Rabbits 
AVANZA 
Laboratories, 
Gaithersburg, 
MD 
Immunogenicity 
Toxicology 
GLP 
New 
Zealand 
White 
Rabbits 
Aluminum 
Hydroxide 
Section 
1.5.2 
1.5.1. Non-GLP Nonclinical Pharmacology/Proof of Concept Studies 
1.5.1.1. 
Evaluation of the Antibody Response in Guinea Pigs Administered Monovalent 
(P[8]) VP8 or P2-VP8 
To improve the vaccine potential of the recombinant VP8 subunit protein, we synthesized a fusion 
protein comprised of the P2 universal CD4+ T-cell epitope (aa 830-844 of tetanus toxin) and a 
truncated VP8 moiety which contains all known VP8 neutralizing epitopes. Increased 
immunogenicity was assessed in guinea pigs inoculated with either VP8 alone or fused to the 
P2 epitope [Dr. T. Hoshino, National Institute of Allergy and Infectious Diseases (NIAID), 
National Institutes of Health (NIH), personal communication]. The sera were tested in a 60% 
plaque neutralization assay against the homologous strain, which showed that inclusion of a 
P2 CD4+ T-cell epitope sequence with VP8 enhances the immunogenicity of VP8 by engendering 
a more rapid rise in neutralizing antibody titers. 

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1.5.1.2. 
Evaluation of the Antibody Response in Guinea Pigs Administered Monovalent 
(P[8]) P2-VP8 with or without Aluminum Phosphate Adjuvant 
Adsorption of monovalent (P[8]) P2-VP8 subunit onto aluminum phosphate increases overall titer 
and promotes an earlier neutralizing antibody titer compared to monovalent (P[8]) P2-VP8 subunit 
vaccine alone in guinea pigs. High neutralizing antibodies titers were observed for up to eight 
months in animals immunized with 10 or 20 µg monovalent (P[8]) P2-VP8 subunit vaccine 
adsorbed to aluminum phosphate. 
 
Aluminum hydroxide [Al(OH)3] was selected as the adjuvant for further preclinical and clinical 
development based on its success in other clinical trials conducted by PVS with other antigens, 
citations in literature that indicate aluminum hydroxide [Al(OH)3] may have greater adjuvant 
activity than aluminum phosphate (27), and availability of clinical-grade material. Similarly, as 
noted in Section 1.5.2.3, rabbits immunized with aluminum hydroxide [Al(OH)3] adjuvanted 
vaccine in the GLP toxicology study displayed higher anti-P2-VP8 IgG binding antibody levels 
than those immunized with non-adjuvanted vaccine. 
1.5.1.3. 
Evaluation of the Rotavirus Neutralizing Antibody Response in Guinea Pigs 
A non-GLP study was conducted in Hartley Guinea Pigs (Elm Hill) to evaluate the magnitude and 
breadth of the immune response following vaccination with research grade monovalent (P[8]) 
P2-VP8 subunit vaccine, adsorbed to aluminum adjuvant, at Covance (Denver, PA).  
 
Five guinea pigs were immunized with 25 µg monovalent (P[8]) P2-VP8 subunit vaccine adsorbed 
to aluminum phosphate adjuvant by intramuscular (IM) injection, the planned route of human 
exposure, on Days 0, 30, and 60. Three doses were used to mimic the EPI schedule. Serum was 
collected for immunological analyses at baseline (Day -7), on Days 0, 30, and 60 (prior to dosing) 
and at termination on Day 74. Animals were observed daily for viability and general health. 
 
With the exception of a single animal that was found with a broken leg that was subsequently 
euthanized, no clinical signs were noted throughout the study. 
 
Rotavirus neutralizing antibodies, which can be directed against either the G (VP7) or P (VP4) 
antigen expressed by rotavirus, were quantified using a neutralization assay run in a blinded 
manner by an independent testing laboratory (M. McNeal, Cincinnati Children’s Hospital Medical 
Center, Cincinnati, OH). To analyze the breadth of the immune response, sera were run against 
six human strains of rotavirus expressing various combinations of G (1/2/3/4 and 9) and P (4/6/8) 
genotypes, which have been found to predominate among human field isolates. The results are 
shown below in Table 2 and Table 3. Immunization with the monovalent (P[8]) P2-VP8 subunit 
vaccine elicited a vigorous antibody response to the Wa, P, and WI61 strains, all of which 
displayed the homologous P[8] genotype (Table 2). However, the post-immunization titers to the 
VA70 strain, which also displays the P[8] genotype were substantially lower. Responses were 
meager to the ST3 strain, which has a P[6] genotype; there were intermediate responses to the 
DS1 strain, which has a P[4] genotype. Overall, immunization resulted in all animals mounting a 
4-fold or greater rise in titer over baseline values, regardless of the P genotype (Table 3). 

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Table 2: 
Geometric Mean Neutralizing Titers to Human Rotavirus Strains: Before and 
After Immunization 
WA 
P  
WI61  
ST3 
VA70 
DS-1  
(G1, P[8]) 
(G3, P[8]) 
(G9, P[8]) 
(G4, P[6]) 
(G4, P[8]) 
(G2, P[4]) 
Pre 
Post 
Pre 
Post 
Pre 
Post 
Pre 
Post 
Pre 
Post 
Pre 
Post 
5 
4,147 
5 
1,459 
5 
1,986 
5 
50 
5 
233 
32 
732 
Table 3: 
Seroconversion to Rotavirus Strains Following Immunization * 
WA 
P  
WI61  
ST3 
VA70 
DS-1  
(G1, P[8]) 
(G3, P[8]) 
(G9, P[8]) 
(G4, P[6]) 
(G4, P[8]) 
(G2, P[4]) 
100 
100 
100 
100 
100 
100 
*Values represent the percentage of animals that showed a ≥4-fold 
rise in titer after immunization. 
These data demonstrate that monovalent (P[8]) P2-VP8 subunit vaccine elicits a good heterotypic 
neutralizing antibody response to strains expressing the P4 but not the P6 genotype. Furthermore, 
the magnitude of the immune response could vary significantly for strains expressing the 
homologous P[8] types, as evidenced by a substantially muted response to strain VA70.  
1.5.1.4. 
Protection from Rotavirus Disease in Neonatal Piglets 
The protective capacity of the P2-VP8 vaccine against a live oral rotavirus challenge was 
evaluated in a gnotobiotic piglet model. This model is widely viewed as the most rigorous and 
relevant animal model for evaluation of rotavirus vaccine efficacy. Three newborn gnotobiotic 
piglets were immunized via the intramuscular route with three doses of research-grade 
monovalent (P[8]) P2-VP8 subunit vaccine (100 µg per dose) adjuvanted with 600 µg aluminum 
phosphate, the first given at birth and the subsequent doses at 10-day intervals. An additional 
nine piglets served as unvaccinated controls. Seven days after the last vaccination, the animals 
were challenged with the Wa strain of rotavirus (homologous P[8] serotype) at a dose of 50 TCID50 
which consistently induces diarrhea in this model (28).  
Titers of P[8] VP4-specific antibodies increased following each vaccination with monovalent (P[8]) 
P2-VP8 subunit vaccine plus aluminum phosphate, with titers first detectable at approximately 
two weeks following the first vaccination. Titers further increased at 10 days post-challenge, 
indicating a boosting effect from natural exposure to a homotypic challenge. 
Titers of rotavirus-neutralizing antibodies increased significantly by Post-immunization Day (PID) 
27, one week following the final vaccination. Titers increased substantially within one week of 
homotypic challenge in monovalent (P[8]) P2-VP8 subunit vaccine treated animals compared to 
concurrent control animals (Figure 3). 

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Figure 3: Rotavirus Geometric Mean Neutralizing Antibody Titers Following 
Vaccination and Challenge  
 
*p < 0.001 
Shedding of the Wa rotavirus was measured after challenge in control unimmunized gnotobiotic 
piglets and piglets receiving three inoculations of monovalent (P[8]) P2-VP8 subunit vaccine. 
Vaccination with monovalent (P[8]) P2-VP8 subunit vaccine induced a drop of more than 10-fold 
in the titer of virus shed in the stools; however, there was no significant difference between the 
P2-VP8 vaccine and control pig groups. 
Monovalent (P[8]) P2-VP8 subunit vaccine significantly reduced post-challenge diarrhea, as 
estimated by the use of a cumulative, 4-point scoring system (0 points = normal stool consistency 
[no diarrhea]; 1 = pasty [mild diarrhea]; 2 = semi liquid [moderate diarrhea]; and 3 = liquid [severe 
diarrhea]). Days to virus shedding and diarrhea onset in addition to the duration of virus shedding 
and diarrhea in control and immunized piglets is shown in Figure 4. The delayed onset and 
shortening of diarrhea was statistically significant in vaccinated animals compared to concurrent 
controls. Immunization did not significantly (p > 0.05) alter the onset of virus shedding nor the 
mean number of days virus was shed post challenge. 
PID 0
PID 6
PID 13
PID 20
PID 27
PCD 2
PCD 7
Control
2
2
2
2
2
2
240
P2-VP8*
2
2
2
9.9
240
240
5054
1
10
100
1,000
10,000
*
*
*  

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Figure 4: Days to Onset of Diarrhea and Viral Shedding by Treatment 
 
Immunization with the monovalent (P[8]) P2-VP8 subunit vaccine conferred significant protection 
against rotavirus diarrhea, as gauged by both time of onset and severity after an oral challenge. 
While the monovalent (P[8]) P2-VP8 subunit vaccine did not completely prevent disease, it was 
superior to a heat-inactivated rotavirus vaccine (IRV) previously tested in the same model (28). 
This model has also been used to test live oral rotavirus vaccines. These vaccines, though 
protective, induce diarrhea when administered to gnotobiotic piglets. Numerous field trials 
evaluating live oral rotavirus vaccines have consistently demonstrated that they do not prevent 
infection or substantially reduce mild illness; rather, their protective efficacy is primarily manifested 
against severe cases of diarrhea. In the piglet study, the monovalent (P[8]) P2-VP8 subunit 
vaccine significantly delayed the onset of diarrhea, significantly reduced the duration of diarrhea, 
and significantly reduced the cumulative diarrhea score. These effects may potentially lead to 
protection against severe diarrhea in infants, the goal of rotavirus immunization. 
1.5.2. GLP Immunogenicity and Toxicology Study in NZW Rabbits 
Monovalent ([P8]) P2-VP8 subunit vaccine plus aluminum hydroxide [Al(OH)3] was well-tolerated 
and immunogenic at 30 or 60 µg. No clinical toxicity was observed during the in-life phase. 
Changes in clinical pathology were transient, expected with immune stimulation associated with 
vaccination, and non-adverse. Microscopic changes observed one day following the final 
administration of monovalent (P[8]) P2-VP8 subunit vaccine were limited to the injection site and 
were resolved or resolving following a four-week recovery period. 
1.5.2.1. 
Study Description 
Saline control or monovalent (P[8]) P2-VP8 subunit vaccine (30 or 60 µg, with or without 0.56 mg 
aluminum hydroxide [Al(OH)3]) was administered in a 0.5 mL volume intramuscularly at 14 day 
intervals on Study Days 1, 15, 29, and 43, and a unique site (left and right quadriceps and dorsal 
lumbar muscles) was used for each injection. 

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Animals were observed for dose-site reactogenicity, clinical observations (including 
ophthalmology), body weight changes, food consumption, body temperatures, and clinical 
pathology (hematology, coagulation parameters, and clinical chemistry). Animals were sacrificed 
two days or 6 weeks following the final dose of vaccine. A full set of tissues were examined 
microscopically at two days (Study Day 45) following the final dose of vaccine. Injection sites and 
underlying muscle tissue were examined six weeks following the final dose of vaccine. 
1.5.2.2. 
Toxicology Results 
Vaccination with monovalent (P[8]) P2-VP8 subunit vaccine with or without aluminum hydroxide 
[Al(OH)3] adjuvant was well-tolerated in NZW rabbits at doses of 30 and 60 µg.  
There were no treatment-related, clinically significant observations on study, and no changes or 
pain assessed by observing reaction to palpation were observed at any dose sites. Following 
immunization, particularly subsequent to the first dose on Study Day 1, slight decreases in body 
weights were observed, but animals generally gained weight on a week to week basis. There 
were no treatment-related significant differences in food consumption, post-dose body 
temperatures, hematology, ophthalmology, or organ weights.  
Slightly, but statistically significant, lower creatinine levels were observed on Study Day 3 in females 
treated with 60 µg monovalent (P[8]) P2-VP8 subunit vaccine and aluminum hydroxide [Al(OH)3] 
adjuvant and on Study Day 8 in females treated with 30 µg monovalent (P[8]) P2-VP8 subunit vaccine 
and aluminum hydroxide [Al(OH)3] or 60 µg monovalent (P[8]) P2-VP8 subunit vaccine with or without 
aluminum hydroxide [Al(OH)3]; however, these changes were not considered toxicologically 
significant. At Study Day 3 and 8 slight, but statistically significant, increases in fibrinogen were 
observed in animals treated with 60 µg monovalent (P[8]) P2-VP8 subunit vaccine with or without 
aluminum hydroxide [Al(OH)3] compared to concurrent controls, which is consistent with inflammatory 
processes associated with vaccination. At Day 45, all treated animals had significantly higher levels 
of fibrinogen in an apparent dose-related manner, with those animals treated with aluminum hydroxide 
[Al(OH)3] having slightly higher fibrinogen levels at each dose than those animals treated with 
monovalent (P[8]) P2-VP8 subunit vaccine alone.  
Males and females treated with 60 µg monovalent (P[8]) P2-VP8 subunit vaccine with or without 
aluminum hydroxide [Al(OH)3] had statistically significantly higher prothrombin time (PT) at Day 
3. For groups immunized with vaccine alone, significantly higher PT was observed on Day 8, Day 
45 (females only), and Day 85 (females only) compared to concurrent controls. No mean or 
individual animal values exceeded the upper limit of the historical range for New Zealand White 
rabbits at AVANZA Laboratories and animals assigned to the vaccinated groups, particularly 
Groups 4 & 5, had substantially higher baseline values for PT than control animals, which 
contributed to the post-vaccination differences from control observed. 
When PT values were compared to the baseline values for each test group, only females treated 
with 60 µg P2-VP8 alone had significant increases over baseline on Day 8 (p = 0.0003). Slight 
elevations in PT over baseline were observed in animals treated with 60 µg P2-VP8 with or without 
aluminum hydroxide [Al(OH)3] on Day 3, although they were not statistically significant. Given the 
modest increases in PT values seen and the temporal nature of any increases over baseline, 
these changes were considered to be non-adverse and clinically irrelevant. 

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With the exception of a single female in Group 4 (60 µg monovalent (P[8]) P2-VP8 subunit 
vaccine) with subcutaneous red discoloration at the Day 43 injection site on Day 45, there were 
no gross observations at either necropsy.  
Test article-related microscopic findings were limited to the injection sites, and were considered 
non-adverse based on minimal to mild severity and apparent reversibility (28). Test article effects 
associated with monovalent (P[8]) P2-VP8 subunit vaccine plus aluminum hydroxide [Al(OH)3] 
were minimal to mild accumulations of macrophages with abundant slightly basophilic, finely 
granular cytoplasm (termed “foamy macrophages”) in the subcutaneous tissue and/or skeletal 
muscle interstitium. These macrophages were observed at each injection site (sites 1-4) in one 
to several animals and are likely related to co-administration of the vaccine with aluminum 
hydroxide [Al(OH)3].  
Minimal to mild inflammation consisting of a mixture of heterophils and mononuclear inflammatory 
cells occurred in the dermis or subcutis of animals in all groups, including controls, suggesting 
these findings are related to mechanical injury from the injection procedure. A few animals in each 
vaccine-treated group had a similar finding in the underlying skeletal muscle.  
The incidence of inflammation was low in all groups at injection sites 1 and 2, indicating a recovery 
of acute changes related to vaccination. At injection sites 3 and 4, the incidence of inflammation 
was slightly higher for groups receiving monovalent (P[8]) P2-VP8 subunit vaccine with or without 
aluminum hydroxide [Al(OH)3]. At injection site 4, the incidence of inflammation was increased in 
a dose-related manner, with the highest incidence found in Groups 4 and 5, which received 60 
µg monovalent (P[8]) P2-VP8 subunit vaccine with or without aluminum hydroxide [Al(OH)3]. All 
other findings had similar incidence in control and treated groups, and/or were common 
spontaneous findings in laboratory rabbits of this age. 
Six weeks after the final dose (Day 85), test article-related residual foamy macrophages were 
present in the dermis and subcutaneous tissue at injection site 3 of one male treated with 60 µg 
monovalent (P[8]) P2-VP8 subunit vaccine plus aluminum hydroxide [Al(OH)3]. Minimal mixed cell 
inflammation and minimal to mild hemorrhage also persisted in one or more animals at each 
injection site. The incidence of these findings was lower than observed at the terminal sacrifice, 
indicating resolution was in progress. A delayed-onset change was minimal mineralization in the 
subcutaneous tissue at injection site 3 in one male vaccinated with 60 µg monovalent (P[8]) 
P2-VP8 subunit vaccine plus aluminum hydroxide [Al(OH)3]. This finding was minimal in extent 
and considered non-adverse.  
1.5.2.3. 
Immunogenicity Results 
The immunogenicity of the monovalent (P[8]) P2-VP8 subunit rotavirus vaccine was 
demonstrated in NZW rabbits in the GLP toxicology study. As described above, animals were 
administered four doses of 30 µg or 60 µg monovalent (P[8]) P2-VP8 subunit vaccine alone or 
adsorbed to aluminum hydroxide [Al(OH)3] adjuvant on Study Days 1, 15, 29, and 43. Serum 
samples for immunological analyses were collected at baseline and two days or six weeks 
following the final vaccination on Study Days 45 and 85, respectively. 
Immunization with 30 or 60 µg of monovalent (P[8]) P2-VP8 subunit vaccine formulations induced 
a 4-fold or greater rise in serum IgG anti-P2-VP8 antibody levels in all treated animals on Study 
Day 45 or Study Day 85. No control animals immunized with saline manifested a similar rise in 

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antibody at any time point, with all samples tested being at the lower limit of quantitation. The 
addition of aluminum hydroxide [Al(OH)3] as an adjuvant potentiated the immune response, as 
determined by comparing GMTs in 3 of 4 analyses performed (Table 4).  
Table 4: 
Geometric Mean Titers by Treatment Following Four Doses of Monovalent 
(P[8]) P2-VP8 Subunit Vaccine with or without Aluminum Hydroxide [Al(OH)3]  
Group 
Treatment 
Geometric Mean Titer (units/mL) 
Day 45 
Day 85 
1 
Vehicle Control  
(0.9% USP Saline) 
<20.41 ± 0 
<20.41 ± 0 
2 
30 µg P2-VP8 subunit vaccine 19,042 ± 13,567 11,404 ± 2,787 
3 
30 µg P2-VP8 subunit vaccine 
+ 0.56 mg aluminum 
hydroxide adjuvant 
22,342 ± 9,694 
21,953 ± 6,381 
4 
60 µg P2-VP8 subunit vaccine 13,096 ± 26,601 6,223 
± 11,697 
5 
60 µg P2-VP8 subunit vaccine 
+ 0.56 mg aluminum 
hydroxide adjuvant 
25,881 ± 18,718 20,215 ± 11,807 
As shown in Figure 5, the presence of aluminum hydroxide [Al(OH)3] did not result in a statistically 
significant rise in GMT on Study Day 45 (two days after the fourth dose of vaccine was given) 
when the 30 µg dose was compared (p = 0.05), but did so for the 60 µg dose (p = 0.041). At Study 
Day 85, the addition of the aluminum hydroxide [Al(OH)3] adjuvant resulted in significantly higher 
GMT both for the 30 µg treated group (p = 0.001) and 60 µg treated group (p = 0.024). 
Figure 5: Geometric Mean Titers by Interval and Treatment 
 
 
p= 0.001 
p = 0.041 

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Rotavirus neutralizing antibody titers were measured against the Wa strain at baseline and on 
Study Day 85, six weeks following the final vaccination. On Study Day 85, neutralizing antibody 
titers were detectable in all animals vaccinated with 60 µg monovalent (P[8]) P2-VP8 subunit 
vaccine with or without aluminum hydroxide [Al(OH)3]. Animals receiving aluminum hydroxide 
[Al(OH)3] adjuvant had higher neutralizing antibody titers than those not receiving adjuvanted 
vaccine. 
1.6. Summary of Nonclinical Studies with Trivalent (P[4], P[6], P[8]) P2-VP8 Vaccine 
The safety and immunogenicity of the trivalent rotavirus P2-VP8 subunit vaccine was evaluated 
in guinea pigs and rabbits. An overview of these preclinical studies supporting the development 
of this vaccine is provided below in Table 5. Information about these studies is presented in the 
subsequent subsections. 
Table 5: 
Preclinical Studies Supporting Trivalent Rotavirus P2-VP8 Subunit Vaccine 
Development 
Study Title 
Study Site 
Study Type & 
Compliance 
Animal Model 
Adjuvant 
Location of 
Summary 
Evaluation of the 
Antibody Response 
in Guinea Pigs 
Administered trivalent 
or monovalent 
vaccines 
Spring Valley 
Laboratories, 
Woodbine, MD 
Immunogenicity 
Non-GLP 
Guinea Pig 
Aluminum 
hydroxide 
Section 
1.6.1 
Repeat Dose 
Intramuscular Safety 
and Immunogenicity 
Study of P2-VP8 
Subunit (with and 
without Aluminum 
Hydroxide Adjuvant) 
in Rabbits 
MPI Research,  
Mattawan, MI 
Immunogenicity 
Toxicology 
GLP 
New Zealand 
White Rabbits 
Aluminum 
Hydroxide 
Section 
1.6.2 
 
1.6.1. Evaluation of the Antibody Response in Guinea Pigs Administered Either Trivalent 
or Monovalent P2-VP8 Vaccine 
Trivalent vaccine was produced by combining the three individual monovalent P2-VP8 vaccines, 
adsorbed onto aluminum hydroxide, and used to immunize guinea pigs. Animals received three 
doses of vaccine at bi-weekly intervals with serum samples taken at baseline and two weeks after 
the third dose for determination of IgG binding and rotavirus neutralizing antibodies. Immunization 
with the individual P2-VP8 proteins evoked not only a robust binding antibody response to the 
homologous antigen but also to the other two vaccine antigens (Table 6). This is not surprising 
given the level of homology (~65-85%) at the DNA level. In all cases, the GMT after immunization 
with the trivalent vaccine was comparable to or slightly higher than that seen with the individual 
components, indicating no blunting of the immune response due to antigen load. However, the 
overall response engendered by the monovalent P2-VP8-P[6] vaccine was lowest both to the 
homologous P[6] and heterologous P[4] and P[8]antigens. A similar trend was noted for the 
neutralizing antibody response (Table 7) where post-immunization titers were lowest to the P[6] 
vaccine component. While the GMTs for the P[8] and P[6] strains were comparable in groups of 

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animals immunized with either the trivalent or monovalent vaccines, that for P[4] strain was higher 
in animals immunized with the monovalent vaccine (338 versus 131, respectively). This could be 
partially attributed to the fact that one animal in the monovalent group mounted a very high 
response (1280) given the relatively small number of animals per group (N=6). 
Table 6:  Serum IgG Binding Antibody Responses in Guinea Pigs following vaccination 
with either Monovalent or Trivalent P-VP8 Vaccines. 
Vaccine 
Group 
Post-immunization Geometric Mean Serum IgG Titer (Range) to Vaccine 
Antigensa  
P2-VP8 [P4] 
P2-VP8 [P6] 
P2-VP8 [P8] 
P2-VP8 [P8] 
50745 (7190-156777) 
13747 (4154-33805) 
81534 (7847-287569) 
P2-VP8 [P6] 
31575 (2588-81094) 
71363 (10510-175924) 
12467 (865-32614) 
P2-VP8 [P4] 
198808 (53675-326972) 
22494 (13429-44099) 
70988 (28188-112081) 
Trivalent  
P2-VP8 [P8]  
+ [P6] + [P4] 
202245 (152332-281906) 
72945 (43079-113257) 
131298 (91187-162513) 
a: Animals (groups of 6) were immunized on days 1, 14 and 28 with 30 µg of each antigen. Pre-immunization titers 
were negative at the lowest dilution tested with a titer <78. 
Table 7.  Neutralizing Antibody titers to Strains of Rotavirus Expressing Various P 
Types 
Vaccine 
Group 
Geometric Mean Neutralizing Antibody Titers (Range)  
Wa (G1P8) 
DS1 (G2P4) 
1076 (G2P6) 
Pre 
Day 43 
Pre 
Day 43 
Pre 
Day 43 
P2-VP8 [P8] 
5 
(5-5) 
453 
(26-1352) 
6 
(5-18) 
41 
(15-125) 
5 
(5-5) 
6 
(5-5) 
P2-VP8 [P4] 
5 
(5-5) 
53 
(27-132) 
10 
(5-37) 
338 
(133-1280) 
5 
(5-5) 
9 
(5-30) 
P2-VP8 [P6] 
5 
(5-5) 
7 
(5-18) 
8 
(5-22) 
12 
(5-35) 
5 
(5-5) 
40 
(5-198) 
Trivalent  
P2-VP8 [P8]  
+ [P6] + [P4] 
5 
(5-5) 
380 
(48-1156) 
18 
(5-78) 
131 
(104-165) 
5 
(5-5) 
37 
(5-118) 
Pre-immunization titers were <10 for all samples and assigned a titer of 5. 
1.6.2. GLP Immunogenicity and Toxicology Study of the Trivalent P2-VP8 Subunit 
Vaccine in NZW Rabbits 
Trivalent P2-VP8 subunit vaccine plus aluminum hydroxide [Al(OH)3] was well-tolerated and 
immunogenic at 180 µg. No clinical toxicity was observed during the in-life phase. Changes in 
clinical pathology were transient, expected with immune stimulation associated with vaccination, 
and non-adverse. Microscopic changes observed two days following the final administration of 
trivalent P2-VP8 subunit vaccine were limited to the injection site and were resolved or resolving 
following a four-week recovery period. 

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1.6.2.1. 
Study Description 
Saline control or trivalent P2-VP8 subunit vaccine (180 µg, comprising of 60 µg of the P[8], P[6], 
and P[4] serotype subunits) with 0.56 mg aluminum hydroxide [Al(OH)3]) was administered in a 
0.5 mL volume intramuscularly at 14 day intervals on Study Days 1, 15, 29, and 43, and a unique 
site (the cranial and caudal aspects of the left and right erector spinae muscle) was used for each 
injection. 
Animals were observed for dose-site reactogenicity, clinical observations (including 
ophthalmology), body weight changes, food consumption, body temperatures, and clinical 
pathology (hematology, coagulation parameters, and clinical chemistry). Animals were sacrificed 
two days or 4 weeks following the final dose of vaccine. A full set of tissues were examined 
microscopically at two days (Study Day 45) following the final dose of vaccine. Injection sites and 
underlying muscle tissue were examined four weeks following the final dose of vaccine. 
1.6.2.2. 
Toxicology Results 
Vaccination with trivalent P2-VP8 subunit vaccine with aluminum hydroxide [Al(OH)3] adjuvant 
was well-tolerated in NZW rabbits at a dose of 180 µg.  
There were no test-article related clinical observations, including ophthalmology, noted on the study. 
One animal assigned to the vaccine group was noted to have a thin body condition and in 
appetence, primarily in the recovery phase of the study. Because this finding was consistent with 
lower food consumption prior to and throughout the study and the animal was able to maintain or 
gain body weight during the study, this finding was not considered to be related to treatment. Dermal 
site scoring was performed at each dose site for three days following each vaccination. No positive 
dermal scores were noted for any animals. There were no test article-related effects on body 
temperatures. There were slight increases in body temperatures at 6 and 24 hours postdose relative 
to predose values for each collection interval; however, these were noted for both control and 
treated groups. There were no adverse effects on body weights throughout the study, and animals 
gained weight. Lower mean food consumption was noted in the vaccine groups; however, these 
reduced values were due to a few treated males with consistently low food consumption values Due 
to the low magnitude and lack of correlating clinical observations or decreases group mean body 
weights, these decreases in food consumption were not considered adverse. 
There were no test article-related effects among hematology or clinical chemistry parameters at 
any interval. All mean and individual values were considered within expected ranges for biological 
and/or procedure-related variation despite occasional mean values that reached statistical 
significance. Two days following the final vaccination (Day 45), in animals of both sexes receiving 
the test article, there were minimal decreases in APTT (up to -11%), relative to pretest means, 
minimal increases in fibrinogen (up to +20%) at termination in treated animals of both sexes, and 
mild increases in CRP (up to 9.1x), relative to pretest means. All of these findings were considered 
test article-related but not adverse; they had generally resolved by the recovery interval. 
Furthermore, increases of CRP, while higher than baseline, were still within normal historical 
range at MPI Research. These findings were typical of a mild immune/inflammatory response 
expected with vaccine administration. All other mean and individual values were considered within 
expected ranges for biological variation. 
There were no consistent test article-related macroscopic observations or differences in organ 
weights. All macroscopic findings and organ weight variations were considered to be incidental to 
test article administration or typical for those seen in rabbits of this sex, strain, and age. Test 
article-related microscopic findings were seen only at the injection sites for all groups. The 
injection site findings were not considered to be adverse, given the low severity, lack of systemic 
findings, and lack of associated clinical findings. 

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Test article-related microscopic findings at the terminal necropsy consisted of increased incidence 
and/or severity of a constellation of findings including granulomatous, acute, and chronic inflammation 
of the muscle; histiocytic infiltration and mixed cell infiltration/inflammation of the subcutaneous 
tissues; hemorrhage of the muscle and subcutaneous tissues; and myofiber degeneration/necrosis. 
Following a 4-week recovery, there was a strong trend towards recovery in all groups, with generally 
reduced incidence and severity of most findings compared to terminal groups. 
1.6.2.3. 
Immunogenicity Results 
The immunogenicity of the trivalent P2-VP8 subunit rotavirus vaccine was demonstrated in NZW 
rabbits in the GLP toxicology study. As described above, animals were administered four doses 
of 180 µg (60 µg [P8] P2-VP8 + 60 µg [P6] P2-VP8 + 60 µg [P4] P2-VP8) subunit vaccine 
adsorbed to aluminum hydroxide [Al(OH)3] adjuvant on Study Days 1, 15, 29, and 43. Serum 
samples for immunological analyses were collected at baseline and two days following the final 
vaccination on Study Day 45. 
At baseline, some animals in the control and vaccine groups had low, but detectable, titers to one 
or more of the P2-VP8 constructs. This may be the result of subclinical rotavirus infections in 
these animals or due to non-specific binding in the assay. The titers are several-fold lower than 
those observed in the treated group post-vaccination. 
All animals vaccinated with trivalent P2-VP8 had substantial titers to all three serotypes on Day 
45, as shown in the table, below. The response to the P4 and P8 vaccine components were 
comparable and roughly 2-fold greater than that attained for the P6 component, similar to what 
was observed in guinea pigs.  
While appreciable titers were observed on Day 45 in some control animals, titers in vaccinated 
animals were significantly higher (p < 0.0000002). 
Table 8. 
Serum IgG Binding Antibody Responses in NZW Rabbits Following 
Vaccination with 180 µg Trivalent P2-VP8 Vaccine (Study Day 45) 
Vaccine Group 
Post-immunization Geometric Mean Serum IgG Titer (Range) to 
Vaccine Antigens* 
P2-VP8 [P4] 
P2-VP8 [P6] 
P2-VP8 [P8] 
1: Control (Saline) 
4.3 
(<LLQ – 955) 
5.9 
(<LLQ – 958) 
3.1 
(<LLQ – 301) 
2: Trivalent P2-VP8 [P8]  
+ [P6] + [P4] 
96639  
(13585 - 283551) 
55561 
(7471 - 148253) 
102963 
(17786 - 252081) 
LLQ < 78 units/mL 
*Note: geometric mean was calculated for controls by setting all values <LLQ to a value of “1” 
1.7. Clinical Studies 
1.7.1. First-in-human testing of monovalent (P[8]) P2-VP8 subunit vaccine in adults 
(VAC 009) 
The first clinical testing of the monovalent (P[8]) P2-VP8 subunit rotavirus vaccine was performed 
in 18-45 year old adults in North America. The trial was a double-blinded, randomized, placebo-

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controlled dose-escalation study, in which study volunteers were randomized into three dose level 
cohorts of 16 individuals (12 vaccine recipients and 4 placebo recipients). Participants in each 
cohort received three intramuscular injections four weeks apart of one of three dose levels of 
vaccine (10 µg, 30 µg and 60 µg) or placebo (saline).  
Overall, the vaccine was well-tolerated at all three dose levels, was associated with only mild 
transient local reactogenicity, and no safety concerns were identified. Thirty-four of the 
36 volunteers allocated to receive vaccine received all 3 injections (11/12 in the 10 µg cohort, 
11/12 in the 30 µg cohort and 12/12 in the 60 µg cohort (10/12 placebo recipients received all 
3 vaccines). The vaccine recipient in the 10 µg cohort who did not receive all three vaccines was 
discontinued due to an unrelated acute illness (pneumonia), and the vaccine recipient in the 30 µg 
cohort who did not receive all three vaccines was discontinued due to reasons unrelated to the 
study.  
Only one volunteer experienced an objective local reaction greater than mild, and that volunteer 
received placebo (Table 9). One vaccine recipient each in the 10 and 60 µg cohorts complained 
of greater than mild pain/tenderness (and none in the 30 µg cohort). 
Table 9:  Maximum Injection Site Reactogenicity per Participant 
Severity 
Pain 
N (%) 
Tenderness  
N (%) 
Itching  
N (%) 
Redness  
N (%) 
Swelling  
N (%) 
Placebo 
None 
10 (83.3) 
8 (66.7) 
9 (75.0) 
11 (91.7) 
11 (91.7) 
Mild 
1 (8.3) 
3 (25.0) 
3 (25.0) 
1 (8.3) 
0 
Moderate 
1 (8.3) 
1 (8.3) 
0 
0 
1 (8.3) 
10 µg 
None 
6 (50.0) 
5 (41.7) 
11 (91.7) 
11 (91.7) 
10 (83.3) 
Mild 
5 (41.7) 
6 (50.0) 
1 (8.3) 
1 (8.3) 
2 (16.7) 
Moderate 
1 (8.3) 
1 (8.3) 
0 
0 
0 
30 µg 
None 
10 (83.3) 
7 (58.3) 
11 (91.7) 
11 (91.7) 
10 (83.3) 
Mild 
2 (16.7) 
5 (41.7) 
1 (8.3) 
1 (8.3) 
2 (16.7) 
Moderate 
0 
0 
0 
0 
0 
60 µg 
None 
8 (66.7) 
 
11 (91.7) 
9 (75.0) 
10 (83.3) 
Mild 
3 (25.0) 
8 (66.7) 
1 (8.3) 
3 (25.0) 
2 (16.7) 
Moderate 
1 (8.3) 
0 
0 
0 
0 
Similarly, systemic reactions were relatively uncommon and restricted to mild (Table 10) No 
volunteers experienced severe reactions, and moderate reactions were limited to fatigue in one 
volunteer in each of the groups receiving placebo, 10 µg or 60 µg. None of the volunteers 
experienced fever.  
 

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Table 10:  Maximum Systemic Reactogenicity per Participant 
Severity 
Fever 
N (%) 
Nausea  
N (%) 
Vomiting  
N (%) 
Arthralgia  
N (%) 
Chills  
N (%) 
Fatigue  
N (%) 
Headache  
N (%) 
Myalgia  
N (%) 
Placebo 
None 
12 (100) 
10 (83.3) 
12 (100) 
10 (83.3) 
11 (91.7) 
10 (83.3) 
10 (83.3) 
11 (91.7) 
Mild 
0 
2 (16.7) 
0 
2 (16.7) 
1 (8.3) 
1 (8.3) 
2 (16.7) 
1 (8.3) 
Moderate 
0 
0 
0 
0 
0 
1 (8.3) 
0 
0 
10 µg 
None 
12 (100) 
11 (91.7) 
11 (91.7) 
11 (91.7) 
11 (91.7) 
9 (75.0) 
10 (83.3) 
10 (83.3) 
Mild 
0 
1 (8.3) 
1 (8.3) 
1 (8.3) 
1 (8.3) 
2 (16.7) 
2 (16.7) 
2 (16.7) 
Moderate 
0 
0 
0 
0 
0 
1 (8.3) 
0 
0 
30 µg 
None 
12 (100) 
12 (100) 
12 (100) 
11 (91.7) 
11 (91.7) 
11 (91.7) 
10 (83.3) 
11 (91.7) 
Mild 
0 
0 
0 
1 (8.3) 
1 (8.3) 
1 (8.3) 
2 (16.7) 
1 (8.3) 
Moderate 
0 
0 
0 
0 
0 
0 
0 
0 
60 µg 
None 
12 (100) 
10 (83.3) 
12 (100) 
9 (75.0) 
11 (91.7) 
10 (83.3) 
9 (75.0) 
9 (75.0) 
Mild 
0 
2 (16.7) 
0 
3 (25.0) 
1 (8.3) 
1 (8.3) 
3 (25.0) 
3 (25.0) 
Moderate 
0 
0 
0 
0 
0 
1 (8.3) 
0 
0 
 
No volunteers experienced a vaccine-related serious adverse event; one participant experienced 
an SAE 123 days after the last vaccination, a hospitalization for severe pneumonia assessed as 
unrelated to the vaccine. That participant had previously experienced a severe, non-serious AE 
that was assessed as unrelated to receipt of vaccine (back pain secondary to trauma). The AE 
data are summarized in Table 11. There were no discernible trends in AEs or safety monitoring 
laboratory values, which include basic clinical chemistry and hematology. 
Table 11:  Maximum Adverse Event Severity per Participant 
AE Severity 
Placebo 
10 µg 
30 µg 
60 µg 
Mild 
5 (41.7%) 
6 (50.0%) 
2 (16.7%) 
6 (50.0%) 
Moderate 
1 (8.3%) 
3 (25.0%) 
3 (25.0%) 
0 
Severe 
0 
0 
0 
1 (8.3%) 
Any 
6 (50.0%) 
9 (75.0%) 
5 (41.7%) 
7 (58.3%) 
 
Sera were assessed for IgG and IgA responses to the P2-VP8 antigen by ELISA (Figure 6/Table 
12 and Figure 7/Table 13, respectively) and for neutralizing antibodies to the homologous 
rotavirus Wa (Figure 8/Table 14) at baseline and one month after each of the three study 
injections. Sera were also assessed for neutralizing antibodies responses at Day 84 to 
heterologous rotavirus strains (Table 15) and for rotavirus-specific antibody in lymphocyte 
supernatant (ALS) to P2-VP8 antigen 7 days after the third study injection (Table 16). 
As noted in Figures 6 and 7, almost all vaccine recipients demonstrated greater than 4-fold rise 
in IgG and IgA response to P2-VP8 antigen by ELISA after three vaccinations: only one vaccine 
recipient did not demonstrate an IgG response (in the 30 µg group) and all vaccine recipients 
demonstrated IgA responses. 
 

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Figure 6:  Serum Anti-P2-VP8 IgG Titers by ELISA 
Vertical lines represent geometric mean titer and corresponding 95% confidence limits 
 
 
Table 12:  Proportion of Participants with ≥4-fold Increase in Anti-P2-VP8 IgG Titers by 
ELISA  
Treatment Arm 
Day 28 
Day 56 
Day 84 
Placebo 
0.0% 
0.0% 
0.0% 
10 µg 
58.3% 
90.9% 
100% 
30 µg 
66.7% 
83.3% 
     91.7% 
60 µg 
58.3% 
91.7% 
100% 
 

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Figure 7:  Serum Anti-P2-VP8 IgA Titers by ELISA 
Vertical lines represent geometric mean titer and corresponding 95% confidence limits 
 
 
Table 13:  Proportion of Participants with ≥4-fold Increase in Anti-P2-VP8 IgA Titers by 
ELISA 
Treatment Arm 
Day 28 
Day 56 
Day 84 
Placebo 
0.0% 
0.0% 
0.0% 
10 µg 
66.7% 
90.9% 
100% 
30 µg 
75.0% 
91.7% 
100% 
60 µg 
66.7% 
91.7% 
100% 
 
Neutralizing antibody responses were also encouraging (Figure 8/Table 14), with clear increases 
in geometric mean titers (GMTs) for all three dose levels at one month post-third study injection 
(Day 84 in Figure 8) compared to pre-vaccination levels. Of particular interest, of the 12 vaccine 
recipients (at any dose level) with baseline titers <100, 10 (83%) demonstrated a >4-fold increase 
in neutralizing antibody; 5 of these 12 were in the 60 µg group, and all demonstrated a >4-fold 
increase. 
Neutralizing antibody responses to heterologous rotavirus strains were most robust to P[8] strains, 
moderate to the P[4] strain and fairly limited to the P[6] strain (Table 11). 
 
 

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Figure 8:  Serum Neutralizing Antibody Titers against Wa Strain 
Vertical lines represent geometric mean titer and corresponding 95% confidence limits 
  
 
Table 14:  Proportion of Participants with ≥4-fold Increase in Neutralizing Antibody Titers 
against Wa Strain 
Treatment Arm 
Day 28 
Day 56 
Day 84 
Placebo 
0.0% 
0.0% 
0.0% 
10 µg 
0.0% 
36.4% 
66.7% 
30 µg 
16.7% 
41.7% 
41.7% 
60 µg 
33.3% 
50.0% 
58.3% 
 
Table 15:  Proportion of Participants with ≥4-fold Increase in Neutralizing Antibody Titers 
at Day 84 against Other Strains 
Strain 
Treatment Arm 
Percent response 
(95% CI) 
89-12 (G1P[8]) 
10 µg 
83 (52, 98) 
30 µg 
67 (35, 90) 
60 µg 
83 (52, 98) 
P (G3P[8]) 
10 µg 
58 (28, 85) 
30 µg 
67 (35, 90) 
60 µg 
83 (52, 98) 
DS1 (G2P[4]) 
10 µg 
0 (0, 26) 
30 µg 
50 (21, 79) 
60 µg 
58 (28, 85) 
ST3 (G4P[6]) 
10 µg 
0 (0, 26) 
30 µg 
8 (0, 38) 
60 µg 
17 (2, 48) 
 

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None of the participants had detectable ALS responses at baseline. As seen in Table 16, there 
was a steady rise in proportion of participants with detectable IgA responses 7 days after the first 
dose of vaccine from lowest to highest dose level. However the sample sizes are too small for 
meaningful analysis of statistical significance. 
Table 16:  Participants with Detectable Antibody in Lymphocyte Supernatant (ALS) to P2-
VP8 Antigen 7 Days after the Third Study Injection 
Treatment Arm 
IgA 
IgG 
Placebo 
0 
0 
10 µg 
4 (33.3%) 
3 (25.0%) 
30 µg 
5 (41.7%) 
3 (25.0%) 
60 µg 
8 (67.7%) 
4 (33.3%) 
1.7.2. Clinical testing of monovalent (P[8]) P2-VP8 subunit vaccine in toddlers and 
infants in South Africa (VAC 013) 
Clinical testing of the monovalent (P[8]) P2-VP8 subunit rotavirus vaccine was initiated in South 
African toddlers and infants in 2014, and that trial is now fully enrolled. Preliminary primary safety 
and immunogenicity results are available and presented below. The trial is a double-blind, 
randomized, placebo-controlled dose-escalation study. Dose escalation, from 10 to 30 to 60 µg, 
was first performed in small groups of toddlers (12 vaccine and 2 placebo recipients per dose 
level), before initiation of similar dose escalation in 6-8 week old infants (12 vaccine and 4 placebo 
recipients per dose level). The two highest tolerated doses, 30 and 60 µg, were selected for 
expanded testing in infants, with simultaneous randomization to each of the two doses or placebo 
(38 infants in each of the three arms). EPI vaccines, excluding oral rotavirus vaccine, were 
co-administered with the three study vaccines in infants (at 6, 10 and 14 weeks of age). 
The vaccine has been generally well-tolerated at all three dose levels. The data presented in 
Tables 17 and 18, for local and systemic reactogenicity are complete, and the data presented for 
adverse events in Table 19 represent the available data through 28 days after the last vaccination 
of all participants. The data are presented by age-group and dose level. In both toddlers and 
infants, when local reactogenicity was reported, it was transient, rarely greater than mild, and 
never severe (Table 17). When present, systemic reactogenicity was also transient and generally 
mild, without discernable dose effect (Table 18). Late in the expanded infant cohort experience, 
two participants were reported to have severe irritability following one of their three study 
injections (co-administered with EPI vaccinations), one of whom received placebo. These events 
occurred after the second dose in one participant and third in the other, and were brief and 
transient (≤24 hours at the severe intensity level in both cases, completely resolved in one day in 
one participant and within 3 days in the other).  
In the dose-escalation phase of the testing in the infant cohorts, the study injections were paused 
temporarily due to findings of severe neutropenia on post-vaccination laboratory monitoring in 
three participants (two infants and one toddler). The Safety Review Committee (SRC, composed 
of the principal investigator, Emmes medical monitor, PATH medical officer and two, independent 
local experts) interpretation of the review of the baseline screening data (including data for those 
who were screened-out), unblinded aggregate data for neutrophil counts at the scheduled 7 day 
post-vaccination time point, the changes relative to baseline, and the unblinded allocation of the 
three participants with severe neutropenia was that the data did not indicate a relationship to 
receipt of the study vaccine, and study injections were resumed. 
 

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Table 17:  Maximum Injection Site Reactogenicity per Participant in VAC 013 
Severity 
Pain/ 
Tenderness 
N (%) 
Erythema/ 
Redness 
N (%) 
Induration/ 
Swelling 
N (%) 
Itching 
N (%) 
Cohort A, 10µg 
None 
11 (91.7) 
12 (100.0) 
12 (100.0) 
11 (91.7) 
Mild 
1 (8.3) 
0 
0 
0 
Moderate 
0 
0 
0 
1 (8.3) 
Severe 
0 
0 
0 
0 
Cohort A, 30µg 
None 
8 (66.7) 
11 (91.7) 
11 (91.7) 
9 (75.0) 
Mild 
4 (33.3) 
0 
1 (8.3) 
3 (25.0) 
Moderate 
0 
1 (8.3) 
0 
0 
Severe 
0 
0 
0 
0 
Cohort A, 60µg 
None 
10 (83.3) 
11 (91.7) 
12 (100.0) 
12 (100.0) 
Mild 
2 (16.7) 
1 (8.3) 
0 
0 
Moderate 
0 
0 
0 
0 
Severe 
0 
0 
0 
0 
Cohort A, Placebo 
None 
5 (83.3) 
6 (100.0) 
6 (100.0) 
6 (100.0) 
Mild 
1 (16.7) 
0 
0 
0 
Moderate 
0 
0 
0 
0 
Severe 
0 
0 
0 
0 
Cohort B1, 10µg 
None 
7 (58.3) 
8 (66.7) 
8 (66.7) 
10 (83.3) 
Mild 
5 (41.7) 
4 (33.3) 
4 (33.3) 
2 (16.7) 
Moderate 
0 
0 
0 
0 
Severe 
0 
0 
0 
0 
Cohort B1, 30µg 
None 
5 (41.7) 
9 (75.0) 
8 (66.7) 
12 (100.0) 
Mild 
6 (50.0) 
3 (25.0) 
4 (33.3) 
0 
Moderate 
1 (8.3) 
0 
0 
0 
Severe 
0 
0 
0 
0 

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Table 17:  Maximum Injection Site Reactogenicity per Participant in VAC 013 (cont’d) 
 
38 
Severity 
Pain/ 
Tenderness 
N (%) 
Erythema/ 
Redness 
N (%) 
Induration/ 
Swelling 
N (%) 
Itching 
N (%) 
Cohort B1, 60µg 
None 
2 (16.7) 
8 (66.7) 
9 (75.0) 
9 (75.0) 
Mild 
8 (66.7) 
4 (33.3) 
3 (25.0) 
3 (25.0) 
Moderate 
2 (16.7) 
0 
0 
0 
Severe 
0 
0 
0 
0 
Pot. LT 
0 
0 
0 
0 
Cohort B1, Placebo 
None 
4 (33.3) 
8 (66.7) 
9 (75.0) 
6 (50.0) 
Mild 
8 (66.7) 
4 (33.3) 
3 (25.0) 
6 (50.0) 
Moderate 
0 
0 
0 
0 
Severe 
0 
0 
0 
0 
Cohort B2, 30µg 
None 
17 (44.7) 
25 (65.8) 
23 (60.5) 
29 (76.3) 
Mild 
19 (50.0) 
13 (34.2) 
15 (39.5) 
9 (23.7) 
Moderate 
2 (5.3) 
0 
0 
0 
Severe 
0 
0 
0 
0 
Cohort B2, 60µg 
None 
18 (47.4) 
27 (71.1) 
29 (76.3) 
32 (84.2) 
Mild 
20 (52.6) 
10 (26.3) 
9 (23.7) 
6 (15.8) 
Moderate 
0 
1 (2.6) 
0 
0 
Severe 
0 
0 
0 
0 
Cohort B2, Placebo 
None 
16 (42.1) 
22 (57.9) 
27 (71.1) 
29 (76.3) 
Mild 
18 (47.4) 
15 (39.5) 
10 (26.3) 
9 (23.7) 
Moderate 
4 (10.5) 
1 (2.6) 
1 (2.6) 
0 
Severe 
0 
0 
0 
0 
 
 

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Table 18: Maximum Systemic Reactogenicity per Participant in VAC 013 
Severity 
Vomiting 
N (%) 
Decreased 
Appetite 
N (%) 
Irritability 
N (%) 
Decreased 
Activity 
N (%) 
Acute Systemic 
Allergic Reaction 
N (%) 
Non-Axillary 
Temperature 
N (%) 
Cohort A, 10µg 
None 12 (100.0) 
9 (75.0) 
11 (91.7) 
11 (91.7) 
12 (100.0) 
12 (100.0) 
Mild 
0 
2 (16.7) 
1 (8.3) 
1 (8.3) 
0 
0 
Moderate 
0 
1 (8.3) 
0 
0 
0 
0 
Severe 
0 
0 
0 
0 
0 
0 
Cohort A, 30µg 
None 
10 (83.3) 
7 (58.3) 
12 (100.0) 
11 (91.7) 
12 (100.0) 
11 (91.7) 
Mild 
2 (16.7) 
5 (41.7) 
0 
1 (8.3) 
0 
1 (8.3) 
Moderate 
0 
0 
0 
0 
0 
0 
Severe 
0 
0 
0 
0 
0 
0 
Cohort A, 60µg 
None 
11 (91.7) 
8 (66.7) 
11 (91.7) 
10 (83.3) 
12 (100.0) 
10 (83.3) 
Mild 
1 (8.3) 
4 (33.3) 
1 (8.3) 
2 (16.7) 
0 
1 (8.3) 
Moderate 
0 
0 
0 
0 
0 
1 (8.3) 
Severe 
0 
0 
0 
0 
0 
0 
Cohort A, Placebo 
None 
6 (100.0) 
5 (83.3) 
6 (100.0) 
6 (100.0) 
6 (100.0) 
5 (83.3) 
Mild 
0 
1 (16.7)1 
0 
0 
0 
0 
Moderate 
0 
0 
0 
0 
0 
1 (16.7) 
Severe 
0 
0 
0 
0 
0 
0 
Cohort B1, 10µg 
None 
9 (75.0) 
8 (66.7) 
7 (58.3) 
9 (75.0) 
12 (100.0) 
6 (50.0) 
Mild 
3 (25.0) 
4 (33.3) 
5 (41.7) 
3 (25.0) 
0 
6 (50.0) 
Moderate 
0 
0 
0 
0 
0 
0 
Severe 
0 
0 
0 
0 
0 
0 
Cohort B1, 30µg 
None 
10 (83.3) 
7 (58.3) 
4 (33.3) 
6 (50.0) 
12 (100.0) 
8 (66.7) 
Mild 
2 (16.7) 
4 (33.3) 
7 (58.3) 
5 (41.7) 
0 
4 (33.3) 
Moderate 
0 
1 (8.3) 
1 (8.3) 
1 (8.3) 
0 
0 
Severe 
0 
0 
0 
0 
0 
0 
Cohort B1, 60µg 
None 
6 (50.0) 
7 (58.3) 
5 (41.7) 
8 (66.7) 
12 (100.0) 
8 (66.7) 
Mild 
6 (50.0) 
5 (41.7) 
6 (50.0) 
4 (33.3) 
0 
3 (25.0) 
Moderate 
0 
0 
1 (8.3) 
0 
0 
0 
Severe 
0 
0 
0 
0 
0 
0 
Pot. LT 
0 
0 
0 
0 
0 
1 (8.3) 

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Table 18:  Maximum Systemic Reactogenicity per Participant in VAC 013 (cont’d) 
 
40 
Severity 
Vomiting 
N (%) 
Decreased 
Appetite 
N (%) 
Irritability 
N (%) 
Decreased 
Activity 
N (%) 
Acute Systemic 
Allergic Reaction 
N (%) 
Non-Axillary 
Temperature 
N (%) 
Cohort B1, Placebo 
None 
5 (41.7) 
5 (41.7) 
3 (25.0) 
7 (58.3) 
12 (100.0) 
6 (50.0) 
Mild 
6 (50.0) 
6 (50.0) 
8 (66.7) 
3 (25.0) 
0 
6 (50.0) 
Moderate 
1 (8.3) 
1 (8.3) 
1 (8.3) 
2 (16.7) 
0 
0 
Severe 
0 
0 
0 
0 
0 
0 
Cohort B2, 30µg 
None 
29 (76.3) 
23 (60.5) 
18 (47.4) 
21 (55.3) 
38 (100.0) 
21 (55.3) 
Mild 
8 (21.1) 
13 (34.2) 
13 (34.2) 
14 (36.8) 
0 
16 (42.1) 
Moderate 
1 (2.6) 
2 (5.3) 
6 (15.8) 
3 (7.9) 
0 
1 (2.6) 
Severe 
0 
0 
1 (2.6) 
0 
0 
0 
Cohort B2, 60µg 
None 
24 (63.2) 
26 (68.4) 
18 (47.4) 
27 (71.1) 
38 (100.0) 
22 (57.9) 
Mild 
13 (34.2) 
11 (28.9) 
19 (50.0) 
10 (26.3) 
0 
15 (39.5) 
Moderate 
1 (2.6) 
1 (2.6) 
1 (2.6) 
1 (2.6) 
0 
1 (2.6) 
Severe 
0 
0 
0 
0 
0 
0 
Cohort B2, Placebo 
None 
23 (60.5) 
20 (52.6) 
15 (39.5) 
23 (60.5) 
38 (100.0) 
23 (60.5) 
Mild 
15 (39.5) 
13 (34.2) 
19 (50.0) 
9 (23.7) 
0 
13 (34.2) 
Moderate 
0 
5 (13.2) 
3 (7.9) 
6 (15.8) 
0 
2 (5.3) 
Severe 
0 
0 
1 (2.6) 
0 
0 
0 
Through 11 September 2015, 19 SAEs have been reported in 15 participants, none of which are 
assessed as related to study product. The study was paused late in the study for the SRC to 
review the death of a participant which occurred the day following the third vaccination. The 
participant had tolerated the first two vaccinations well, but had been hospitalized two weeks 
before the third vaccination with a severe respiratory infection. The infant had been put down to 
rest while the mother took a bath, and when the mother returned the infant had died. Laboratory 
results of nasopharyngeal secretions during the hospitalization only became available after the 
infant’s death, with PCR revealing respiratory syncytial virus B, cytomegalovirus and 
Pneumocystis jiroveci (in substantial quantity). A minimally invasive autopsy did not reveal any 
major organ pathology, however the lung tissue was inadequate for investigation. The SRC 
assessment after review of the available data was that there was not a reasonable possibility that 
the study product caused the event. 
As stated above, the aggregate unblinded primary safety data will be submitted for review to the 
MCC, FDA, ECs/IRBs, SRC and DSMB prior to study initiation.  
 

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Table 19: Maximum Adverse Event Severity per Participant in VAC 013 
 
Cohort A 
Cohort B1 
Cohort B2 
AE 
Severity 
10 µg 
N (%) 
30 µg 
N (%) 
60 µg 
N (%) 
Placebo 
N (%) 
10 µg 
N (%) 
30 µg 
N (%) 
60 µg 
N (%) 
Placebo 
N (%) 
30 µg 
N (%) 
60 µg 
N (%) 
Placebo 
N (%) 
Mild 7 (58.3) 6 (50.0) 10 (83.3) 
2 (33.3) 
11 (91.7) 10 (83.3) 
11 (91.7) 
10 (83.3) 
32 (84.2) 33 (86.8) 33 (86.8) 
Moderate 
1 (8.3) 
1 (8.3) 
0 
1 (16.7) 
1 (8.3) 
0 
1 (8.3) 
1 (8.3) 
4 (10.5) 
2 (5.3) 
2 (5.3) 
Severe 
0 
0 
0 
0 
0 
1 (8.3) 
0 
1 (8.3) 
2 (5.3) 
1 (2.6) 
0 
Any 8 (66.7) 7 (58.3) 10 (83.3) 
3 (50.0) 12 (100.0) 11 (91.7) 12 (100.0) 12 (100.0) 38 (100.0) 36 (94.7) 35 (92.1) 
Excludes 19 SAEs in 15 subjects. One from cohort B1 and 18 from cohort B2 
 
Tables 20-22 present the primary serology results in the infant cohorts. Whether IgG responses 
were adjusted for maternal antibody or not (Table 20), there were close to universal responses, 
with substantial increases in titer as demonstrated by mean-fold increase in GMT. In infants 
receiving the 30 µg dose, the GMT rose from a baseline value of 107 to a post-vaccination value 
of 9,583. Although not as dramatic as the IgG responses, there were good IgA responses to P2-
VP8, with the seroresponse rate over 80% in infants receiving 30 µg of vaccine. The 60 µg dose 
did not appear to provide any better response than the 30 µg dose, although the study was not 
powered for that comparison. 
 
Table 20: Anti-P2-VP8 IgG Responses in Infants 
 
 
Unadjusted 
Adjusted* 
Study 
arm 
N 
Mean fold-increase 
in GMT 
4-fold increase 
n (%) 
Mean fold-increase 
in GMT 
4-fold increase 
n (%) 
Placebo 
45 
0.3 
1 (2.2%) 
1.4 
9 (20.0%) 
10µg 
12 
131 
12 (100%) 
556 
12 (100%) 
30µg 
47 
89 
46 (97.9%) 
369 
46 (97.9%) 
60µg 
47 
58 
46 (97.9%) 
239 
47 (100.0%) 
*Adjusted for maternal antibody 
 
Table 21: Anti-P2-VP8 IgA Responses in Infants 
Study arm 
N 
Mean fold-increase 
4-fold increase 
n (%) 
Placebo 
45 
1.9 
9 (20.0%) 
10µg 
12 
7.1 
7 (58.3%) 
30µg 
47 
10.0 
38 (80.9%) 
60µg 
47 
6.7 
32 (68.1%) 
 
Although unadjusted neutralizing antibody responses to the Wa strain from which the 
vaccine is derived were modest, when adjusted for maternal antibody a high proportion 
of infants demonstrated a seroresponse. This was the case both when the protocol 
stipulated 4-fold increase was used to define seroresponse and when the laboratory’s 
more recent validated cut-off of 2.7-fold was used. Again, although not powered to 
compare responses to the two dose level, the 60 µg dose did not appear to provide any better 
response than the 30 µg dose. 
 
 

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Table 22: Neutralizing Antibody Responses to Wa Strain in Infants 
 
 
Unadjusted 
Adjusted 
Study arm 
N 
Mean fold-
increase 
4-fold 
increase 
n (%) 
2.7-fold 
increase 
n (%) 
Mean fold-
increase 
4-fold 
increase 
n (%) 
2.7-fold 
increase 
n (%) 
Placebo 
45 
0.2 
0 (0%) 
1 (2.2%) 
0.9 
4 (8.9%) 
5 (11.1%) 
10µg 
12 
4.2 
7 (58.3%) 
7 (58.3%) 
24.9 
11 (91.7%) 
12 (100%) 
30µg 
47 
2.5 
17 (36.2%) 
21 (44.7%) 
14.1 
38 (80.9%) 
42 (89.4%) 
60µg 
47 
1.6 
7 (14.9%) 
10 (21.3%) 
9.1 
37 (78.7%) 
42 (89.4%) 
*Adjusted for maternal antibody 
 
As a test of concept, shedding of vaccine virus was assessed 5, 7 and 9 days following 
administration of the first dose of Rotarix after completion of the study vaccine series (Table 23). 
In this proxy model of efficacy, there were substantial reductions in shedding (at any time) in 
infants who had received vaccine compared to infants who had received placebo. The reduction 
was most pronounced for infants receiving 30 µg, with a 65% reduction in shedding (p-value = 
0.009). In contrast, the reduction in shedding in infants receiving 60 µg of vaccine was 49%. 
 
Table 23: Rotarix Shedding in Infants 
Study arm 
N 
n (%) 
Placebo 
44 
17 (38.6%) 
30µg 
45 
6 (13.3%) 
60µg 
46 
9 (19.6%) 
30µg + 60µg 
91 
15 (16.5%) 
 
In summary, the P2-VP8 monovalent vaccine was generally well-tolerated in healthy South 
African infants, and there was no evidence that the higher vaccine dose (60 µg) provided any 
benefit in serologic responses or impact on shedding of Rotarix compared to the lower dose 
(30 µg). 
2. HYPOTHESIS, SCIENTIFIC RATIONALE, OBJECTIVES AND STUDY DESIGN 
2.1. Study Hypothesis 
Primary Hypotheses:  
Safety 
The trivalent P2-VP8 subunit rotavirus vaccine is safe and well-tolerated in healthy South African 
adults, toddlers and infants. 
Immunogenicity 
The trivalent P2-VP8 subunit rotavirus vaccine is immunogenic in healthy South African infants 
and will induce neutralizing immune responses to at least 2 of the 3 strains from which vaccine 
antigens are derived in 60% or more of participants in at least one of the study groups. 
2.2. Scientific Rationale  
Live oral rotavirus vaccines have not performed optimally in developing-country populations. The 
discrepancy in performance of these vaccines in developed versus developing countries is not a 
unique feature of rotavirus vaccines; it has also been observed with other oral, live attenuated 
enteric vaccines, such as those targeted against cholera and poliomyelitis, for which efficacy is 
diminished in resource poor countries. Strategies targeting the development of inactivated rotavirus 
vaccines have recently been proposed [3], which are supported by the successful use of parenteral 
vaccines to control disease caused by several orally transmitted pathogens including poliovirus, 

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Vibrio cholera and Salmonella typhi. Most approaches for developing inactivated rotavirus vaccines 
have focused on eliciting rotavirus neutralizing antibodies directed at either the VP4 or VP7 rotavirus 
neutralizing antigens. Several direct and indirect observations support the possibility that 
parenterally-administered, non-replicating rotavirus vaccines will likely be successful, including 
observations derived from natural rotavirus infection studies in infants, rotavirus challenge studies 
in adult volunteers, human rotavirus vaccine trials, and animal rotavirus vaccine studies. The 
induction of non-neutralizing antibody responses may result in mucosal protection, either through 
the induction of local IgA responses or, more likely, through “leaking” into the gut of IgG antibodies 
generated by adjuvanted parenteral injection of non-replicating vaccines. This possibility is 
supported by the finding that high titers of circulating anti-rotavirus IgG are transmitted trans-
placentally in humans. Maternally derived IgG antibody appears to modulate vaccine takes and be 
protective against early rotavirus disease, and passively administered antibody may prevent or 
diminish disease [32, 33]. 
Clinical testing of the monovalent (P[8]) P2-VP8 subunit vaccine has demonstrated the vaccine 
to be well-tolerated, without safety signals. In adults, neutralizing antibody results against P[8] 
strains were promising, but less so against P[4] strains and disappointing against P[6] strains. 
Therefore, a trivalent vaccine, comprising antigens from all 3 strains has been developed to 
optimize responses against all three strains, which together are responsible for the vast majority 
of global disease burden. Preclinical testing indicates improved responses to P[4] and P[6] with 
the trivalent vaccine. 
The dose levels to be assessed in this protocol are based on results in infants in VAC 013 (see 
section 1.7.2), in which the 60 µg dose did not seem to provide any better responses in terms of 
immune response or impact on Rotarix shedding, compared to the 30 µg dose. The highest dose 
to be assessed in this protocol is an aggregated dose of 90 µg, composed of 30 µg of each of the 
three antigens.  
Further, as in VAC 013, the impact of the trivalent vaccine on the replication of Rotarix, which will 
be administered to the infants after completion of the 3-dose regimen of P2-VP8, will be assessed. 
Impact on shedding of Rotarix will be interpreted as a surrogate of protection. This assessment 
will provide an indication of whether the addition of two more antigens alters the impact observed 
in VAC 013 with the monovalent vaccine. 
2.3. Overall Clinical Development Strategy 
The ultimate goal of the P2-VP8 subunit rotavirus vaccine clinical development plan is to 
demonstrate the safety and efficacy of this vaccine approach in a series of studies to gain 
sufficient information to achieve both local registration of the vaccine in the country(ies) where 
the pivotal trials will be conducted and prequalification by WHO to support product acquisition by 
GAVI, UNICEF or others for distribution, in general, to low income countries. Briefly, the overall 
clinical development plan consists of: 1) the first in human phase 1 safety trial of the monovalent 
(P[8]) P2-VP8 subunit vaccine performed in the US in 2013; 2) the phase 1-2 descending age 
safety and immunogenicity trial of the monovalent (P[8]) P2-VP8 subunit vaccine in toddlers and 
infants currently approaching completion in South Africa; 3) the proposed phase 1-2 descending 
age safety and immunogenicity trial of the trivalent (P[4], P[6] and P[8]) P2-VP8 subunit vaccine 
in adults, toddlers and infants in South Africa; and 4) phase 3 efficacy trials to evaluate efficacy 
of the trivalent P2-VP8 subunit vaccine against severe rotavirus diarrhea. Additional clinical trial 
assessments could include: clinical assessment of lot-to-lot consistency (potentially could be 
combined with the efficacy trials); assessment of interference of the vaccine with the immune 
responses to routine EPI vaccinations; and assessment of vaccine safety and immunogenicity in 
other populations.  

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2.4. Study Design 
The trial is a double-blind, randomized, placebo-controlled dose-escalation study in which two 
dose-levels (30 µg and 90 µg) of vaccine will be tested in adults and toddlers and then three dose-
levels will be assessed in infants (15 µg, 30 µg and 90 µg). In Group A, cohorts of 15 adults (12 
vaccine and 3 placebo recipients) per dose level will receive three study injections at 4 week 
intervals (consistent with the regimen in the adult study of the monovalent vaccine), advancing 
from the first to the second dose-level after assessment of safety data through the first week after 
the first injection (A1). Based on the safety data in the adult group, Group B cohorts of 15 toddlers 
(12 vaccine recipients and 3 placebo recipients) per dose level will receive a single intramuscular 
injection (consistent with the regimen administered to toddlers in the current study of the 
monovalent vaccine in toddlers), advancing from the first to the second dose-level after 
assessment of safety data through the first week after the injection in the lower dose toddler group 
(B1) and all available data for the adult group. Based on the safety data in the toddler cohorts, 
Group C cohorts of 16 infants (12 vaccine recipients and 4 placebo recipients) per dose level will 
receive three study injections at 4 week intervals (concomitant with EPI vaccinations, with the 
exception of rotavirus vaccination), starting at ≥6 and <8 weeks of age. Advancing from the first 
to the second dose-level in Group C will occur after assessment of safety data through the first 
week after the first injection (C1), as well as all available data for toddlers and adults. Advancing 
from the second to the third dose-level (90 µg) in Group C will occur after assessment of safety 
data through the first week after the first injection of the second dose-level (30 µg). If all three 
dose levels are tolerated in the infant dose-escalation phase (Part C), they will be assessed in an 
expanded infant cohort (Group D) of 552 infants (138 at each dose level and 138 placebo 
recipients). All infants will receive Rotarix at 4, 8 and 12 weeks after the third study injection (study 
Days 84, 112 and 140), and fecal shedding of Rotarix will be assessed during the week after the 
first dose in a subset of infants. Three doses of Rotarix are to be administered rather than two to 
counterbalance the remote theoretical possibility that the P2-VP8 subunit vaccine could reduce 
response to Rotarix. 
2.4.1. Primary Objectives 
Safety 
 
To evaluate the safety and tolerability of the trivalent P2-VP8 subunit rotavirus vaccine at 
escalating dose levels in healthy South African adults, toddlers and infants 
Immunogenicity 
 
To evaluate the immunogenicity of three doses of the trivalent P2-VP8 subunit rotavirus 
vaccine at different dose levels in healthy South African infants 
2.4.2. Secondary Objectives 
Safety 
 
To evaluate the longer term safety (through 6 months after the last vaccination) of the trivalent 
P2-VP8 subunit rotavirus vaccine at escalating dose levels in healthy South African adults, 
toddlers and infants 
Immunogenicity 
 
To evaluate the immunogenicity of two doses of the trivalent P2-VP8 subunit rotavirus vaccine 
at different dose levels in healthy South African infants 
 
To evaluate the immunogenicity of the trivalent P2-VP8 subunit rotavirus vaccine at different 
dose levels in healthy South African adults and toddlers 

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2.4.3. Exploratory Objective 
Efficacy 
 
To evaluate the impact of the trivalent P2-VP8 subunit rotavirus vaccination on shedding of 
Rotarix subsequently administered in healthy South African infants as a test of concept 
2.4.4. Primary Endpoints 
Safety 
 
Number of SAEs through 28 days after the last study injection 
 
Number of AEs through 28 days after the last study injection 
 
Number of vaccine-induced local and systemic reactions 
Immunogenicity (infants): 
 
Proportion of infants with anti-P2-VP8 IgG & IgA seroresponses by ELISA (four-fold increase 
in antibody titers between baseline and 4-weeks post-third study injection) in assays using 
each of the three vaccine antigens (P[4], P[6] and P[8]) 
 
Proportion of infants with neutralizing antibody responses (2.7-fold increases in antibody titers 
between baseline and 4-weeks post-third study injection) to each of the three rotavirus strains 
from which the vaccine antigens are derived, as well as heterologous strains 
 
Anti-P2-VP8 IgG & IgA geometric mean titers (GMT) (baseline and 4 weeks post third study 
injection) in ELISA assays using each of the three vaccine antigens (P[4], P[6] and P[8]) 
 
Neutralizing antibody GMT (baseline and 4 weeks post-third study injection) to each of the 
three rotavirus strains from which the vaccine antigens are derived, as well as heterologous 
strains 
 
Proportion of infants with neutralizing antibody responses to at least two of the three strains 
from which the vaccine antigens are derived (4-weeks post-third study injection) 
2.4.5. Secondary Endpoints: 
Safety (all 3 age-groups) 
 
Number of SAEs at any time during the study 
 
Number of AEs at any time during the study 
Immunogenicity (infants) 
 
Proportion of infants with anti-P2-VP8 IgG & IgA seroresponses by ELISA (four-fold increase 
in antibody titers between baseline and 4-weeks post-second study injection) in assays using 
each of the three vaccine antigens (P[4], P[6] and P[8]) 
 
Proportion of infants with neutralizing antibody responses (2.7-fold increases in antibody titers 
between baseline and 4-weeks post-second study injection) to each of the three rotavirus 
strains from which the vaccine antigens are derived, as well as heterologous strains 
 
Anti-P2-VP8 IgG & IgA geometric mean titers (GMT) (baseline and 4 weeks post-second study 
injection) in ELISA assays using each of the three vaccine antigens (P[4], P[6] and P[8]) 
 
Neutralizing antibody GMT (baseline and 4 weeks post-second study injection) to each of the 
three rotavirus strains from which the vaccine antigens are derived, as well as heterologous 
strains 
Immunogenicity (adults and toddlers) 

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 
Proportion of adults and toddlers with anti-P2-VP8 IgG & IgA seroresponses by ELISA (four-
fold increase in antibody titers between baseline and 4-weeks post-final study injection) in 
assays using each of the three vaccine antigens (P[4], P[6] and P[8]) 
 
Proportion of adults and toddlers with neutralizing antibody responses (2.7-fold increases in 
antibody titers between baseline and 4-weeks post-final study injection) to each of the three 
rotavirus strains from which the vaccine antigens are derived, as well as heterologous strains 
 
Anti-P2-VP8 IgG & IgA geometric mean titers (GMT) (baseline and 4 weeks post final study 
injection) in ELISA assays using each of the three vaccine antigens (P[4], P[6] and P[8]) 
 
Neutralizing antibody GMT (baseline and 4 weeks post-final study injection) to each of the three 
rotavirus strains from which the vaccine antigens are derived, as well as heterologous strains 
 
Proportion of adults and toddlers with neutralizing antibody responses to at least two of the 
three strains from which the vaccine antigens are derived 
2.4.6. Exploratory Endpoint 
 
Proportion of participants shedding Rotarix after administration of Rotarix “challenge” 
 
3. STUDY PRODUCT 
3.1. Product Description 
The P2-VP8 subunit rotavirus vaccine is made by inserting a codon optimized synthetic gene 
encoding the VP8 region of rotavirus VP4 fused to the P2 T-cell epitope coding sequence of 
tetanus toxin into the Pj411 proprietary cloning vector developed by DNA 2.0, Menlo Park, CA. 
The vector carries a kanamycin resistance gene as a selection marker. The vector was 
transfected into the BL21 strain of E. coli. The fusion protein is purified from Isopropyl β-D-1-
thiogalactopyranoside (IPTG)-induced and physically lysed cultures using standard column 
chromatographic techniques employing Q-Sepharose and Butyl 650 as resins in addition to 
ultrafiltration and diafiltration. The purified protein is co-formulated with aluminum hydroxide 
[Al(OH)3] (see Section 3.3). 
3.2. Products and Manufacturers 
Trivalent P2-VP8 subunit rotavirus vaccine is manufactured and supplied by the Walter Reed 
Army Institute of Research (WRAIR) Pilot Bioproduction Facility (BPF).  
Sterile aluminum hydroxide adjuvant Lot Nr 1862 (2 mg of aluminum per mL) was prepared, 
labeled and sampled for QC release at WRAIR and will be used as a diluent to prepare the 60 µg 
vaccine dose. The vials were stored at 2-8°C at WRAIR. 
Sodium Chloride 0.9%, USP for Injection will be used to dilute the active P2-VP8 vaccine to final 
dosing concentration and will be used for the placebo for the study. The research pharmacy at 
each site will obtain enough supply of the same lot of Sodium Chloride 0.9% for Injection, USP 
from Pharma Logistical Solutions (Pty) Ltd to complete the study.  
3.3. Presentation and Formulation 
The trivalent P2-VP8 vaccine is formulated as a sterile suspension containing a total of 360 µg of 
protein (120 µg of each P type) per mL adsorbed to aluminum hydroxide (1.125 mg of aluminum 
per mL in a phosphate buffer, pH 7). The vaccine is contained within a 2 mL borosilicate glass 
vial sealed with a butyl rubber stopper and a crimped metal collar. The vaccine is stored at 2-8°C. 

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To produce the 15, 30 and 90 µg doses (containing 5, 10 and 30 µg of each P2-VP8 vaccine 
component, respectively), the vaccine is diluted with the aluminum hydroxide adjuvant diluent 
within 6 hours of administration (aluminum hydroxide diluted to a concentration of 1.125 mg/mL 
before being used to dilute the vaccine).  
The doses of the trivalent P2-VP8 subunit vaccine will be prepared in the pharmacy. Blinded study 
syringes will be prepared for the study coordinator for administration to the volunteers. 
3.4. Stability and Storage 
The trivalent P2-VP8 subunit vaccine is stored at 2-8°C until used. A pilot lot of vaccine has been 
shown to be stable when stored for 6 months at 2-8°C (study ongoing). The P2-VP8 trivalent 
vaccine to be used in this study (Lot Nr 1917) is being monitored for stability at 2-8°C per ICH 
guidelines. The vaccine has been shown to be stable for 3 months (latest time point available). 
Stability will be evaluated for a minimum of 24 months. The aluminum hydroxide adjuvant 
(Lot Nr 1862) to be used as a diluent to prepare the 60 µg dose of vaccine has been shown to be 
stable for 6 months when stored at 2-8°C. Stability studies are ongoing and will be conducted for 
a minimum of 24 months. Sodium chloride 0.9% for Injection, USP, will be used to prepare the 
aluminum hydroxide diluent. These 10 mL vials will be stored at room temperature. 
3.5. Preparation and Administration 
In Groups A and B, at each dose level 12 participants will be randomized to receive vaccine and 
three (3) to receive placebo; in Group C, at each dose level 12 participants will be randomized to 
receive vaccine and four (4) to receive placebo. In Group D, the allocation for the four arms (three 
dose levels and placebo) will be 1:1:1:1. Vaccine is administered as a 0.5 mL intramuscular 
injection: in Groups A, C and D, study injections will be administered on Day 0, Day 28 and Day 
56; and in Group B, the single injection will be administered on Day 0. Study injections will be 
administered with 25 gauge, 25 mm needles into muscle at 90°.  
 
 

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Table 24 illustrates the dosing schedule. 
Table 24: Dosing Schedule 
Group 
P2-VP8 dose 
N  
Study Day 
0 
28 
56 
A 
Adults 
30 µg 
12 
X 
X 
X 
Placebo 
3 
X 
X 
X 
90 µg 
12 
X 
X 
X 
Placebo 
3 
X 
X 
X 
B 
Toddlers 
30 µg 
12 
X 
 
 
Placebo 
3 
X 
 
 
90 µg 
12 
X 
 
 
Placebo 
3 
X 
 
 
C 
Infants 
15 µg 
12 
X 
X 
X 
Placebo 
4 
X 
X 
X 
30 µg 
12 
X 
X 
X 
Placebo 
4 
X 
X 
X 
90 µg 
12 
X 
X 
X 
Placebo 
4 
X 
X 
X 
D 
Infants 
15 µg* 
138 
X 
X 
X 
30 µg* 
138 
X 
X 
X 
90 µg* 
138 
X 
X 
X 
Placebo 
138 
X 
X 
X 
X = Injection day 
*If dose tolerated in Group C 
 
3.5.1. Dose Preparation and Administration 
Dose preparation, as described in detail in the Pharmacy Preparation Manual, will be carried out 
by a qualified unblinded research pharmacist and witnessed by another un-blinded study staff 
member. Investigational product will be dispensed in a masked syringe with administration 
needle, labeled with participant number, and will be administered by blinded clinic staff.  
Investigational product is prepared at the research pharmacy at each of the trial sites. The 
prepared vaccine will be stored in the research pharmacy refrigerator (2°C – 8°C) for up to 6 hours 
after preparation prior to administration. 
The study product will be administered intramuscularly: to the deltoid in adults and to the 
anterolateral thigh muscle in toddlers and infants.  
3.5.2. Accountability and Disposal 
The site pharmacist is required to maintain complete records of all study products received from 
the Sponsor and will be responsible for maintaining an accurate record of the randomization 
codes, inventory, and an accountability record of vaccine and placebo supplies for this study. The 
site pharmacist will also be responsible for ensuring the security of these documents. The site will 
receive instruction from the IND Sponsor regarding the final disposition of any remaining study 
products. Partially used vials will not be used for human administration or for in vitro experimental 
studies. All unused study products must be disposed of according to the Sponsor’s instructions. 

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4. STUDY POPULATION 
4.1. Clinical Trial Sites 
This study is a multisite clinical trial, to be performed in South Africa at the Family Clinical 
Research Unit (FAM-CRU) at Tygerberg Children’s Hospital, the Respiratory and Meningeal 
Pathogens Research Unit (RMPRU) in Soweto, and the Shandukani Research Centre in Hillbrow, 
Johannesburg. The study population will be recruited, screened and qualified by site staff under 
the direction of the PIs at each site. 
4.2. Study Population 
The study population will consist of healthy adults, toddlers and infants from the communities in 
the vicinity of the study clinics. Adult participants and parents of toddler and infant participants will 
be fully informed about the study, after which they will provide consent for participation. 
4.3. Eligibility 
Participants will be in general good health: in the adult group, participants will be >18 and <45 
years of age; in the toddler cohort, participants will be >2 and <3 years of age; and in the infant 
cohorts will be >6 and <8 weeks old, at the time of enrollment. Potential volunteer families of 
infants may be contacted from before the babies are born through the noted targeted age. Final 
eligibility determination will depend on the results of the medical history, clinical examination, 
screening laboratory tests and fulfillment of all the inclusion and absence of any of the exclusion 
criteria, and appropriate understanding of the study and completion of the consent process by 
adult participants and parents of toddler and infant participants. 
Investigators should always use good clinical judgment in considering a participant’s overall 
fitness for inclusion in the trial. Some participants may not be appropriate for the study, even if 
they meet all inclusion/exclusion criteria. For instance, medical, occupational or other conditions 
present in the parents may make safety evaluations difficult or make toddlers and infants poor 
candidates for retention. All toddlers and infants targeted for enrollment will need to have parents 
that can comprehend the purpose of the study and provide written informed consent. In addition, 
the adult participants and families of toddler and infant participants should be resident in the area 
without plans to leave the study site during the course of the study. 
A sufficient number of healthy adults, toddlers and infants will be screened, with consent, to enroll 
30 adults, 30 toddlers and 600 infants in the study.  
4.4. Inclusion Criteria  
Fulfillment of all of the following criteria is required to accept an adult, toddler or infant in the study: 
1. Healthy adults, toddlers and infants as established by medical history and clinical 
examination before entering the study 
2. Age:  
a. Adults: ≥18 and ≤45 years old at time of enrollment 
b. Toddlers: ≥2 and <3 years old at the time of enrollment 
c. Infants: ≥6 and <8 weeks at the time of enrollment 
3. Participant (adults) or parental (toddlers and infants) ability and willingness to provide 
written informed consent 
4. Intention for the participant (and for toddlers and infants, the parent) to remain in the area 
with the child during the study period 
5. If female and of childbearing potential, be not breastfeeding and not pregnant (based on 
a negative serum pregnancy test at screening and a negative urine pregnancy test at each 

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study injection visit, prior to injection), planning to avoid pregnancy for at least 4 weeks 
after the last injection, and willing to use an adequate method of contraception consistently 
and have repeated pregnancy tests prior to second and third injections. 
4.5. Exclusion Criteria 
Any of the following will exclude an adult, toddler or infant from the study:  
1. Presence of fever on the day of enrollment (axillary or oral temperature >37.6oC) 
2. Acute disease at the time of enrollment 
3. Concurrent participation in another clinical trial throughout the entire timeframe for this 
study 
4. Presence of malnutrition or any systemic disorder (cardiovascular, pulmonary, hepatic, 
renal, gastrointestinal, hematological, endocrine, immunological, dermatological, 
neurological, cancer or autoimmune disease) as determined by medical history and/or 
physical examination that would compromise the participant’s health or is likely to result 
in nonconformance to the protocol 
5. For infant cohort, history of premature birth (<37 weeks gestation) 
6. History of congenital abdominal disorders, intussusception, or abdominal surgery 
7. Known or suspected impairment of immunological function based on medical history and 
physical examination 
8. For infant cohort only, prior receipt of rotavirus vaccine 
9. A known sensitivity or allergy to any components of the study vaccine 
10. History of anaphylactic reaction 
11. Major congenital or genetic defect 
12. Parents of participating toddlers or infants not able, available or willing to accept active 
weekly follow-up by the study staff 
13. Receipt of any immunoglobulin therapy and/or blood products in the last 6 months or 
planned administration during the study period 
14. History of chronic administration (defined as more than 14 days) of immunosuppressant 
medications, including corticosteroids, in the last 6 months (those on inhaled or topical 
steroids may be permitted to participate in the study) 
15. Any medical condition in the participant (or parents of toddler and infant participants) that, 
in the judgment of the investigator, would interfere with or serves as a contraindication to 
protocol adherence or a participant’s (or parents’) ability to give informed consent 
16. Clinically significant screening laboratory value* 
17. HIV infection 
a. For adults and toddlers, to be assessed by HIV ELISA 
b. For infants, to be assessed by PCR, if mother is not known to be negative (negative 
test result between 24 weeks gestation and screening)  
*Grade 1 laboratory abnormalities (see toxicity table in Appendix I) will not be considered to be 
exclusionary at screening unless judged to be clinically significant by the PI. Potential participants 

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with laboratory values of grade 2 or higher are not to be randomized without concurrence of the 
Emmes medical monitor and PATH medical officer. 
After informed consent has been obtained and the potential participant is identified as meeting 
inclusion and exclusion criteria for enrollment, the (s)he will be enrolled in the study and assigned 
a randomization number.  
5. STUDY PROCEDURES 
5.1. Recruitment  
Adults will be invited for screening by means of advertisements in the local community. All 
advertisements will be approved the Ethics Committee before implementation. 
Toddlers will be identified from hospital birth registers. The parent will be contacted and invited to 
bring the child to RMPRU for screening. 
Infants will be identified in the postnatal wards at Chris Hani Baragwanath Hospital after delivery, 
and the mother will be invited to bring her infant to the RMPRU prior to the first clinic visit for 
screening and potential enrolment. 
At FAMCRU, in Tygerberg, pregnant women will be informed of the study at the Kraaifontein 
Midwife obstetric unit’s antenatal clinic. Those interested will then be followed up for delivery at 
the midwife obstetric unit. Potential participants will also be identified at the Tygerberg hospital 
antenatal clinic and followed up at the labour ward of Tygerberg hospital with delivery of the infant.  
At the Shandukani Research Centre, pregnant women at antenatal clinics and mothers of infants 
attending the Day 3 visit will be approached. Recruitment activity will also occur at surrounding 
City of Johannesburg Region F clinics. The study will be discussed with the parents, and, if they 
agree, permission will be obtained to collect their names and contact details. Closer to the 
potential enrollment date, parents will be contacted and invited to visit the Shandukani Research 
Centre. At that time interested parents will be also asked not to give their infants the 6 week 
Rotarix vaccine. Recruitment activity will also occur at the 6 week EPI vaccination visit. The site 
will develop a study-specific pamphlet for parents considering participation in the study.  
5.2. Screening, Randomization, and Masking Procedures (for all age-groups) 
5.2.1. Initial and Continuing Informed Consent  
Informed consent is the process of ensuring that study participants, or, in the case of toddlers and 
infants, their parents, fully understand the purpose of the study and what will and may happen to 
them/their children while participating in a research study. Initial written informed consent is 
required before performance of any study-related procedures. The informed consent process 
continues throughout the study. Key study concepts are reviewed with the study 
participants/participants’ parents at designated times and as needed, and the review is 
documented. Additionally, if any new information becomes available that, in the judgment of PVS 
and/or PIs, may affect the participants’/parents’ decision to continue in the trial, such information 
will be shared with the participants/parents, and they may be asked to sign a new consent form. 
5.2.2. Screening – Day -7 (infants) or Day -28 (adults and toddlers) to Day -1 
After the site PI or designee has obtained informed consent from participants/parents, the 
following procedures will be completed during screening to determine study eligibility and may 
occur over multiple screening visits. Additional screening visits may be scheduled for any follow 
up as needed, but are not required. At the screening visit(s), the site PI or designee will provide 
prospective participants (adults) and parents of prospective participants (toddlers and infants) a 

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detailed description of the study objectives and study participation requirements, as well as 
potential health risks and benefits associated with study participation. Baseline data are obtained 
during screening, which may occur over the course of several contacts/visits, between 7 days 
prior to and on Day 0, the day of first injection for infants and from 28 days prior to Day 0 for 
toddlers and adults. All inclusion/exclusion criteria must be assessed from data obtained within 
that period, unless otherwise specified in the eligibility criteria. After study information has been 
provided and the appropriate informed consent has been obtained, the following procedures are 
performed before enrollment: 
a) Confirm written informed consent has been obtained and solicit/discuss any remaining 
questions the participant/parent may have; 
b) Assign participant ID once study specific consent form has been signed; 
c) Obtain demographic and contact (e.g., address, telephone, email) information; 
d) Obtain medical history; 
e) Obtain vaccination history for toddlers and infants (for adults, vaccination history for past 
30 days); 
f) Obtain history of medication use in the past 30 days; 
g) Measure height/length & weight; 
h) Perform full physical exam (including vital signs); and 
i) Collect blood samples for screening laboratory testing: complete blood count (CBC) 
(including WBC, hemoglobin, platelet count), alanine transaminase (ALT), total bilirubin, 
albumin (obtained only at screening) and creatinine (Appendix I).* Serum pregnancy test 
will be performed on women of childbearing potential. 
a. For adults toddlers, HIV ELISA 
b. For infants, HIV PCR (unless mother has negative test result between 24 weeks 
gestation and screening)  
j) Collect blood samples for immunological testing.  
* Grade 1 laboratory abnormalities will not be considered to be exclusionary at screening, unless 
judged to be clinically significant by the site PI. 
5.2.3. Randomization  
Enrollment will be performed online using the enrollment module of AdvantageEDCSM, the Emmes 
Corporation’s electronic data capture system. Randomization will occur on the day participants 
are to receive their first study injection, after confirmation of eligibility and immediately prior to 
injection.  
Participants will be assigned to a coded treatment assignment after demographic and eligibility 
data have been entered into the system. The unblinded research pharmacist will be provided with 
the treatment assignment codes for preparation of the vaccine or placebo to be given to each 
participant. The unblinded research pharmacist will maintain the treatment code list in a secure 
place. 
5.2.4. Masking procedures 
The research pharmacist will not reveal the randomization code to any other study staff member, 
participant or parent. Investigational study product will be prepared by a qualified unblinded 
research pharmacist and witnessed by another unblinded study staff member. Investigational 

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product will be dispensed in a masked syringe (opaque sheath over the syringe barrel) with 
administration needle.  
5.2.5. Group A (adults) 
5.2.5.1. 
Day 0 – First Dose 
Prior to Study Injection 
a) Provisionally eligible participants are scheduled to arrive at the study clinic on the morning 
of Day 0;  
b) Interval medical history is obtained, including medications; 
c) Vital signs are measured prior to dosing; 
d) A full physical exam is performed; 
e) New health information is reviewed to determine qualification for enrollment; 
f) Inclusion/Exclusion criteria are reviewed to assess continued eligibility; 
g) Urine pregnancy test is performed for women of childbearing potential. 
h) The prospective injection site (deltoid) is examined visually and by palpation, and the 
axillary lymph nodes are examined. When possible, the first and third injections are to be 
administered to the deltoid of the non-dominant arm and the second injection to the 
dominant arm; and 
i) Participants are randomized. 
Study Injection 
a) If a qualified participant selected for enrollment is disqualified prior to randomization and 
dose administration (e.g., participant withdraws consent or the site PI reconsiders and 
disqualifies for a documented reason), another qualified participant will be selected for 
randomization in place of the disqualified participant.  
b) The injection site will be observed prior to dose administration (as above). 
c) The prospective injection site will be cleaned with an alcohol swab and allowed to dry 
completely. The study injection will be administered into the deltoid by inserting the needle 
(25 gauge, 25 mm) into muscle at 90°, holding the muscle mass stable with the non-
injecting hand, drawing back to ensure that the needle is not in a blood vessel, and slowly 
depressing the plunger. The needle is removed and the injection site gently rubbed with 
cotton wool. 
Post Dose – Observation Period 
a) Participants will remain at the site for at least 30 minutes post-injection.  
b) After at least 30 minutes: Vital signs and the injection site will be assessed, redness and/or 
induration will be measured and recorded. Any complaints or reactogenicity signs or 
symptoms will be assessed and documented. 
c) The site PI (or designee) may determine that a participant requires further on-site 
observation; additional site or clinical assessments may be completed as needed.  
d) When all study related procedures are complete and the site PI (or designee) determines 
that a participant’s condition is acceptable, the participant will be discharged from the 
study clinic.  

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e) Before discharge, the participant will be provided with supplies (including a thermometer, 
a transparent plastic tool to assess size of injection site redness and swelling, and a 
Memory Aid); instructed in their use; and provided with written materials, follow-up visit 
information and contact telephone numbers for study staff. Participants are to be 
instructed to contact study staff for any reaction greater than mild. 
5.2.5.2. 
Day 7, 35 and 63 ± 1 day (to be defined by 7 days after most recent study 
injection day) – Clinic visit follow-up 
On the 7th day after each injection (±1 day), the participant will return to the study clinic for the 
following procedures: 
a) Study staff will review and record the participant’s interval health history, medication use 
and the participant’s assessment of the post-injection experience through personal 
interview, assisted by the Memory Aid (See Appendix III). 
b) Vital sign measurement, injection site and draining lymph node assessment and, if 
indicated, a symptom-directed physical examination will be performed. 
c) Local and systemic reaction and AE review will be performed by the site PI (or designee). 
The highest oral temperature written in the Memory Aid each day will be recorded in 
source document as peak temperature. 
d) Findings of site PI or study staff that suggest inaccuracy of reported self-assessments will 
be clearly documented. 
e) Any topics or new information considered by site PI to be important to continued informed 
consent will be shared with the participant. 
f) Blood samples will be obtained for safety monitoring laboratory tests on Day 7 
(Appendix I). 
g) Study staff will instruct the participant regarding continued health assessment (without a 
Memory Aid) and need to contact study staff (a) in follow-up of specific AEs, (b) if injection 
site changes worsen or do not resolve, (c) if systemic symptoms worsen or do not resolve, 
and (d) if other AEs occur.  
h) The next visit is scheduled. 
5.2.5.3. 
Day 28 (-2 to +7 days) and Day 56 (-2 to +7 days) days (to be defined by 28 days 
after the previous study injection) – Study injection days 
Every effort should be made to adhere to the protocol injection schedule windows. However, 
injections may be given outside the specified window on a case-by-case basis, if approved by the 
Sponsor, provided that the injection will not have any adverse effect on the participant and that it 
is in the best interest of the scientific soundness of the research plan. The appropriate follow-up 
and subsequent injection schedule for each participant with delayed injections will be determined 
by the Sponsor. 
Prior to Dose 
The participant returns to the study clinic and the following procedures will occur. 
a) Review of interval medical history, including AEs and medication use 
b) Vital signs measurement prior to dosing 
c) Review of inclusion/exclusion criteria to assure continued eligibility 

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d) Urine pregnancy test for women of childbearing potential 
e) A targeted physical exam, if indicated 
f) Examination of the prior injection site and corresponding draining lymph nodes 
g) Review of safety laboratory test results from Day 7 at the Day 28 visit before vaccination 
(and discussed with participants assuming there has not been occasion to do so earlier). 
h) Blood samples will be obtained for immunological assays (see Appendix I). 
i) Examination (visually and by palpation) of the prospective injection site (deltoid), and 
axillary lymph nodes  
Study Injection 
a) The injection site is observed before dose administration. 
b) The prospective injection site will be cleaned with an alcohol swab and allowed to dry 
completely. The study injection will be administered into the deltoid by inserting the needle 
(25 gauge, 25 mm) into muscle at 90°, holding the muscle mass stable with the non-
injecting hand, drawing back to ensure that the needle is not in a blood vessel, and slowly 
depressing plunger. The needle is removed and the injection site gently rubbed with cotton 
wool. 
Post Dose – Observation Period 
a) Participants will remain at the site for at least 30 minutes post-injection.  
b) After at least 30 minutes: Vital signs and the injection site will be assessed, redness and/or 
indurations will be measured and recorded. Any complaints or reactogenicity signs or 
symptoms will be assessed and documented. 
c) The site PI (or designee) may determine that a participant requires further on-site 
observation; additional site or clinical assessments may be completed as needed.  
d) When all study related procedures are complete and the site PI (or designee) determines 
that a participant’s condition is acceptable, the participant will be discharged from the 
study clinic. 
e) Before discharge, the participant will be provided with supplies including a thermometer, 
ruler for measuring injection site redness and swelling, and a Memory Aid; instructed in 
their use; and provided with written materials, follow-up visit information and contact 
telephone numbers for study staff. Participants are to be instructed to contact study staff 
for any reaction greater than mild. 
5.2.5.4. 
Day 84 ± 4 days (to be defined by 28 days post-3rd study injection day) – Clinic 
follow-up visit and immunogenicity sampling 
On Day 84 (plus or minus 4 calendar days if necessary for participant compliance), the participant 
will return to the study clinic and the following procedures will occur:  
a) Interval medical history will be obtained, including medication use. 
b) Vital sign measurement, injection site and draining lymph node assessment and, if 
indicated, a symptom-directed physical examination will be performed. 
c) AEs will be evaluated. 
d) Blood samples will be obtained for immunological assays (see Appendix I). 

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e) The participant will be reminded to contact the study clinic with any new information about 
chronic illnesses, serious health events, and/or hospitalizations, and the Day 224 follow-
up will be scheduled.  
5.2.5.5. 
Day 224 ± 14 days (to be defined by 168 days post-3rd study injection day) – 
Final participant contact (telephone or clinic visit) 
On Day 224 (plus or minus 14 calendar days if necessary for participant compliance), the 
participant will be contacted by telephone or return to the study clinic for a final follow-up visit and 
the following procedures occur:  
a) Interval medical history will be obtained for SAEs and other major or new chronic 
medical conditions. 
b) A targeted physical exam will be performed, if indicated (request to come to clinic for 
those being contacted by telephone). 
5.2.6. Group B (toddlers) 
5.2.6.1. 
Day 0 – Study Injection Day 
Prior to Study Injection 
a) Provisionally eligible participants are scheduled to arrive at the study clinic on the morning 
of Day 0;  
b) Interval medical history is obtained and inclusion/exclusion criteria reviewed to determine 
continuous qualification for enrollment, including medications; 
c) Vital signs are measured prior to dosing; 
d) A full physical exam is performed; 
e) The prospective injection site (anterolateral thigh) is examined visually and by palpation, 
and the axillary lymph nodes are examined; and 
f) Participants are randomized. 
Study Injection 
a) If a qualified participant selected for enrollment is disqualified prior to randomization and 
dose administration (e.g., parent withdraws consent or the site PI reconsiders and 
disqualifies for a documented reason), another qualified participant will be selected for 
randomization in place of the disqualified participant.  
b) The injection site will be observed prior to dose administration (as above). 
c) The prospective injection site will be cleaned with an alcohol swab and allowed to dry 
completely. The study injection will be administered into the anterolateral thigh by inserting 
the needle (25 gauge, 25 mm) into muscle at 90°, holding the muscle mass stable with 
the non-injecting hand, drawing back to ensure that the needle is not in a blood vessel, 
and slowly depressing plunger. The needle is removed and the injection site gently rubbed 
with cotton wool.  
Post Dose – Observation Period 
a) Participants will remain at the site for at least 30 minutes post-injection.  

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b) After at least 30 minutes: Vital signs and the injection site will be assessed, redness and/or 
induration will be measured and recorded. Any complaints or reactogenicity signs or 
symptoms will be assessed and documented. 
c) The site PI (or designee) may determine that a participant requires further on-site 
observation; additional site or clinical assessments may be completed as needed.  
d) When all study related procedures are complete and the site PI (or designee) determines 
that a participant’s condition is acceptable, the participant will be discharged from the 
study clinic.  
e) Before discharge, the participant’s parents will be provided with supplies (including a 
thermometer, a transparent plastic tool to assess size of injection site redness and 
swelling, and a Memory Aid); instructed in their use; and provided with written materials, 
follow-up visit information and contact telephone numbers for study staff. Participants’ 
parents are to be instructed to contact study staff for any reaction greater than mild. 
Participants are to be assessed in clinic for any local site reaction greater than mild. 
5.2.6.2. 
Day 3 (to be defined by 3 days after study injection day) – Clinic follow-up visit 
On the third day after the study injection, the participants will return to the study clinic for the 
following procedures: 
a) Study staff will review and record the participant’s interval health history, medication use 
and the parent’s assessment of the post-injection experience through personal interview, 
assisted by the Memory Aid (See Appendix III). 
b) Vital sign measurement, injection site and draining lymph node assessment and, if 
indicated, a symptom-directed physical examination will be performed. 
c) Local and systemic reaction and AE review will be performed by the site PI (or designee). 
The highest axillary temperature written in the Memory Aid each day will be recorded in 
source document as peak temperature. 
d) Findings of site PI or study staff that suggest inaccuracy of reported parental assessments 
will be clearly documented. 
e) Any topics or new information considered by the site PI to be important to continued 
informed consent will be shared with the parent. 
f) Study staff will instruct the parent regarding continued assessment of the child’s health 
(and use of Memory Aid) and need to contact study staff (a) in follow-up of specific AEs, 
(b) if any injection site changes worsen or do not resolve, (c) if systemic symptoms worsen 
or do not resolve, and (d) if other AEs occur.  
g) The next visit for Day 7 is scheduled. 
5.2.6.3. 
Day 7 ± 1 day (to be defined by 7 days after study injection day) – Clinic follow-
up visit 
On the 7th day after the study injection (±1 day), the participant will return to the study clinic for 
the following procedures: 
a) Study staff will review and record the participant’s interval health history, medication use 
and the parent’s assessment of the post-injection experience through personal interview, 
assisted by the Memory Aid (See Appendix III). 
b) Vital sign measurement, injection site and draining lymph node assessment and, if 
indicated, a symptom-directed physical examination will be performed. 

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c) Local and systemic reaction and AE review will be performed by the site PI (or designee). 
The highest axillary temperature written in the Memory Aid each day will be recorded in 
source document as peak temperature. 
d) Findings of site PI or study staff that suggest inaccuracy of reported self-assessments will 
be clearly documented. 
e) Any topics or new information considered by site PI to be important to continued informed 
consent will be shared with the parent. 
f) Blood samples will be obtained for safety monitoring laboratory tests on Day 7 
(Appendix I). 
g) Study staff will instruct the parent regarding continued assessment of the child’s health 
(without a Memory Aid) and need to contact study staff (a) in follow-up of specific AEs, (b) 
if injection site changes worsen or do not resolve, (c) if systemic symptoms worsen or do 
not resolve, and (d) if other AEs occur.  
h) The next visit is scheduled. 
5.2.6.4. 
Day 28 ± 4 days (to be defined by 28 days after study injection day) – Clinic 
follow-up 
On Day 28 (plus or minus 4 calendar days if necessary for participant compliance), the participant 
will return to the study clinic and the following procedures occur:  
a) Interval medical history will be obtained, including medication use. 
b) Vital sign measurement, injection site and draining lymph node assessment and, if 
indicated, a symptom-directed physical examination will be performed. 
c) AEs will be evaluated and recorded. 
d) Blood samples will be obtained for immunological assays (see Appendix I). 
e) The participant’s parent will be reminded to contact the study clinic with any new 
information about chronic illnesses, serious health events, and/or hospitalizations, and the 
Day 168 follow-up will be scheduled.  
5.2.6.5. 
Day 168 ± 14 days (to be defined by 168 days post-injection day) – Final 
participant contact (telephone or clinic visit) 
On Day 168 (plus or minus 14 calendar days if necessary for participant compliance), the 
participant’s parents will be contacted by telephone or return to the study clinic with the participant 
for a final follow-up visit and the following procedures will occur:  
a) Interval medical history will be obtained for SAEs and other major or new chronic 
medical conditions. 
b) A targeted physical exam will be performed, if indicated (request to come to clinic for those 
being contacted by telephone). 
5.2.7. Groups C and D (infants) 
5.2.7.1. 
Day 0 – First Dose 
Prior to Study Injection 
a) Provisionally eligible participants are scheduled to arrive at the study clinic on the morning 
of Day 0;  
b) Interval medical history is obtained, including medications; 

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c) Vital signs are measured prior to dosing; 
d) A full physical exam is performed; 
e) New health information is reviewed to determine qualification for enrollment; 
f) Inclusion/Exclusion criteria are reviewed to assess continued eligibility; 
g) The prospective injection site (anterolateral thigh) is examined visually and by palpation, 
and the inguinal lymph nodes are examined. When possible, the first and third injections 
are to be administered to the left anterolateral thigh and the second injection to the right 
anterolateral thigh; and 
h) Participants are randomized. 
Study Injection 
a) If a qualified participant selected for enrollment is disqualified prior to randomization and 
dose administration (e.g., parent withdraws consent or the site PI reconsiders and 
disqualifies for a documented reason), another qualified participant will be selected for 
randomization in place of the disqualified participant.  
b) The injection site will be observed prior to dose administration (as above). 
c) The prospective injection site will be cleaned with an alcohol swab and allowed to dry 
completely. The study injection will be administered into the outer aspect of the 
anterolateral thigh by inserting needle (25 gauge, 25 mm) into muscle at 90°, holding the 
muscle mass stable with the non-injecting hand, drawing back to ensure that the needle 
is not in a blood vessel, and slowly depressing plunger. The needle is removed and the 
injection site gently rubbed with cotton wool. 
Post Dose – Observation Period 
a) Participants will remain at the site for at least 30 minutes post-injection.  
b) After at least 30 minutes: Vital signs and the injection site will be assessed, redness and/or 
induration will be measured and recorded. Any complaints or reactogenicity signs or 
symptoms will be assessed and documented. 
c) The site PI (or designee) may determine that a participant requires further on-site 
observation; additional site or clinical assessments may be completed as needed.  
d) When all study related procedures are complete and the site PI (or designee) determines 
that a participant’s condition is acceptable, the participant will be discharged from the 
study clinic.  
e) Before discharge, the participant’s parents will be provided with supplies (including a 
thermometer, a transparent plastic tool to assess size of injection site redness and 
swelling, and a Memory Aid); instructed in their use; and provided with written materials, 
follow-up visit information and contact telephone numbers for study staff. Participants’ 
parents are to be instructed to contact study staff for any reaction greater than mild. 
Participants are to be assessed in clinic for any local site reaction greater than mild. 
5.2.7.2. 
Day 3, 31 and 59 (to be defined by 3 days after most recent study injection day) 
– Clinic visit follow-up 
On the third day after each study injection, the participants will return to the study clinic for the 
following procedures: 

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a) Study staff will review and record the participant’s interval health history, medication use 
and the parent’s assessment of the post-injection experience through personal interview, 
assisted by the Memory Aid (See Appendix III). 
b) Vital sign measurement, injection site and draining lymph node assessment and, if 
indicated, a symptom-directed physical examination will be performed. 
c) Local and systemic reaction and AE review will be performed by the site PI (or designee). 
The highest axillary temperature written in the Memory Aid each day will be recorded in 
source document as peak temperature. 
d) Findings of site PI or study staff that suggest inaccuracy of reported parental assessments 
will be clearly documented. 
e) Any topics or new information considered by the site PI to be important to continued 
informed consent will be shared with the parent. 
f) Study staff will instruct the parent regarding continued assessment of the child’s health 
(and use of Memory Aid) and need to contact study staff (a) in follow-up of specific AEs, 
(b) if any injection site changes worsen or do not resolve, (c) if systemic symptoms worsen 
or do not resolve, and (d) if other AEs occur.  
g) The next visit is scheduled. 
5.2.7.3. 
Day 7, 35 and 63 ± 1 day (to be defined by 7 days after most recent study 
injection day) – Clinic visit follow-up 
On the 7th day after each study injection (±1 day), the participant will return to the study clinic for 
the following procedures: 
a) Study staff will review and record the participant’s interval health history, medication use 
and the parent’s assessment of the post-injection experience through personal interview, 
assisted by the Memory Aid (See Appendix III). 
b) Vital sign measurement, injection site and draining lymph node assessment and, if 
indicated, a symptom-directed physical examination will be performed. 
c) Local and systemic reaction and AE review will be performed by the site PI (or designee). 
The highest axillary temperature written in the Memory Aid each day will be recorded in 
source document as peak temperature. 
d) Findings of site PI or study staff that suggest inaccuracy of reported self-assessments will 
be clearly documented. 
e) Any topics or new information considered by site PI to be important to continued informed 
consent will be shared with the parent. 
f) Blood samples will be obtained for safety monitoring laboratory tests on Day 7 for 
participants in Group C only (Appendix I). 
g) Study staff will instruct the parent regarding continued assessment of the child’s health 
(without a Memory Aid) and need to contact study staff (a) in follow-up of specific AEs, (b) 
if injection site changes worsen or do not resolve, (c) if systemic symptoms worsen or do 
not resolve, and (d) if other AEs occur.  
h) The next visit is scheduled. 

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5.2.7.4. 
Day 28 (-2 to +7 days) (to be defined by 28 days after the previous study 
injection) – Study injection day 
Every effort should be made to adhere to the protocol injection schedule windows. However, study 
injections may be given outside the specified window on a case-by-case basis, if approved by the 
Sponsor, provided that the injection will not have any adverse effect on the participant and that it 
is in the best interest of the scientific soundness of the research plan. The appropriate follow-up 
and subsequent injection schedule for each participant with delayed injections will be determined 
by the Sponsor. 
Prior to Dose 
The participant returns to the study clinic and the following procedures will occur. 
a) Review of interval medical history, including AEs and medication use. 
b) Vital signs measurement prior to dosing. 
c) Review of inclusion/exclusion criteria to assure continued eligibility. 
d) A full physical exam 
e) Examination of the prior injection site and corresponding draining lymph nodes 
f) Review of safety laboratory test results from Day 7 at the Day 28 visit before vaccination 
(and discussed with parents assuming there has not been occasion to do so earlier). 
g) Examination (visually and by palpation) of the prospective injection site (anterolateral 
thigh), and inguinal lymph nodes  
Study Injection 
a) The injection site is observed before dose administration. 
b) The prospective injection site will be cleaned with an alcohol swab and allowed to dry 
completely. The study injection will be administered into the outer aspect of the 
anterolateral thigh by inserting the needle (25 gauge, 25 mm) into muscle at 90°, holding 
the muscle mass stable with the non-injecting hand, drawing back to ensure that the 
needle is not in a blood vessel, and slowly depressing plunger. The needle is removed 
and the injection site gently rubbed with cotton wool. 
Post Dose – Observation Period 
a) Participants will remain at the site for at least 30 minutes post-injection.  
b) After at least 30 minutes: Vital signs and the injection site will be assessed, redness and/or 
indurations will be measured and recorded. Any complaints or reactogenicity signs or 
symptoms will be assessed and documented. 
c) The site PI (or designee) may determine that a participant requires further on-site 
observation; additional site or clinical assessments may be completed as needed.  
d) When all study related procedures are complete and the site PI (or designee) determines 
that a participant’s condition is acceptable, the participant will be discharged from the 
study clinic. 
e) Before discharge, the participant’s parents will be provided with supplies including a 
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instructed in their use; and provided with written materials, follow-up visit information and 
contact telephone numbers for study staff. Participants’ parents are to be instructed to 
contact study staff for any reaction greater than mild. Participants are to be assessed in 
clinic for any local site reaction greater than mild. 
5.2.7.5. 
Day 56 (-2 to +7 days) (to be defined by 28 days after the previous study 
injection) – Study injection day 
Every effort should be made to adhere to the protocol injection schedule windows. However, study 
injections may be given outside the specified window on a case-by-case basis, if approved by the 
Sponsor, provided that the injection will not have any adverse effect on the participant and that it 
is in the best interest of the scientific soundness of the research plan. The appropriate follow-up 
and subsequent injection schedule for each participant with delayed injections will be determined 
by the Sponsor. 
Prior to Dose 
The participant returns to the study clinic and the following procedures will occur. 
a) Review of interval medical history, including AEs and medication use. 
b) Vital signs measurement prior to dosing. 
c) Review of inclusion/exclusion criteria to assure continued eligibility. 
d) A full physical exam 
e) Blood samples will be obtained for immunological assays (see Appendix I). 
f) Examination of the prior injection site and corresponding draining lymph nodes 
g) Review of safety laboratory test results from Day 7 at the Day 28 visit before vaccination 
(and discussed with parents assuming there has not been occasion to do so earlier). 
h) Examination (visually and by palpation) of the prospective injection site (anterolateral 
thigh), and inguinal lymph nodes  
Study Injection 
a) The injection site is observed before dose administration. 
b) The prospective injection site will be cleaned with an alcohol swab and allowed to dry 
completely. The study injection will be administered into the outer aspect of the 
anterolateral thigh by inserting the needle (25 gauge, 25 mm) into muscle at 90°, holding 
the muscle mass stable with the non-injecting hand, drawing back to ensure that the 
needle is not in a blood vessel, and slowly depressing plunger. The needle is removed 
and the injection site gently rubbed with cotton wool. 
Post Dose – Observation Period 
a) Participants will remain at the site for at least 30 minutes post-injection.  
b) After at least 30 minutes: Vital signs and the injection site will be assessed, redness and/or 
indurations will be measured and recorded. Any complaints or reactogenicity signs or 
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c) The site PI (or designee) may determine that a participant requires further on-site 
observation; additional site or clinical assessments may be completed as needed.  
d) When all study related procedures are complete and the site PI (or designee) determines 
that a participant’s condition is acceptable, the participant will be discharged from the 
study clinic. 
e) Before discharge, the participant’s parents will be provided with supplies including a 
thermometer, ruler for measuring injection site redness and swelling, and a Memory Aid; 
instructed in their use; and provided with written materials, follow-up visit information and 
contact telephone numbers for study staff. Participants’ parents are to be instructed to 
contact study staff for any reaction greater than mild. Participants are to be assessed in 
clinic for any local site reaction greater than mild. 
5.2.7.6. 
Day 84 ± 4 days (to be defined by 28 days post-3rd Study injection day) – Clinic 
follow-up visit, immunogenicity sampling and first dose of Rotarix 
On Day 84 (plus or minus 4 calendar days if necessary for participant compliance), the participant 
will return to study clinic and the following procedures occur:  
a) Interval medical history will be obtained, including medication use. 
b) Vital sign measurement, injection site and draining lymph node assessment and, if 
indicated, a symptom-directed physical examination will be performed. 
c) AEs will be evaluated. 
d) Blood samples will be obtained for immunological assays (see Appendix I). 
e) The first dose of Rotarix will be administered. 
f) Parents will be provided an empty container to obtain stool sample on the morning of Day 
89 (RMPRU only). 
g) The participant’s parent will be reminded to contact the study clinic with any new 
information about chronic illnesses, serious health events, and/or hospitalizations.  
h) The next clinic visit is scheduled. 
5.2.7.7. 
Day 89 – Clinic visit for stool sampling (RMPRU only) 
The parent will return to the clinic with a stool sample obtained that morning. If the parent is unable 
to bring the specimen to the clinic, a clinic staff member will visit the home to collect the sample. 
If the infant does not pass stool during the morning, a specimen will be obtained by rectal swab. 
Parents will be provided an empty container to obtain stool sample on the morning of Day 91. 
5.2.7.8. 
Day 91 – Clinic visit for stool sampling (RMPRU only) 
The parent will return to the clinic with a stool sample obtained that morning. If the parent is unable 
to bring the specimen to the clinic, a clinic staff member will visit the home to collect the sample. 
If the infant does not pass stool during the morning, a specimen will be obtained by rectal swab. 
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5.2.7.9. 
Day 93 – Clinic visit for stool sampling (RMPRU only) 
The parent will return to the clinic with a stool sample obtained that morning. If the parent is unable 
to bring the specimen to the clinic, a clinic staff member will visit the home to collect the sample. 
If the infant does not pass stool during the morning, a specimen will be obtained by rectal swab. 
The next clinic visit is scheduled. 
5.2.7.10. Day 112 and 140 + 14 days (to be defined by 28 days after the previous Rotarix 
vaccination) – Clinic visit for second and third dose of Rotarix 
On Day 112 and Day 140 (up to plus 14 calendar days), the participant will return for the second 
and third doses of Rotarix, and the following procedures will occur: 
a) Interval medical history will be obtained, including medication use. 
b) Vital signs will be measured. 
c) AEs will be evaluated and recorded. 
d) A targeted physical exam will be performed, if indicated. 
The participant’s parent will be reminded to contact the study clinic with any new information 
about chronic illnesses, serious health events, and/or hospitalizations, and the Day 224 follow-
up will be scheduled. 
5.2.7.11. Day 224 ± 14 days (to be defined by 168 days post-3rd injection day) – Final 
contact (telephone or clinic visit) 
On Day 224 (plus or minus 14 calendar days if necessary for participant compliance), the 
participant’s parents will be contacted by telephone or return to study clinic with the participant for 
a final follow-up visit and the following procedures will occur:  
a) Interval medical history will be obtained for SAEs and other major or new chronic 
medical conditions. 
b) A targeted physical exam will be performed, if indicated (request to come to clinic for 
those being contacted by telephone). 
5.3. Interim Contacts and Unscheduled Visits  
Interim contacts and visits (those between regularly scheduled follow up visits) may be performed 
at participants’/parents’ request, or as deemed necessary by the site PI or designee for diagnosis 
and/or management of a finding or AE, at any time during the study. All interim contacts and visits 
will be documented in participants’ study records and on applicable case report forms. 
5.4. Stool Samples from Infants with Diarrhea  
Parents of infants are to be encouraged to contact study staff if infants develop diarrhea. Should 
diarrhea be reported (defined as ≥3 looser than normal stools in a 24 hour period), a single stool 
sample is to be obtained to test for rotavirus, as early as possible after onset, but no later than 5 
days after onset. 
5.5. Early Termination 
If a participant is withdrawn from the study for any reason prior to the planned final visit, every 
attempt is made to perform the following. 
a) Review AEs by site PI (or designee).  
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c) Physical examination, including vital signs and examination of the injection site(s). 
d) Safety laboratory testing if withdrawal occurs prior to scheduled safety laboratory testing. 
5.6. Procedure Methods 
Study procedures are performed and recorded in source documents as outlined in the Schedule 
of Events (Appendix I) and according to the following subsections. 
5.6.1. Vital Signs 
 
Temperature in degrees Celsius (recorded to the nearest 0.1 degree) will be measured by 
oral thermometer for adults and axillary thermometer for toddlers and infants.  
 
Respiratory rate in breaths per minute.  
 
Heart rate in beats per minute will be measured by automated device or manually. 
5.6.2. Height/length and Weight 
 
Height/length is measured in cm and recorded to the nearest 0.1 cm.  
 
Weight is measured in kg and recorded to the nearest 0.1 kg.  
5.6.3. Physical Examination 
Full physical examination will include assessment of vital signs, head, eyes, ears, nose, 
oropharynx, neck, chest (auscultation), lymph nodes (neck, supraclavicular, axillary, inguinal), 
abdomen (auscultation and palpation), genitourinary (for toddlers and infants), musculoskeletal, 
skin (especially injection sites), and neurological.  
5.6.4. Medical History 
A comprehensive medical history will be collected including details of any previous vaccinations 
(toddlers and infants – for adults, in the past 30 days) and reaction to vaccinations, birth (toddlers 
and infants), participation in clinical trials, surgery, previous hospitalization, allergy to food/drugs, 
current medication and history of any chronic or recurrent medical conditions. 
An interval medical history will consist of inquiring regarding changes since the last medical 
history discussion (healthcare events, signs, symptoms and changes in use of prescription or 
nonprescription drugs or herbal preparations).  
5.6.5. Injection Site Examination 
Injection site assessment will be done by trained study personnel. Local reactions will be graded 
according the toxicity grading scale in Appendix II. 
 
Erythema/redness will be examined under standardized lighting conditions and measured. 
Severity of the reaction will be determined by staff on the basis of the severity grading 
table (Appendix II).  
 
Swelling/induration will be examined by palpation and visual inspection under 
standardized lighting conditions; the examiner may temporarily mark skin at margins of 
visible swelling/induration, then measure maximum diameter (as per Appendix I and as 
above).  
 
Pain/tenderness will be assessed by (a) observing signs of discomfort by gentle palpation 
of the injection site and (b) inquiring whether the parents have noted discomfort. 
 
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5.6.6. Reactogenicity Assessment 
The following parameters for reactogenicity will be assessed during the 7 days after each study 
injection. 
 
Local reactogenicity (adults, toddlers and infants) 
o Pain 
o Tenderness 
o Erythema 
o Induration 
o Pruritus 
 
Systemic reactogenicity 
 
Adults 
o Fever 
o Headache 
o Vomiting 
o Nausea 
o Fatigue 
o Chills 
o Myalgia 
 
Toddlers and infants 
o Fever 
o Vomiting 
o Irritability 
o Decreased activity 
o Decreased appetite 
5.6.7. Clinical Laboratory Testing 
 
Laboratory evaluations for screening (hematology, chemistry, testing for HIV) and safety 
monitoring (hematology and chemistry) are outlined in Appendix I, Schedule of Events. 
5.6.8. Withdrawal from Further Study Injection and Early Termination from Study 
An enrolled/vaccinated participant may be terminated from the study for any of these reasons.  
a) Participant or parent withdraws consent for any reason. 
b) Site PI, Emmes medical monitor, Safety Review Committee (SRC), Data Safety 
Monitoring Board (DSMB) or PATH medical officer decides that termination is in the best 
interest of the participant. 
c) Site PI, Emmes medical monitor, SRC, DSMB or PATH medical officer decides that 
termination is necessary to protect the integrity of the study or achieve the objectives of 
the study.  
d) Interruption of study schedule makes the participant’s data unusable according to protocol 
requirements. 
A participant may be withdrawn from receiving further study injections, but participants/parents 
will be encouraged to complete the safety-related follow-up. If the participant/parent agrees, other 
study procedures (e.g., blood sampling for measuring levels of antibodies) may be continued. 
Withdrawal from further study injection may occur based on the local investigator decision at the 
time of the event or based on the SRC review of these events on a cumulative basis if: 
(a) Participant experiences objective local reactogenicity (erythema/induration) of Grade 2 or 
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(b) Participant experiences an event that contributes to study stopping rules (see Section 9.4). 
The final decision on withdrawing a participant from further study injections due to a and b will be 
made once the SRC reviews all the available data.  
(c) For any other safety-related reasons at the discretion of the site PI, Emmes medical 
monitor, SRC, PATH medical officer, or the participant/parent. 
5.6.9. Interim Contacts and Visits 
Interim contacts and visits (those between regularly scheduled follow up visits) may be performed 
at participants’/parents’ request, or as deemed necessary by the site PI or designee, at any time 
during the study. All interim contacts and visits will be documented in participants’ study records 
and on applicable case report forms. 
5.6.10. Contraception and Pregnancy 
Contraception status is assessed and documented prior to enrollment and each study injection 
for female participants who are of childbearing potential. Prior to enrollment and at each injection 
visit, staff will ask volunteers to verbally confirm their use of adequate contraception methods if 
they are able to become pregnant. Adequate methods of contraception include barrier 
contraception, hormonal birth control, IUD, or surgical sterility; abstinence is an acceptable form 
of birth control. If a female participant becomes pregnant following randomization, study injections 
will be discontinued and she will be encouraged to complete remaining visits and study 
procedures unless medically contraindicated. Any participant who becomes pregnant during the 
period between first injection and visit Day 84 will continue to be followed for pregnancy outcome. 
6. LABORATORY EVALUATIONS 
Blood samples will be obtained to examine vaccine safety and immunogenicity. 
6.1. Sample collection, distribution and storage  
Samples to evaluate vaccine safety will be obtained and processed at the clinical trial site and 
transported to each site’s designated laboratory for clinical testing. Research specimens collected 
for the immunogenicity time points will be separated into aliquots by per study specific process 
and stored at -20° C before being shipped to the Laboratory for Specialized Clinical Studies in the 
Division of Infectious Diseases at Cincinnati Children’s Hospital Medical Center. Research 
specimens may be retained for elective analysis at the discretion of PVS. Samples will be stored 
properly in controlled-temperature refrigerators/freezers. Backup generators are available for 
proper sample storage.  
Stool samples obtained from infants will be processed at the clinical trial site and transported  
to the laboratory at the National Institute for Communicable Disease, Sandringham, 
Johannesburg. Samples will be stored at the clinical trial sites and transported to the National 
Institute for Communicable Diseases (NICD) at 2-8° C. Following testing, residual specimens will 
be stored at NICD at -20° C. 
6.2. Safety clinical laboratory assays  
Protocol mandated screening and clinical safety laboratory tests will be conducted in real time. 
Refer to the Schedule of Events (Appendix I). The clinical laboratory for each site maintains proper 
accreditation and subscribes to a proficiency testing program.  
Laboratory results will be reviewed promptly by the site PI or designee. Participants/parents will 
be notified of any clinically significant abnormalities. If clinically significant abnormalities are 
identified during screening, participants will be referred to their primary health provider or 
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the study clinic for further evaluation, including clinical evaluation and repeat or additional 
laboratory testing, as warranted. 
At the discretion of PVS, GCLP audits may be conducted to ascertain adequate processing of 
immunogenicity specimens. 
6.3. Immunological assays  
The immunological assays to be performed include: 
 
Antibody (IgG & IgA by ELISA) to P2-VP8 analysis on sera from the screening visit and 
Days 56, 84 and 140 in the infant groups, the screening visit and Day 28 in the toddler 
group, and the screening visit and 28 days after each study injection in the adult group. 
Magnitude (GMTs) of the ELISA IgG and IgA titers will be determined in serum samples 
for each of these time points; frequency of seroresponse rate will be determined for each 
post-study injection time point. Magnitude (GMTs) of the ELISA IgG and IgA will be 
determined 28 days after the second dose of Rotarix in the infant cohort (Day 140). 
 
Rotavirus neutralization analysis on sera from the screening visit and Days 56, 84 and 
140 in the infant groups, the screening visit and Day 28 in the toddler group, and the 
screening visit and 28 days after each study injection in the adult group. Neutralization 
assays against the strains from which the vaccine was based will be conducted. 
Neutralization against several divergent rotavirus strains will be assessed if homologous 
neutralization is detected. 
 
Anti-rotavirus IgA (whole viral lysate) by ELISA will be determined in serum samples 
collected at baseline and one month after the final study injection. 
 
Additional immunogenicity assays may be performed by research laboratories to further 
evaluate post-study injection immune responses and to explore whether correlates of 
protection may be identified. 
6.4. Assay qualification, standardization and validation  
Serum anti-rotavirus IgA by ELISA (to whole viral lysate) and neutralizing antibody responses will 
be assessed in validated assays. Anti-P2-VP8 IgA and IgG by ELISA will be assessed in qualified 
assays. 
6.5. Stool testing for rotavirus 
Diagnosis of rotavirus infection in infants experiencing diarrhea during the study will be 
determined by stool detection of rotavirus using a validated antigen detection assay by ELISA at 
NICD. ELISA positive specimens will be confirmed and genotyped by PCR amplification of the 
VP7 and VP4 genes with a panel of G- and P-specific primers, respectively (WHO Manual of 
rotavirus detection and characterization methods, Methods 14 and 15 with minor modifications). 
The primer sets include: G-specific primer set (Beg9, End9, aAT8v, aBT1, aCT2, aDT4, mG3, 
mG9, mG10, mG12b and EndA) for the detection of G1, G2, G3, G4, G8, G9, G10 and G12 
rotavirus strains and P-specific primer set (con2, con3, 1T-1v, 2T-1, 3T-1, 4T-1, 5T-1, mP11, 
p4943 and VP4F) for the detection of P[4], P[6], P[8], P[9], P[10], P[11] and P[14] rotavirus strains. 
Where the genotype cannot be obtained using the G or P-specific primers, sequencing of the VP7 
or VP4 genes will be performed. 
6.6. Rotarix shedding 
At one study site, RMPRU, stool samples will be obtained 5, 7 and 9 days following the first dose 
of Rotarix for qualitative assessment of vaccine virus shedding at NICD. 

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All stool samples will be tested for the presence of rotavirus using the commercially available 
ProsPecT™ Rotavirus Microplate Assay (Oxoid Ltd, Ely, United Kingdom), according to the 
manufacturer’s instructions.  
ELISA positive specimens may be confirmed and genotyped by PCR amplification of the VP7 and 
VP4 genes with a panel of G- and P-specific primers, respectively (WHO Manual of rotavirus 
detection and characterization methods, Methods 14 and 15 with minor modifications). The primer 
sets include: G-specific primer set (Beg9, End9, aAT8v, aBT1, aCT2, aDT4, mG3, mG9, mG10, 
mG12b and EndA) for the detection of G1, G2, G3, G4, G8, G9, G10 and G12 rotavirus strains 
and P-specific primer set (con2, con3, 1T-1v, 2T-1, 3T-1, 4T-1, 5T-1, mP11, p4943 and VP4F) 
for the detection of P[4], P[6], P[8], P[9], P[10], P[11] and P[14] rotavirus strains. Genotype G1 
and/or P[8] rotavirus strains will be sequenced to determine if the strain is vaccine derived or a 
wild-type case. The VP7 (1062bp) and VP4 (876 bp) genes amplified by PCR will be purified and 
sequenced using the BigDye® Terminator v 3.1 cycle sequencing kits (Applied Biosystems). 
Reactions will be run on a 3500 Genetic Analyzer (Applied Biosystems) and data analyzed with 
manufacturer supplied software. Sequences will be directly compared to the VP7 and VP4 genes 
of the Rotarix strains (sequences available on Genbank; JN849114.1 VP7 and JN849113.1 VP4) 
and submitted to GenBank for BLASTn analyses to establish whether the VP7 and VP4 genes 
are from the vaccine or from wild-type G1P[8] strains. 
6.7. Biohazard Containment  
As transmission of blood-borne pathogens can occur through contact with contaminated needles, 
blood, and blood products, appropriate blood and secretion precautions will be employed by all 
personnel in the drawing of blood and processing of blood, processing of stool, and shipping and 
handling of all specimens for this study. All biological specimens will be transported using 
packaging mandated by 42 CFR Part 72. All dangerous goods materials, including diagnostic 
specimens and infectious substances, must be transported according to instructions detailed in 
the International Air Transport Association (IATA) Dangerous Goods Regulations. 
Biohazardous waste will be contained according to institutional, transportation/carrier, and all 
other applicable regulations. 
7. STATISTICAL DESIGN AND ANALYSIS 
7.1. Overview and General Design  
This study is a Phase 1/2 randomized, double-blind, dose-escalation, descending age clinical trial 
to assess the safety and immunogenicity of the trivalent rotavirus P2-VP8 subunit rotavirus 
vaccine (adsorbed onto aluminum hydroxide [Al(OH)3]) as compared to a placebo (NS). The 
primary objectives are to evaluate the safety, tolerability and immunogenicity of the P2-VP8 
subunit rotavirus vaccine with Al(OH)3 in infants. Immunogenicity is to be assessed through 
detection of vaccine-specific anti-P2-VP8 IgG and IgA antibodies by ELISA (to the P[4], P[6] and 
P[8] antigens in the vaccine) and neutralization of both homologous (the three strains from which 
the vaccine antigens are derived) and heterologous rotavirus strains.  
The primary hypothesis of safety will be fulfilled if no participants develop a vaccine related SAE 
and the vaccine is well-tolerated.  
The second hypothesis of immunogenicity will be fulfilled if at least 60% of the infants in at least 
one dose level demonstrate neutralizing responses to at least two of three strains from which the 
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A detailed statistical analysis plan for preparation of the final study report will be created and 
made final prior to database lock and unblinding for each age group. All statistical analyses will 
be performed using SAS® software Version 9.2 or later. 
Medical history and AEs will be coded using MedDRA dictionary Version 16.1 or later. The 
frequency count and percentage of participants will be summarized according to the coded terms 
of system organ class and preferred term. Participant-wise data listings will be provided. 
7.2. Randomization and blinding procedures 
The randomization scheme will be generated and maintained by the Statistical and Data 
Management Group (SDMG) at The Emmes Corporation, Rockville, MD. Participants will be 
enrolled into the study cohorts online and randomized using the enrollment module of The Emmes 
Corporation’s AdvantageEDCSM electronic data capture system. Each participant enrolled into the 
trial will be assigned a treatment code based on the cohort he/she is enrolled into after 
demographic and eligibility data have been entered into the system. 
The site pharmacists with primary responsibility for dispensing study products are charged with 
maintaining security of the treatment assignments.  
7.3. Objectives and Endpoints  
7.3.1. Primary Objectives: 
Safety 
To evaluate the safety and tolerability of the trivalent P2-VP8 subunit rotavirus vaccine at 
escalating dose levels in healthy South African adults, toddlers and infants 
Endpoints:  
 
Number of SAEs through 28 days of last study injection 
 
Number of AEs through 28 days of last study injection 
o AEs within four weeks after each injection. AEs will be categorized by MedDRA 
SOC and MedDRA PT and analyzed by study cohort, severity, duration and 
relationship to vaccine. 
 
Maximum severity per participant 
 
Proportion of AEs related to vaccine 
 
Number of Grade 2 or greater local reactogenicity events (injection site pain/ 
tenderness, redness, swelling, itching, local lymphadenopathy) within 1 week (Day 0 
to Day 7), after each injection and for the three combined injections. 
 
Number of Grade 2 or greater systemic reactogenicity (fever, vomiting, nausea, 
fatigue, chills and myalgia for adults; fever, vomiting, irritability, decreased activity, and 
decreased appetite for toddlers and infants) per cohort and product received, within 
1 week (Day 0 to Day 7) after each injection and for the three combined injections and 
maximum severity per participant. 
Immunogenicity 
To evaluate the immunogenicity of three doses of the trivalent P2-VP8 subunit rotavirus vaccine 
at different dose levels in healthy South African infants 

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Endpoints: 
 
Proportion of infants with anti-P2-VP8 IgG & IgA seroresponses by ELISA (four-fold 
increase in antibody titers between baseline and 4-weeks post-third study injection) in 
assays using each of the three vaccine antigens (P[4], P[6] and P[8]) 
 
Proportion of infants with neutralizing antibody responses (2.7-fold increases in antibody 
titers between baseline and 4-weeks post-third study injection) to each of the three 
rotavirus strains from which the vaccine antigens are derived, as well as heterologous 
strains 
 
Anti-P2-VP8 IgG & IgA geometric mean titers (GMT) (baseline and 4 weeks post third 
study injection) in ELISA assays using each of the three vaccine antigens (P[4], P[6] and 
P[8]) 
 
Neutralizing antibody GMT (baseline and 4 weeks post-third study injection) to each of 
the three rotavirus strains from which the vaccine antigens are derived, as well as 
heterologous strains 
 
Proportion of infants with neutralizing antibody responses to at least two of the three 
strains from which the vaccine antigens are derived 
7.3.2. Secondary Objectives 
Safety 
To evaluate the longer term safety (through 6 months after the last vaccination) of the trivalent 
P2-VP8 subunit rotavirus vaccine at escalating dose levels in healthy South African adults, 
toddlers and infants 
Endpoints: 
 
Number of SAEs at any time during the study 
 
Number of AEs at any time during the study  
 
Immunogenicity 
To evaluate the immunogenicity of two doses of the trivalent P2-VP8 subunit rotavirus vaccine at 
different dose levels in healthy South African infants 
Endpoints: 
 
Proportion of infants with anti-P2-VP8 IgG & IgA seroresponses by ELISA (four-fold 
increase in antibody titers between baseline and 4 weeks post-second study injection) in 
assays using each of the three vaccine antigens (P[4], P[6] and P[8]) 
 
Proportion of infants with neutralizing antibody responses (2.7-fold increases in antibody 
titers between baseline and 4 weeks post-second study injection) to each of the three 
rotavirus strains from which the vaccine antigens are derived, as well as heterologous 
strains 
 
Anti-P2-VP8 IgG & IgA geometric mean titers (GMT) (baseline and 4 weeks post-second 
study injection) in ELISA assays using each of the three vaccine antigens (P[4], P[6] and 
P[8]) 
 
Neutralizing antibody GMT (baseline and 4 weeks post-second study injection) to each of 
the three rotavirus strains from which the vaccine antigens are derived, as well as 
heterologous strains 

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 
Proportion of infants with neutralizing antibody responses to at least two of the three 
strains from which the vaccine antigens are derived (4 weeks post-second study injection) 
To evaluate the immunogenicity of the trivalent P2-VP8 subunit rotavirus vaccine at different dose 
levels in healthy South African adults and toddlers 
Endpoints: 
 
Proportion of adults and toddlers with anti-P2-VP8 IgG & IgA seroresponses by ELISA 
(four-fold increase in antibody titers between baseline and 4-weeks post-final study 
injection) in assays using each of the three vaccine antigens (P[4], P[6] and P[8]) 
 
Proportion of adults and toddlers with neutralizing antibody responses (2.7-fold increases 
in antibody titers between baseline and 4-weeks post-final study injection) to each of the 
three rotavirus strains from which the vaccine antigens are derived, as well as 
heterologous strains 
 
Anti-P2-VP8 IgG & IgA geometric mean titers (GMT) (baseline and 4 weeks post final 
study injection) in ELISA assays using each of the three vaccine antigens (P[4], P[6] and 
P[8]) 
 
Neutralizing antibody GMT (baseline and 4 weeks post-final study injection) to each of the 
three rotavirus strains from which the vaccine antigens are derived, as well as 
heterologous strains 
 
Proportion of adults and toddlers with neutralizing antibody responses to at least two of 
the three strains from which the vaccine antigens are derived 
7.3.3. Exploratory Objective 
Efficacy 
To evaluate the impact of the trivalent P2-VP8 subunit rotavirus vaccine on shedding of Rotarix 
administered following P2-VP8 subunit rotavirus vaccination of healthy South African infants as a 
test of concept 
Endpoint: 
 
Proportion of participants with Rotarix vaccine virus detected in stools on Days 5-9 
following the first dose of Rotarix, which is to be administered 28 days after the third study 
injection 
7.4. Sample Size  
7.4.1. Primary Safety Endpoints 
Safety will be assessed by analyses of the following primary endpoints (events), where the unit 
of analysis in each case will be the proportion of participants with at least one event: 
 
Moderate or greater solicited local reactions 
 
Moderate or greater solicited systemic reactions 
 
Moderate or greater adverse events 
 
Moderate or greater adverse events where there is a reasonable possibility that the 
study product caused the event, i.e., are suspected adverse reactions 
 
Serious adverse events 

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It is assumed that almost all participants enrolled will provide data for safety analysis and at least 
90% of enrolled participants will be evaluable for immunogenicity assessments.  
Safety 
For Cohorts A and B, 12 vaccine recipients per dose group and 24 vaccine recipients for the two 
dose groups combined will provide a greater than 90% chance of observing an AE that has a 
17.5% and 9.2% rate of occurrence, respectively. For the two infant cohorts combined (C and D), 
with 150 vaccine recipients per dose group and 450 vaccine recipients for the three dose groups 
combined will provide a greater than 90% chance of observing an AE that has a 1.6% and 0.5% 
rate of occurrence, respectively. 
Conversely, if no SAEs are observed in 24 and 450 vaccine recipients, the study will be able to 
rule out SAEs occurring at a rate of approximately 11.7% and 0.7%, respectively based on the 
upper bounds of the one-sided 95% confidence. 
 
 

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Table 25:  Probabilities of observing AEs 
Cohort 
 
Sample Size 
Underlying Event 
Rate 
Probability to 
observe an AE 
A (Adults) 
 
OR 
 
B (toddlers) 
Each Dose 
Group 
12 
1.0% 
11% 
2.0% 
22% 
5.0% 
46% 
17.5% 
90% 
Both Doses 
Combined 
24 
1.0% 
21% 
2.0% 
38% 
5.0% 
71% 
6.2% 
90% 
C+D (Infants) 
Each Dose 
Group 
150 
1.0% 
78% 
1.6% 
91% 
2.0% 
95% 
5.0% 
>99% 
All 3 Doses 
Combined 
450 
0.2% 
59% 
0.3% 
74% 
0.4% 
84% 
2.0% 
90% 
 
7.4.2. Primary Immunogenicity Endpoints 
Based on the results in South African infants who received monovalent P2-VP8 vaccine or 
placebo (VAC-013), the strain specific seroresponse rates for both P2-VP8 vaccine doses are 
expected to be ≥80% for at least one of the three P2-VP8 vaccine doses and <20% for the placebo 
group. For the two infant cohorts combined with 135 evaluable vaccine recipients per dose level 
(assuming 10% loss), this study is designed to provide at least 74% and 95% power (Fisher’s 
exact test) to detect 15 and 20 percentage points difference (e.g. 65% vs. 80 and 60% vs. 80%%), 
respectively, in seroresponse rates between any two P2-VP8 dose groups. For comparisons 
between a P2-VP8 dose group and the combined placebo groups, this study is designed to 
provide ≥99% power to detect ≥30 percentage point difference (e.g. 50% vs. 20%). 
For the sample calculations of the Geometric Mean Titer (GMT) endpoint based on the (VAC 013 
study, the LOG10 standard deviations were estimated to be <0.6 (range: 0.24 – 0.56) for the P2-
VP8 doses and the placebo group. Using a conservative LOG10 standard deviation of 0.6, this 
study with 135 evaluable infants per group (assuming 10% loss) is designed to provide at least 
98% power to detect as low as a 2.0-fold difference between any two P2-VP8 dose groups or 
between any P2-VP8 vaccine dose (15 µg, 30 µg or 90 µg dose) and the placebo group. 
 
 

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Table 26:  Power calculations for Comparing GMT in Two Groups of Infants (Cohorts C 
and D combined) 
LOG10 Standard 
Deviation 
Difference to 
detect 
Power** 
0.3 
1.5-fold 
>99% 
2-fold 
>99% 
4-fold 
>99% 
0.4 
1.5-fold 
95% 
2-fold 
>99% 
4-fold 
>99% 
0.5 
1.5-fold 
82% 
2-fold 
>99% 
4-fold 
>99% 
0.6 
1.5-fold 
67% 
2-fold 
98% 
4-fold 
100% 
*  
Between two P2-VP8 dose groups or between a P2-VP8 vaccine dose and 
the placebo group 
**  Two-Sample T-Test Assuming Equal Variance using PASS 13. 
 
7.5. Analytical Methodology 
The different populations to be used in data analysis are the following: 
 
The per-protocol population (PP), which is defined as all randomized participants who 
adhere to the protocol, complete all their scheduled visits, and present no major protocol 
violations, as defined prior to database lock. A “major” violation is defined as a protocol 
deviation that is considered to have an impact on the immunogenicity results of the study. 
Volunteers are analyzed according to randomized treatment arm. 
 
The safety population, which is defined as all participants who are randomize and receive 
at least one injection of the investigational product. Volunteers are analyzed according to 
treatment received rather than randomized treatment arm. 
The primary objectives of the study are to assess the safety, tolerability and immunogenicity of 
the study vaccine. All safety data (solicited local and systemic reactions and unsolicited AEs and 
SAEs) collected after participants are exposed to the study product will be included in the primary 
analysis of safety.  
To assess safety, the number and percentage of participants experiencing at least one AE, and 
the number and percentage of participants experiencing each specific AE, categorized by body 
system and preferred term, will be tabulated by study cohort and product received along with their 
corresponding 95% confidence intervals. Overall summaries by cohort and by product received 
include the number and percentage of participants experiencing: (1) any SAE; (2) any adverse 
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Grade 2 or greater AE judged related to study product. In addition, AEs will be summarized by 
grade.  
To assess tolerability, the number and percentage of participants with Grade 2 or higher local or 
systemic reactions will be tabulated by study cohort and by product received along with their 
corresponding 95% confidence intervals. Local and systemic reactions will be tabulated 
separately. Overall summaries by cohort and by product received will include the number and 
percentage of participants experiencing: (1) each specific reaction at Grade 2 or greater after 
each injection; (2) each specific reaction at Grade 2 or greater after any injection; (3) any reaction 
at Grade 2 or greater after each injection; and (4) any reaction at Grade 2 or greater after any 
injection. Descriptive statistics will be used to summarize the duration of local and systemic 
reactions. In addition, local and systemic reactions will be summarized by grade.  
Changes in clinical laboratory values between baseline and one week after vaccination will be 
analyzed descriptively. 
Immunogenicity analysis will compare the rate of responses and geometric mean titers (GMTs) 
between the various treatment groups based on injections received per protocol. To adjust for the 
decay in maternal antibody occurring concurrently with the IgG and neutralizing antibody immune 
responses to the vaccine, an analysis of the adjusted seroresponse rates utilizing the exponential 
decay function based on the estimated half-life of the maternal antibody will be performed in 
addition to the analysis based on the unadjusted raw values. For each assay, an estimated 
maternal antibody half-life will be derived from linear regression of LOG transformed titers in all 
infant placebo recipients, combined. Multiple components within each assay will be pooled (e.g. 
categorical class variable in the regression analysis) for the regression analysis to obtain a single 
half-life estimate per assay. Any placebo recipient with suspected community acquired rotavirus 
infection as identified by having a higher post vaccination titer higher than that at baseline, as well 
as those with values below the detectable limit of the assay at baseline will be excluded from the 
regression analysis.  
The following formula will be used to calculate the adjusted fold-rise and titer: 
frma = 2^[(LOG2(titerx / titer0) + LOG2(2x/MH)) 
titerma = 2^[LOG2(titerx) + LOG2(2x/MH)]  
  where frma = maternal antibody adjusted fold-rise 
  titerma = maternal antibody adjusted post-vaccination titer 
  titerx = titer at Day x post Dose One 
  titer0 = titer at pre-Dose One 
  x = number of days post Dose One 
  MH = maternal half-life in LOG2 scale 
For each immune assay, the proportion of participants with a positive response at a specific time 
point or with any positive response at any time point will be evaluated using the 2-sided exact 
binomial (Clopper-Pearson) 95% CI for each treatment group and compared between 2 treatment 
groups (i.e., between vaccine dose groups or between vaccine and placebo groups) using 
Fisher’s exact test. For continuous variables (i.e., titer/level of response), 2-sided 95% CI for the 
GMT for each treatment group using a t-distribution on log transformed titers/levels and 2-sample 
t-test on log transformed titers/levels will be used to compare between 2 treatment groups 
(i.e., between vaccine dose groups or between vaccine and placebo groups). A non-parametric 
method (e.g., bootstrap or Wilcoxon rank sum test) will be used for continuous variables if the 
data are not consistent with the normal distribution.  

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Assessment of shedding of Rotarix virus will be performed for each of the three specified post-
vaccination days and for shedding on any of the three days. Rates of shedding between the 
placebo and (1) each dose group and (2) the combined three dose groups will be compared using 
the Fisher’s exact test. 
When the use of descriptive statistics to assess group characteristics or differences is required, 
the following methods may be used: for categorical variables, the number and percent of 
participants in each category; for continuous variables, the mean, median, standard deviation, 
quartiles and range (minimum, maximum). Within-group assessment of the change from the 
baseline measurement to a follow-up measurement will be analyzed using McNemar’s test (for 
categorical response variables) or the paired t-test or Wilcoxon signed-rank test (for continuous 
variables). The following methods will be used for formal testing of differences between the study 
vaccine and placebo: for binomial response variables, Fisher’s exact test and/or logistic 
regression; for continuous variables, analysis of variance and/or Student’s t-test. The specific 
summary statistics and methods to be used for each endpoint will be described in the Statistical 
Analysis Plan. 
All testing and inference will be performed with 2-sided p-value of 0.05. For each immunogenicity 
endpoint, a multiplicity adjustment for 4 treatment groups will be applied using a sequential testing 
approach by performing an overall comparison of all treatment groups prior to pairwise 
comparisons. No multiplicity adjustment for multiple immunogenicity endpoints is planned, since 
the dose selection for the Phase 3 will be based on comprehensive statistical and clinical 
evaluation of all immunogenicity endpoints including safety results. 
8. SAFETY ASSESSMENT AND REPORTING 
This section defines the types of safety events that should be reported and outlines the 
procedures for appropriately collecting, grading, recording and reporting them. 
8.1. Safety Events 
All safety events observed under this protocol will be reported through the AdvantageEDC data 
system throughout the study. Information about vaccine reactogenicity (during the 7 days 
following vaccination) are collected on study-specific forms and are not captured on an Adverse 
Event form. Reactogenicity data (solicited signs or symptoms) will be collected through visit Day 
7 post-study injection; if a solicited sign or symptom has started during the 7 days post study 
injection and continues beyond Day 7 it will continue to be reported as a reactogenicity symptom. 
Any symptom starting after 7 days post any study injection will be recorded as an AE. Only when 
a solicited sign or symptom is considered an SAE, as defined below, will it be reported on an 
AE/SAE form set, in addition to the reactogenicity form. All other safety events that meet the 
definition of an AE or SAE that occur throughout the study are reported on the AE/SAE form set. 
AE's will be collected through study visit Day 84 in Groups A, C and D, and through study Day 28 
in Group B. SAEs and new major medical diagnoses will be assessed through Day 224 in Groups 
A, C and D, and through Day 168 in Group B, and will be reported on the AE/SAE form set. 
8.2. Reporting Period 
Safety events are reported from the time of the first study injection through completion of the study 
at 6 months after the final injection (as above). For participants who withdraw or are withdrawn 
from the study, an early termination visit should occur 5-7 days after the last injection or study 
visit to elicit occurrence of AEs (serious and non-serious).  

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8.3. Definitions 
8.3.1. Adverse Event (AE) or Medical Event  
An adverse event is any untoward medical occurrence in humans, whether or not considered 
related to study product, that occurs during the conduct of a clinical trial. Any change from baseline 
assessment of clinical status, ECGs, routine laboratory tests, x-rays, physical examinations, etc., 
that is considered clinically significant by the site PI is considered an AE.  
Suspected adverse drug reaction is any AE for which there is a reasonable possibility that the 
study product caused the AE. A reasonable possibility implies that there is evidence that the study 
product caused the event. 
Adverse reaction is any AE caused by the vaccine.  
8.3.2. Serious Adverse Events (Defined as Serious Adverse Events, Serious Suspected 
Adverse Reactions or Serious Adverse Reactions)  
An SAE, including a serious suspected adverse reaction or serious adverse reaction as 
determined by the site PI or the Sponsor, is any event that results in any of the following outcomes: 
1. Death 
2. Life-threatening AE (Life-threatening means that the study participant was, in the opinion 
of the site PI or Sponsor, at immediate risk of death from the event as it occurred.)  
3. Inpatient hospitalization or prolongation of existing hospitalization  
4. Persistent or significant incapacity or substantial disruption of the ability to conduct normal 
life functions  
5. Congenital abnormality or birth defect 
6. Important medical event that may not result in one of the above outcomes but may 
jeopardize the health of the study participant or require medical or surgical intervention to 
prevent one of the outcomes listed in the above definition of serious event  
8.3.3. Unexpected Adverse Event  
An AE is “unexpected” when its nature (specificity) or severity is not consistent with applicable 
product information, such as safety information provided in the Investigators’ Brochure (IB), the 
investigational plan or the protocol.  
8.4. Toxicity Grading  
The study clinic assigns toxicity grades to indicate the severity of adverse experiences and 
toxicities. The toxicity grading criteria provided in Appendix II grade AEs from Mild (grade 1) to 
Life Threatening (grade 4). All AEs leading to death are Grade 5 events. AEs are graded with the 
worst severity grade during the illness/symptoms. AE severity will be graded using the attached 
grading scale (Appendix II), which has been developed on the basis of Division of AIDS (DAIDS) 
Table for Grading the Severity of Adult and Pediatric Adverse Events, version 2.0, November 
2014, of the US National Institutes of Health, with modifications to reflect local population norms. 
For laboratory values not included in Appendix II, grading will be based on the Division of AIDS 
(DAIDS) Table for Grading the Severity of Adult and Pediatric Adverse Events, version 2.0, 
November 2014, available at webpage:  
http://rsc.tech-res.com/safetyandpharmacovigilance/gradingtables.aspx 

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8.4.1. Guidelines for Determining Causality of an Adverse Event 
All AEs, and SAEs, will be assessed for a causal relationship with the study product by the site 
PI. Appropriate medical judgment should be used to determine the relationship, considering all 
relevant factors, including pattern of reaction, temporal sequence and relationship, biological 
plausibility, confounding factors such as concomitant medication, concomitant diseases and 
relevant history. Assessment of causal relationship should be recorded in the AE and in SAE 
forms. 
The site PI should consider the following question when assessing causality of an AE to study 
product:  
Is there a reasonable possibility that the product caused the event? 
Reasonable possibility implies there is evidence that the study product caused the reported event. 
An affirmative answer designates the event as a suspected adverse reaction, and the AE is 
therefore considered “related.” If the answer is no, then the AE is considered “unrelated.” 
8.5. Adverse Event Identification, Resolution and Reporting 
To improve the quality and precision of acquired AE data, the site PI should observe the following 
guidelines: 
 
Whenever possible, use recognized medical terms when recording AEs on the AE CRF. 
Do not use colloquialisms and/or abbreviations. 
 
If known, record the diagnosis (i.e., disease or syndrome) rather than component signs, 
symptoms and laboratory values on the AE CRF (e.g., record congestive heart failure 
rather than dyspnea, rales, and cyanosis). However, signs and symptoms that are 
considered unrelated to an encountered syndrome or disease should be recorded as 
individual AEs on the CRF (e.g., if congestive heart failure and severe headache are 
observed at the same time, each event should be recorded as an individual AE). 
 
AEs occurring secondary to other events (e.g., sequelae) should be identified by the 
primary cause. A “primary” AE, if clearly identifiable, generally represents the most 
accurate clinical term to record on the AE CRF. If a primary serious AE (SAE) is recorded 
on an SAE CRF, events occurring secondary to the primary event should be described in 
the narrative description of the case. 
 
Grade 2 or higher abnormal laboratory test results will be entered as AEs with the toxicity 
grade associated with the abnormal laboratory test and attribution of the abnormality 
relative to the study product. Grade 1 abnormal laboratory test results will be entered as 
AEs if the site PI determines them to be clinically significant. 
 
Death is an outcome of an event. The event that resulted in the death should be recorded 
and reported on the SAE CRF. 
 
For hospitalizations for surgical or diagnostic procedures, the illness leading to the surgical 
or diagnostic procedure should be recorded as the SAE, not the procedure itself. The 
procedure should be captured in the case narrative as part of the action taken in response 
to the illness. 
AEs may be discovered through any of these methods. 
 
Observing the participant. 
 
Questioning the participant’s parent, which should be done in an objective manner. 
 
Receiving an unsolicited complaint from the participant’s parent. 
 
Review of medical records/source documents. 

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Each participant will have a scheduled observation following each study injection, including a 
symptom-directed physical examination, if indicated. Participants will be monitored in the clinic for 
at least 30 minutes after each study injection. On Days 0 to 7 after each study injection, participants 
and parents will be instructed to assess and record daily in a participant memory aid any signs and 
symptoms at the study injection site (injection site pain/tenderness, redness, swelling, itching), as 
well as systemic signs and symptoms (fever, headache, vomiting, nausea, fatigue, chills and 
myalgia for adults; and fever, vomiting, decreased appetite, irritability, and decreased activity for 
toddlers and infants). Follow-up visits will be conducted on post-study injection Days 3 and 7 in 
toddlers and infants and Day 7 in adults, including verification of memory aid notes and directed 
assessment. On Day 7 in Groups A, B and C, blood will be drawn for monitoring hematology and 
chemistry tests. Participants/parents will be instructed to call the study team if they observe 
significant injection site or systemic signs or symptoms, and then to continue monitoring and 
reporting their children’s condition. All participants will be followed for safety for six months after the 
last injection, with follow-up by telephone or clinic visit. 
8.5.1. AE Resolution 
All reported AEs should be followed until resolution or stabilization, or until completion of the 
study. Participants who have an ongoing study product-related SAE at study completion or at 
discontinuation from the study will be followed by the site PI or designee until the event is resolved 
or determined to be irreversible, chronic, or stable by the site PI. 
8.5.2. General Recording and Reporting Procedures 
A multi-page AE/SAE form set will be used, allowing all AEs to be submitted through a single 
reporting mechanism. SAEs will require additional information reported on additional pages within 
the AdvantageEDC data system. As appropriate or per request of the Emmes medical monitor or 
PATH medical officer, source documents (e.g., hospitalization discharge summaries) may be 
uploaded to the AE/SAE form set, as well (with care taken to remove personal identifiers). The 
site PI will treat or refer, as appropriate, participants experiencing AEs and observe them at 
suitable intervals until their symptoms resolve or their status stabilizes. 
8.5.3. SAE Recording and Reporting Procedures 
SAEs will be recorded on the AE case report form (CRF). The site is obligated to report SAEs to 
the Emmes Coordinating Center within 24 hours of the site’s knowledge of the event. The 
following attributes will be assigned by the site PI or assignment by designee will undergo 
documented review by the site PI:  
 
Description 
 
Date of onset and resolution (if known when reported) 
 
Severity 
 
Assessment of relatedness to test article  
 
Action taken 
The site PI will apply clinical judgment to determine whether an AE is of sufficient severity to 
require discontinuation of administration of study injection for that participant. If necessary, the 
site PI will suspend any trial procedures and institute the necessary medical therapy to protect a 
participant from any immediate danger. 

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8.5.4. Site Reporting of Events 
 
Notify the site PI. 
 
Complete and transmit an AE Form through the AdvantageEDC data system. Information 
regarding a SAE report must be recorded in the participant’s medical chart and entered in 
the AdvantageEDC data system. 
 
SAE reports should include hospital admittance notes, hospital discharge summary, 
clinical notes, resolution date, treatment, and any other pertinent information regarding the 
event. If not immediately available, reporting should not be delayed to provide these 
documents. 
 
In the event of a death, the SAE Form must be completed and transmitted along with other 
supporting data (e.g., death certificate, medical notes). 
Figure 9: Reporting Decisions for Adverse Events 
 
 
 
 
 
 
 
 
 
 
 
 
8.6. Serious Adverse Event Notification 
8.6.1. Notifications and Review 
The site PIs will provide the Emmes Coordinating Center with data for all SAEs, as defined per 
the protocol, on an ongoing basis by entering the information in AdvantageEDC. The Emmes 
Coordinating Center is responsible for notifying the Sponsor and the CRO performing site 
monitoring and will do so simultaneously with the reporting to the clinical database. Notification of 
reported events will be generated at the time of entry into the data system, and all SAEs will be 
reviewed promptly by the Emmes medical monitor within the data system. As noted above, this 
notification should be within 24 hours of site awareness of the event. Site personnel will be trained 
in reporting AEs and SAEs.  
The Emmes medical monitor will review all unanticipated events involving risk to participants or 
others, SAEs and all participant deaths associated with the protocol and will provide a written 
report. At a minimum, the Emmes medical monitor must comment on the outcomes of the event 
or problem and, in case of an SAE or death, comment on the relationship to participation in the 
study. The Emmes medical monitor must also indicate whether he/she concurs with the details of 
the report provided by the site PI. 
Summary review of all reported AEs and SAEs will be compiled on a weekly basis to identify any 
safety trends. Review of reactogenicity and safety monitoring laboratory tests will also be 
conducted on a weekly basis during the periods such monitoring is being performed. 
No 
Yes 
Safety Event requiring AE Form Submission: 
All adverse events and all serious adverse events 
SAE? 
Expedited Reporting 
(within 24 hours of site awareness) 
Use the Adverse Event Form 
Submit to Emmes Coordinating Center 
(also notify ethics board/IRB) 
Standard Reporting 
(within 7 days) 
Use the Adverse Event Form  
and other CRFs 
Submit to Emmes Coordinating Center 

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8.6.2. Expedited Reporting 
The Emmes Coordinating Center will be responsible for expedited Safety Reports and IND Annual 
reports to the US Food and Drug Administration (FDA). SCT Consulting will be responsible for 
reporting to the South African Medicines Control Council (MCC). 
FDA Reporting 
When expedited reports as defined below are required, the cover memorandum, 
MedWatch Form FDA 3500A, and any pertinent attachments will be processed by the 
Emmes medical monitor and a copy of the completed report will be submitted by fax or 
courier delivery before the regulatory reporting deadline, to the following persons: 
 FDA medical officer as appropriate (submitted as an amendment to the applicable IND) 
 Site PI (who is responsible for forwarding the report to the local EC/IRB) 
 SRC independent medical monitors  
 PATH medical officer 
If relevant follow-up information becomes available, the Emmes medical monitor will be 
responsible for obtaining and reviewing details from the site. A follow-up MedWatch form 
will be completed and forwarded to all parties that received the earlier SAE report. A copy 
of the safety sections for annual FDA reports will be forwarded to PVS.  
Suspected adverse reactions that are serious and unexpected will be reported to the FDA 
within 15 days, or for deaths and life threatening events that are both suspected adverse 
reactions and unexpected, within 7 days (per 21 CFR 312.32). 
MCC Reporting 
SCT Consulting will be responsible for reporting to the MCC. 
All fatal and life-threatening, unexpected adverse drug reactions should be reported within 
7 calendar days after first knowledge by the applicant. The initial notification must be 
followed by as complete a report as possible, within an additional 8 calendar days.  
Serious, unexpected adverse drug reactions that are not fatal or life-threatening must be 
reported as soon as possible, and not later than 15 calendar days after first knowledge by 
the applicant. 
The MCC must be notified, within 15 calendar days after first knowledge by the applicant, 
when there is a suggestion of a change in the nature, severity or frequency of expected 
adverse drug reactions or when new risk factors are identified. The basis on which these 
assessments are made should be included.  
Any information, that may in any way influence the benefit-risk assessment of a medicine 
or that would be sufficient to consider changes in the administration of the medicine or in 
the overall conduct of a clinical trial, must be reported to the MCC. The applicant must 
submit this information to the MCC within three calendar days of first knowledge by the 
applicant.  
Subsequent review by the MCC, FDA, the SRC, EC/IRB, or the Sponsor may suspend further 
study product administration or procedures. The study Sponsor, MCC, FDA and SRC retain the 
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8.6.3. Notifying the Protocol Safety Review Committee 
The Emmes Coordinating Center will provide the SRC with listings of all SAEs on an ongoing 
basis. Furthermore, the SRC will be informed of expedited reports of SAEs. 
8.6.4. Notifying the Ethics Committee/Institutional Review Board 
The site PIs will ensure the timely dissemination of required SAE information, including expedited 
reports, to their respective local ethics committees in compliance with applicable local regulations 
and guidelines. The site PIs are responsible for submitting the safety report (initial and follow up 
SAE reports) or other safety information (e.g., revised IB) to their respective ethics committees 
and for retaining a copy in the site’s study file. 
WIRB Guidelines 
All SAEs will be reported to WIRB by PVS according to WIRB guidelines and using the WIRB Ten 
Day Adverse Event Form. 
WIRB Phone: 800-562-4789, Fax: 360-252-2498.  
9. SAFETY MONITORING 
Extensive safety monitoring will be provided for this protocol. The site PIs and/or designated site 
staff will be responsible for continuous close safety monitoring of all study participants and for 
alerting the Sponsor if unexpected concerns arise or stopping criteria are met.  
9.1. Safety Review Committee  
A SRC, comprised of the site PIs from each study site, the Sponsor medical monitor, the Emmes 
medical monitor and two independent local medical experts, will monitor safety throughout the 
duration of the study (in addition to the Data Safety Monitoring Board described in section 8.2). 
The responsibilities and procedures of the SRC will be defined in the SRC Charter. The study 
statistician with assistance of the data management staff will prepare safety reports as needed 
for SRC discussions. In addition to routine review of safety information, a central role of the SRC 
is the review of safety data for the recommendation of whether to progress to the next dose level 
in each age cohort and to the next age cohort. Cumulative safety data will be available 
continuously for review by SRC members, and safety reports will be prepared for each SRC 
deliberation of dose escalation (see Section 9.4, Dose Escalation) and progression to subsequent 
cohorts (adults to toddlers and toddlers to infants). 
The SRC members will review the safety data throughout the study to determine whether pause 
criteria have been met. The SRC will be notified by the Emmes Coordinating Center and convene 
by conference call if pause criteria may have been met. The SRC may request review of unblinded 
safety data by the independent SRC members in deliberating whether to refer concerns and 
events to the DSMB. If pause criteria are met, the DSMB is to be notified and convened, as 
described in section 9.4. 
The SRC may make recommendations regarding permanent discontinuation of administration of 
study product in individual participants based on careful review of all relevant data.  
SRC reviews will be summarized with recommendations to the study Sponsor. 
9.2. Data and Safety Monitoring Board (DSMB)  
A DSMB formed by independent vaccine, pediatric and infectious disease experts, as well as a 
biostatistician, will be established to periodically review cumulative data, as needed. The 
responsibilities and procedures of the DSMB will be defined in the DSMB Charter. The DSMB will 
be responsible for safeguarding the interests of trial participants, assessing safety during the 
trial, and for monitoring the overall conduct of the clinical trial. The DSMB will provide 

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recommendations about continuing, modifying or stopping the trial. Items reviewed by the DSMB 
will include: study participant accrual and demographic information; interim/cumulative safety 
data; discontinuations of study injections; factors that might affect the study outcome or 
compromise the confidentiality of the trial data (such as treatment and endpoint unbinding); data 
quality, completeness, and timeliness; and factors external to the study, such as scientific or 
therapeutic developments that may impact participant safety or the ethics of the study.  
The DSMB will convene prior to study initiation and then at least every six months. In addition to 
routinely scheduled calls, if the protocol team or SRC has serious safety concerns or study pause 
criteria are met, the DSMB will convene by teleconference to jointly review the data. The DSMB 
reviews will be summarized with recommendations to the study Sponsor as to whether there are 
safety concerns and whether the study should continue without change, be modified, or be 
terminated.  
If at any time, a decision is made to permanently discontinue administration of study product in 
all participants, expeditious notification will be provided by PVS to the U.S. FDA (via Emmes 
Coordinating Center) and WIRB, to the MCC (via SCT Consulting) and by the site PIs to their 
respective local ECs.  
9.3. Withdrawal for Safety 
A participant may be withdrawn from further study injections for safety-related reasons, at the 
discretion of the site PIs, Emmes medical monitor, PATH medical officer, SRC independent 
members, or the participant/participant’s parent. 
Participants/parents may withdraw from study product administration for any other reason. If 
withdrawal is requested, the participants/parents should be asked to continue (at least limited) 
scheduled evaluations, complete a study termination form, and be given appropriate care under 
medical supervision until symptoms of any AE resolve or any condition becomes stable. 
9.4. Study Pause  
Study pause is defined as a decision to cease, temporarily or permanently, enrollment and all 
study injections. Study pause will not eliminate any safety follow-up procedures specified by 
protocol. The Sponsor will pause the study if the SRC determines that study pause criteria have 
been met. The DSMB will be convened by teleconference to review study pauses and provide 
recommendations to the Sponsor regarding continuation or permanent discontinuation of study 
vaccinations, or study modification. 
9.4.1. Study Pause Rules  
The following study pause rules will automatically pause or halt further study injections. These 
stopping rules refer to suspected adverse reactions, as defined below: 
Study pause rules: Meeting one or more of the following criteria will automatically pause or halt 
further study injections in the study. These pause rules refer to suspected adverse reactions 
across all cohorts, as defined below: 
 
One participant with a vaccine-related serious AE (SAE). 
 
Two participants with ≥grade 3 (severe) localized inflammatory reaction at the injection 
site.  
 
The same objective ≥grade 3 (severe) systemic reactogenicity signs or symptoms (fever, 
vomiting), within seven days following study injection in three participants in Groups A, B 
or C, and ≥5% of participants in Group D. 

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 
Two participants with a systemic rash, including, but not limited to, generalized urticaria, 
generalized petechiae, or erythema multiforme, within seven days following study 
injection. 
 
Two participants with the same ≥grade 3 (severe) vaccine-related abnormal clinical 
laboratory evaluation, within seven days following study injection. 
9.4.2. Study Pause Procedure 
 
If the site PI (or designee), the Emmes medical monitor or any member of the SRC 
identifies that a pause criterion may have been met or proposes that the study be paused 
on a discretionary basis, all study injections and enrollment will be suspended. The DSMB 
will be notified and will convene expeditiously by conference call to review all available 
and relevant information, confirm whether pause criteria have been met, and provide 
recommendations to the Sponsor to permanently halt, re-start or otherwise modify the 
study. In the event that the study is permanently stopped, the HREC and WIRB will be 
notified within 48 hours. 
 
At scheduled study review meetings, the SRC may determine that pause criteria have 
been met, and the DSMB will be notified, as above.  
 
If the Sponsor re-starts the study after DSMB review and recommendation to restart, study 
injections may resume. Study injections that are delayed beyond the protocol-specified 
window may be administered. The appropriate follow-up and subsequent injection 
schedule for participants with delayed study injections will be determined by the Sponsor 
on a case-by-case basis. 
9.5. Dose Escalation 
The SRC will review available safety data through post-study injection Day 7 for all Group A 
participants in the 30 µg dose group (including safety monitoring laboratory tests) and determine 
whether to proceed with enrollment of the 90 µg dose group.  
The SRC’s evaluation of the safety data through Day 7 of all Group A participants in the 30 µg 
dose group, and any other safety data from Cohort A available at that time, will also be the basis 
for determining whether it is safe to proceed with enrollment of the 30 µg dose group in Group B. 
As above, the SRC will review safety data through post-study injection Day 7 in both the group A 
90 µg dose and the group B 30 µg dose in deciding whether to progress from the 30 µg dose 
group to the 90 µg dose in Group B. 
Similarly, the SRC’s evaluation of the safety data through Day 7 of all Group B participants in the 
30 µg dose group, and any other safety data from Groups A and B available at that time, will also 
be the basis for determining whether it is safe to proceed with enrollment of the 15 µg dose group 
in Group C. As above, the SRC will review safety data through post-study injection Day 7 in 
deciding whether to progress from the 15 µg dose group to the 30 µg dose in Group C, and then 
from the 30 µg dose group to the 90 µg dose. 
The SRC will review the safety data through Day 7 for all participants in Groups A, B and C, and 
any additional data available at the time of deliberation, and determine whether to proceed with 
enrollment of Group D. 
 
 

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10. 
DATA MANAGEMENT 
The site PIs are responsible for ensuring the accuracy, completeness, and timeliness of the data 
reported. Data collection is the responsibility of the clinical trial staff at the study site under the 
supervision of the site PIs. The Emmes Corporation is responsible for data management activities, 
including quality review, analysis, and reporting of the study data according to SOPs. 
10.1. Case Report Form Development and Completion 
Electronic Data Capture (EDC) will be the method of data collection in this study. The eCRFs will 
be developed by The Emmes Corporation and approved by PVS. Clinical data (including AEs, 
concomitant medications, and reactogenicity data) and clinical laboratory data will be entered into 
the 21 CFR Part 11-compliant Internet Data Entry System (IDES) provided by The Emmes 
Corporation. The data system includes password protection and internal quality checks, such as 
automatic range checks, to identify data that appear inconsistent, incomplete, or inaccurate. 
Clinical data will be entered directly from the source documents.  
Data for each participant will be recorded in the eCRF. It is the site PIs’ responsibility to ensure 
the accuracy, completeness, and timeliness of the data reported in the participant’s eCRF and 
any supporting documentation. All source documents should be completed in a neat, legible 
manner to ensure accurate interpretation of data. Source documentation supporting the eCRF 
data should document the dates and details of study procedures, AEs and participant status. The 
site PIs/institutions will maintain all information in the eCRFs and all source documents that 
support the data collected from each participant. 
The site PIs or designated representatives should complete the eCRFs as soon as possible after 
information is collected. Completed eCRFs must be submitted for each screened participant who 
signs (or whose parent signs) the study-specific informed consent form (ICF). The site PIs will 
retain all essential documents and a CD-ROM copy of the eCRF data (after the study is closed 
and unblinded and the Clinical Study Report is completed or at such time that the site no longer 
has access to the electronic data system). 
10.2. Details of Data Management 
A Data Management Handbook (DMH) will be written by The Emmes Corporation and contain all 
study-specific requirements.  
10.2.1. Data Validation 
The Emmes Corporation will inspect the data entered into the database for completeness and 
consistency.  
10.2.2. Source Data Verification 
For source data verification (SDV), the monitor (on behalf of the study Sponsor) must have direct 
access to source documents that support the data recorded, e.g., medical records, original 
laboratory records and ICFs. If source data are electronic, these data must be printed, signed and 
dated by the site PI and stored in the participant’s study file. Clinical laboratory data will remain in 
study participant records. Essential documents, including ICFs, must be filed and kept in the study 
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10.2.3. Definition of Source Data  
Source data are all information in original records or certified copies of original records of clinical 
findings, observations, or other activities in a clinical study. Source data are contained in source 
documents.  
10.2.4. Definition of Source Document 
Original source documents include data and records, e.g., hospital records, medical notes, 
laboratory notes, evaluation checklists, pharmacy dispensing records, records kept at the 
pharmacy and at the laboratory, documentation of shipments. Note that, for this protocol, the 
participant data collection tool (i.e., the Memory Aid) is not considered a source document (Refer 
to Appendix III) Data reported in the eCRF derived from source documents should be consistent 
with the source documents or the discrepancies should be explained. 
10.3. Database Locking Procedures 
A final database lock for the primary analysis will occur after all participants have completed all 
follow-up visits, including the 6 month safety follow-up call, a case-by-case review of the severity 
of any AEs has been performed and finalized, all data anomalies have been resolved and 
monitoring is complete.  
Remaining immunology data will be maintained in a separate immunology database.  
10.4. Record Archival  
The site PIs are responsible for retaining study records for a period of 2 years following the date 
that a marketing application is approved for the product or, if no application is to be filed or, if a 
file application is not approved, until 2 years after the investigation is discontinued and the FDA 
is notified. The Sponsor will be responsible for providing the site with date of vaccine approval or 
IND withdrawal. 
These records are also to be maintained in compliance with local EC and local authority medical 
records retention requirements, whichever is longest. Storage of all trial-related documents will 
be such that confidentiality will be strictly maintained to the extent provided by US and local law.  
10.5. Screen Failures 
If a participant or participant’s parent signs the ICF but the participant is not randomized because 
of ineligibility (a screen failure), the reason for his/her ineligibility should be entered in the medical 
records/notes/charts. Also, a screening log must be kept. Data from participants who fail 
screening will not be recorded in the eCRF, with the exception of demographic data and the 
reason for screen fail.  
10.6. Protocol Deviations 
A protocol deviation is any noncompliance with the clinical trial protocol, GCP, or site SOP 
requirements. The noncompliance may be either on the part of the participant, the site PI, or the 
study site staff. As a result of deviations, corrective actions are to be developed by the site and 
implemented promptly.  

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These practices are consistent with ICH E6: 
 
Compliance with Protocol, Sections 4.5.1, 4.5.2, and 4.5.3 
 
Quality Assurance and Quality Control, Section 5.1.1 
 
Noncompliance, Sections 5.20.1 and 5.20.2. 
It is the responsibility of the site to exercise continuous vigilance to identify and report deviations 
within 5 working days after identification of the protocol deviation, or within 5 working days of the 
scheduled protocol-required activity. All deviations must be promptly reported via the appropriate 
eCRF within the electronic data system.  
All deviations from the protocol must be addressed in study participant source documents. A 
completed copy of WIRB reportable Protocol Deviation forms must be maintained in the regulatory 
file, as well as in the participant’s source document. Protocol deviations must be sent to the local 
EC per its guidelines. The site PI/study staff is responsible for knowing and adhering to the 
respective EC’s requirements. 
11. STUDY MONITORING 
Sponsor monitoring responsibilities will be provided through a CRO, SCT Consulting, experienced 
in monitoring clinical site activity. A site initiation visit will be conducted prior to beginning the 
study, and monitoring will be conducted at initiation, during, and at closeout of the study by the 
study monitor or designee.  
During the course of the study, the monitor will visit the clinical site at intervals to verify compliance 
to the protocol; completeness, accuracy, and consistency of the data and study product 
accountability; adherence to CFR and MCC regulations, and any additional regulations and 
requirements, including GCP, of the conduct of clinical research. The monitor should have access 
to participant medical records, study product accountability and other study-related records 
needed to verify the entries on the eCRFs. 
The site PIs and the monitor must agree to cooperate to ensure that any problems detected in 
the course of these monitoring visits, including eCRF completion and query resolution, are 
resolved in a predefined timeframe.  
To ensure the quality of clinical data across all participants at the site, a clinical data management 
review will be performed on participant data received at The Emmes Corporation. During this 
review, participant data will be checked for consistency, omissions, and any apparent 
discrepancies. In addition, the data will be reviewed for adherence to the protocol and GCP. To 
resolve any questions arising from the clinical data management review process, data queries 
and/or site notifications will be sent to the site for resolution as soon as possible; all queries must 
be resolved prior to database lock. 
Essential documents must be filed in the site study file on an ongoing basis and available for 
review by the Sponsor’s contracted site monitor. Monitoring visits will be performed according to 
the Clinical Monitoring Plan. 
11.1. Independent Auditing 
PVS representatives may audit the study to ensure that study procedures and data collected 
comply with the protocol and applicable SOPs at the clinical site and The Emmes Corporation, 
and that data are correct and complete. The site PIs will permit auditors (employees of the 
Sponsor or an external company designated by the Sponsor) to verify source data validation of 
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source data and evaluate the study site for its adherence to the clinical study protocol and GCP 
guidelines and applicable regulatory requirements. 
11.2. Regulatory Agency Auditing 
The site PIs must be aware that representatives from regulatory authorities or WIRB and their 
local ECs may wish to inspect the eCRFs and associated study records. The site PIs will notify 
the Sponsor within 24 hours following contact by a regulatory agency. The site PIs and study 
coordinators must make the relevant records available for inspection and will be available to 
respond to reasonable requests and audit queries made by authorized representatives of 
regulatory agencies. The site PIs will provide the Sponsor with copies of all correspondence that 
may affect the review of the current study or his qualification as an investigator in clinical studies 
conducted by the Sponsor. The Sponsor will provide any needed assistance in responding to 
regulatory audits or correspondence. 
12. OBLIGATIONS AND ROLES OF THE SPONSOR, SITE PIS AND STUDY PERSONNEL  
This study will be conducted according to GCP and in accordance with all US federal regulations 
regarding the protection of human subjects in research including US 21 CFR Part 50 and US 21 
CFR Part 312, as well in accordance with South African MCC regulations.  
The Sponsor will assure the trial is conducted in compliance with the protocol, GCP, and 
regulatory authority requirements. The Sponsor will provide the investigators with the funding and 
information needed to conduct the trial properly, ensuring proper monitoring of trial activities and 
that the trial is conducted in accordance with the general investigational plan and protocols 
contained in the submissions to the regulatory authorities. The Sponsor will ensure that regulatory 
authorities and the investigators are promptly informed of significant new adverse effects or risks 
with respect to the study vaccine. 
The site PIs agree to perform the research in strict accordance with this protocol, the International 
Conference on Harmonisation (ICH) Guideline for Good Clinical Practice (E6), as well as in 
conformity with any US or local regulations regarding the conduct of clinical studies. 
In addition, the site PIs must follow local and institutional requirements including, but not limited 
to, investigational product, clinical research, informed consent and local EC requirements. The 
Sponsor will provide notification to the site PIs of protocol and amendment approvals by regulatory 
authorities when applicable. Any modifications to the research protocol, the ICF, and/or the 
questionnaires or change in site PIs must be submitted to WIRB and respective local EC for 
review and approval prior to implementation. The site PIs may deviate from the protocol without 
prior approval only when the deviation is necessary to eliminate an apparent immediate hazard 
to the participant.  
The site PIs will notify their respective ECs of SAEs and protocol deviations according to their EC 
requirements. Any deviation to the protocol that may have an effect on the safety or rights of the 
participant, or the integrity of the study, must be reported to the Sponsor by the site PIs as soon 
as the deviation is identified. In that event, the site PIs will notify the Sponsor immediately by 
phone, notify their respective ECs, enter the deviation into the appropriate eCRF, and confirm 
notification to the Sponsor in writing within 10 working days after the change is implemented. All 
deviations will be noted in the continuing review report to the respective ECs, the annual report 
to the Sponsor, and in the final study report for the ECs. 
Except where the site PIs’ signatures are specifically required, it is understood that the term 
"investigator" as used in this protocol and on the electronic Case Report Forms (eCRFs) refers to 
the site PIs or appropriate study personnel that the site PIs designate to perform a certain duty. 
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investigators or other appropriate study personnel are eligible to sign for the site PIs on designated 
eCRFs. 
The Emmes medical monitor will be responsible for reviewing all serious and unexpected AEs 
and providing an unbiased written report of the event. 
13. ETHICAL CONSIDERATIONS AND INFORMED CONSENT 
13.1. Informed Consent Process 
Before any study-related activities and in agreement with applicable regulatory requirements, the 
site PI must ensure that the participant’s parent is fully informed about the aims, procedures, 
potential risks, and potential benefits of the study. The participant/participant’s parent will be given 
the written, local EC/WIRB approved ICF, allowed ample time to read the consent form, 
encouraged to ask questions about the study, have the questions answered and then be given 
time to decide if s/he would like to participate or have her/his child participate in the study. It will 
be emphasized that participation is voluntary, and that the participant/parent has the right to 
withdraw from the study at any time without prejudice. 
The site PIs or designees must obtain the participant’s/parent’s voluntary, signed and dated ICF 
(or, if the participant/parent is unable to sign, independently witnessed and documented consent) 
before any study-related procedures are performed. Study staff must document the informed 
consent process. The original, signed ICF must be kept in the site study file.  
13.2. Risk/Benefit  
No benefits can be guaranteed to participants for their participation in this research study.  
Previous experience with the study product is limited to the first in human trial of the monovalent 
(P[8]) P2-VP8 subunit vaccine conducted in the United States and the ongoing study in toddlers 
and infants in South Africa. In those trials, the vaccine has been well-tolerated, with reactogenicity, 
when it occurred, predominantly mild in nature and no vaccine related SAEs were observed. See 
Section 1.6 for details. 
As with any vaccine, severe allergic reaction is a potential rare event. None were observed in two 
clinical trials with the monovalent vaccine. 
In this trial, participants in the infant groups are to receive Rotarix following completion of the 
study product series, to provide the protection that licensed vaccine provides. As it cannot yet be 
demonstrated that the P2-VP8 subunit vaccine does not interfere with response to Rotarix, 
participants will be provided a third dose of Rotarix. Additional risk mitigation will be provided by 
clinical monitoring and access to clinical evaluation and management. 
13.3. Protocol Review Process 
Scientific review of this protocol will be conducted by PATH. The protocol will be submitted the 
existing IND at the US FDA and to the MCC. The IND Sponsor will be PVS. Protocol ethical review 
and oversight will be performed by each site’s local EC and WIRB. Continuing review will be 
undertaken in accordance with existing regulations. The Sponsor will be responsible for trial 
registration at ClinicalTrials.gov. 
Copies of the approved continuing review and final study reports, along with the respective local 
IRB approval notifications, will be submitted to the Sponsor as soon as these documents become 
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13.4. Participant Confidentiality 
The site PIs must ensure that participant confidentiality is maintained. Personal identifiers will not 
be included in any study reports. All study records will be kept confidential to the extent provided 
by national and local laws.  
All study procedures will be conducted per GCP guidelines, and every effort will be made to 
protect participant privacy and confidentiality to the extent possible.  
All study-related information will be stored securely at the study site or at a designated, secure 
off-site location. When not in use and under immediate control of study staff, all participant 
information will be stored in locked areas with access limited to study staff. Data collection, 
process, and administrative forms, laboratory specimens, and other reports will be identified 
exclusively by a coded number to maintain participant confidentiality. All local databases will be 
secured with password-protected access systems. Participants’ study information will not be 
released without written parental permission, except as necessary for monitoring or compliance 
with legal or regulatory requirements. 
Medical records containing identifying information may be made available for review when the 
study is monitored by the Sponsor or an authorized regulatory agency. Direct access may include 
examining, analyzing, verifying, and reproducing any records and reports that are important to 
the evaluation of the study. 
13.5. 
Reimbursement  
Pending local EC approval, participants/parents of participants will be compensated for their time 
and effort in this study, and be reimbursed for travel to study visits. The study ICF will state the 
plan for reimbursement. Participants/parents of study participants will not be charged for study 
injections, research clinic visits, research-related examinations, or research-related laboratory 
tests. 
13.6. Storage of Specimens  
Stored study research samples (including samples retained for elective analysis) will be labeled 
by a code that only the study site can link to the participant. All stored research samples will be 
logged into a secure database and any use documented. Samples may be stored at several 
different repositories and laboratories to complete the analyses required to meet study primary, 
secondary and exploratory analyses. As a part of the informed consent process, participants will 
be informed of and asked to agree to long-term storage of specimens for use in future, related 
research.  
14. APPENDICES 
14.1. APPENDIX I: Schedule of Events 
14.1.1. Schedule of Events for Group A (Adults) 
14.1.2. Schedule of Events for Group B (Toddlers) 
14.1.3. Schedule of Events for Groups C & D (Infants) 
14.2. APPENDIX II: Toxicity Grading Scales 
14.3. APPENDIX III: Memory Aid 
14.4. APPENDIX IV: Literature Cited 
 

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14.1. 
APPENDIX I: Schedule of Events 
14.1.1. Schedule of Events for Group A (Adults) 
 
Screen 
Day 0 
Day 7 
(±1) 
Day 28 
(-2 to +7) 
Day 35 
(±1) 
Day 56 
(-2 to +7) 
Day 63 
(±1) 
Day 84 
(±4) 
Day 224 
(±14) 
Visit Number 
1 
2 
3 
4 
5 
6 
7 
8 
9 
Assessment 
Day 
-28 to -1 
* 
0 
>30 
min 
 
* 
0 
>30 
min 
 
* 
0 
>30 
min 
 
 
 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical History Update 
 
X 
 
 
X 
X 
 
 
X 
X 
 
 
X 
X 
X 
Prior and Concurrent Medications 
X 
X 
 
 
X 
X 
 
 
X 
X 
 
 
X 
X 
 
Inclusion & Exclusion Criteria 
X 
X 
 
 
 
X 
 
 
 
X 
 
 
 
 
 
Physical Examination 
X 
X 
 
 
XC 
XC 
 
 
XC 
XC 
 
 
XC 
XC 
XC 
Study injections 
 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
 
Injection Site Assessment 
 
X 
 
X 
X 
X 
 
X 
X 
X 
 
X 
X 
X 
 
AE Assessment 
 
X 
 
X 
X 
X 
 
X 
X 
X 
 
X 
X 
X 
X 
Memory Aid / reminders 
 
 
 
X 
X 
 
 
X 
X 
 
 
X 
X 
 
 
Vital Signs 
X 
X 
 
X 
X 
X 
 
X 
X 
X 
 
X 
X 
X 
 
HematologyA 
X 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
Clinical ChemistryB 
X 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
Pregnancy testD 
X 
X 
 
 
 
X 
 
 
 
X 
 
 
 
 
 
HIV 1/2 Ab 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Serum for IgG&A/Neutralizing Ab 
X 
 
 
 
 
X 
 
 
 
X 
 
 
 
X 
 
Safety Blood Volume† 
20 
 
 
 
10 
 
 
 
 
 
 
 
 
 
 
Immunogenicity Blood Volume† 
10 
 
 
 
 
 
10 
 
 
 
10 
 
 
10 
 
Cumulative Blood Volume† 
30 
 
 
 
40 
 
50 
 
 
 
60 
 
 
70 
 
*  Prior to study injection 
† 
All blood volume units mL 
A. Complete blood count (CBC), including WBC, hemoglobin, and platelet count 
B. Chemistry Panel: ALT, bilirubin, albumin, and creatinine (albumin at screening only) 
C. Targeted physical exam, as indicated 
D. Serum pregnancy test at screening and urine pregnancy test on the day of each vaccination for female participants (prior to vaccination) 
 

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14.1.2. Schedule of Events for Group B (Toddlers) 
 
Screen 
Day 0 
Day 3 
Day 7 
(±1) 
Day 28 
(±4) 
Day 168 
(±14) 
Visit Number 
1 
2 
3 
4 
5 
6 
Assessment 
Day 
-28 to -1 
* 
0 
>30
min 
 
 
 
 
Informed Consent 
X 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
Medical History 
X 
 
 
 
 
 
 
 
Medical History Update 
 
X 
 
 
X 
X 
X 
X 
Prior and Concurrent Medications 
X 
X 
 
 
X 
X 
X 
 
Inclusion & Exclusion Criteria 
X 
X 
 
 
 
 
 
 
Physical Examination 
X 
X 
 
 
 
XC 
XC 
XC 
Study injections 
 
 
X 
 
 
 
 
 
Injection Site Assessment 
 
X 
 
X 
X 
X 
X 
 
AE Assessment 
 
X 
 
X 
X 
X 
X 
X 
Memory Aid / reminders 
 
 
 
X 
X 
X 
 
 
Vital Signs 
X 
X 
 
X 
X 
X 
X 
 
HematologyA 
X 
 
 
 
 
X 
 
 
Clinical ChemistryB 
X 
 
 
 
 
X 
 
 
HIV 1/2 Ab 
X 
 
 
 
 
 
 
 
Safety Blood Volume† 
5 
 
 
 
 
5 
 
 
Immunogenicity Blood Volume† 
5 
 
 
 
 
 
5 
 
Cumulative Blood Volume† 
10 
 
 
 
 
15 
20 
 
* 
Prior to study injection 
† 
All blood volume units mL 
A. Complete blood count (CBC), including WBC, hemoglobin, and platelet count 
B. Chemistry Panel: ALT, bilirubin, albumin, and creatinine (albumin at screening only) 
C. Targeted physical exam, as indicated 
 
 

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14.1.3. Schedule of Events for Groups C & D (Infants) 
 
Screen 
Day 0 
Day 3 
Day 7 
(±1) 
Day 28 
(-2 to +7) Day 31 
Day 35 
(±1) 
Day 56  
(-2 to +7) 
Day 59 
Day 63 
(±1) 
Day 84 
(±4) 
Days 89, 
91, 93 
Day 112 
(±14) 
Day 140 
(±14) 
Day 224 
(±14) 
Visit Number 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
 
12 
13 
14 
Assessment 
Day 
-7 to -1 * 
0 >30 
min 
 
 
* 
0 >30 
min 
 
 
* 
0 
>30 
min 
 
 
 
 
 
 
 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical History Update 
 
X 
 
 
X 
X 
X 
 
 
X 
X 
X 
 
 
X 
X 
X 
 
X 
X 
X 
Prior and Concurrent Medications 
X 
X 
 
 
X 
X 
X 
 
 
X 
X 
X 
 
 
X 
X 
X 
 
 
 
 
Inclusion & Exclusion Criteria 
X 
X 
 
 
 
 
X 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
Physical Examination 
X 
X 
 
 
 
XC 
X 
 
 
 
XC 
X 
 
 
 
XC 
XC 
 
XC 
XC 
XC 
Study injections 
 
 
X 
 
 
 
 
X 
 
 
 
 
X 
 
 
 
 
 
 
 
 
EPI vaccinations (except rotavirus) 
 
 
X 
 
 
 
 
X 
 
 
 
 
X 
 
 
 
 
 
 
 
 
Rotarix administration 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
X 
X 
 
Injection Site Assessment 
 
X 
 
X 
X 
X 
X 
 
X 
X 
X 
X 
 
X 
X 
X 
X 
 
 
 
 
AE Assessment 
 
X 
 
X 
X 
X 
X 
 
X 
X 
X 
X 
 
X 
X 
X 
X 
 
X 
X 
X 
Memory Aid / reminders 
 
 
 
X 
X 
X 
 
 
X 
X 
X 
 
 
X 
X 
X 
 
 
 
 
 
Vital Signs 
X 
X 
 
X 
X 
X 
X 
 
X 
X 
X 
X 
 
X 
X 
X 
X 
 
X 
X 
 
HematologyA 
X 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Clinical ChemistryB 
X 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HIV PCRD 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Serum for IgG&A/Neutralizing Ab 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
X 
 
 
 
 
Stool for Rotarix shedding (RMPRU) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
Safety Blood Volume, Group C† 
5 
 
 
 
 
5 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Safety Blood Volume, Group D† 
5 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Immunogenicity Blood Volume† 
5 
 
 
 
 
 
 
 
 
 
 
5 
 
 
 
 
5 
 
 
 
 
Cumulative Blood Volume, Group C† 
10 
 
 
 
 
15 
 
 
 
 
 
20 
 
 
 
 
25 
 
 
 
 
Cumulative Blood Volume, Group D† 
10 
 
 
 
 
 
 
 
 
 
 
15 
 
 
 
 
20 
 
 
 
 
*  
Prior to study injection 
† 
All blood volume units mL 
A. 
Complete blood count (CBC), including WBC, hemoglobin, and platelet count 
B. 
Chemistry Panel: ALT, bilirubin, albumin, and creatinine (albumin at screening only) 
C. 
Targeted physical exam, as indicated 
D. 
If mother not known to be negative (negative test result between 24 weeks gestation and screening) 
E. 
Stool samples to be obtained from infants with diarrhea to test for rotavirus 
 

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14.2. APPENDIX II: Toxicity Grading Scales  
Systemic Illness 
Mild  
(Grade 1) 
Moderate 
(Grade 2) 
Severe  
(Grade 3) 
Potentially  
Life Threatening 
(Grade 4) 
Illness or clinical 
AE (as defined  
according to 
applicable  
regulations)  
No or minimal 
interference with 
usual activities; no 
medical 
intervention/ 
therapy required  
Greater than 
minimal 
interference with 
usual activities; no 
or minimal medical 
intervention/ 
therapy required  
Marked limitation in 
ability to perform 
usual activities; 
medical 
intervention/ 
therapy required 
Inability to perform 
basic functions OR 
Medical or operative 
intervention indicated 
to prevent permanent 
impairment, persistent 
disability, or death  
 
Local Reaction to 
Injectable Product 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially  
Life Threatening 
(Grade 4) 
Injection site pain 
(pain without 
touching) 
OR 
Tenderness (pain 
when area 
touched) 
Pain/tenderness 
causing no or 
minimal limitation 
of use of limb 
Pain or tenderness 
causing greater 
than minimal 
limitation of use of 
limb 
Pain/tenderness 
causing inability to 
perform usual 
activities 
Pain/tenderness 
causing inability to 
perform basic 
functions OR 
Hospitalization 
indicated 
Injection site 
erythema or 
induration 
 
Infants and 
toddlers 
≤ 2.5 cm in 
diameter 
> 2.5 cm in 
diameter with < 
50% surface area 
of the extremity 
segment involved 
(thigh) 
≥ 50% surface area 
of the extremity 
segment involved 
(thigh) OR 
Ulceration OR 
Secondary infection 
OR Phlebitis OR 
Sterile abscess OR 
Drainage 
Potentially life-
threatening 
consequences (e.g., 
abscess, exfoliative 
dermatitis, necrosis 
involving dermis or 
deeper tissue) 
Injection site 
erythema or 
induration 
 
Adults 
2.5 to < 5 cm in 
diameter OR 6.25 
to < 25 cm2 
surface area AND 
Symptoms 
causing no or 
minimal 
interference with 
usual social & 
functional 
activities 
≥ 5 to < 10 cm in 
diameter OR ≥ 25 
to < 100 cm2 
surface area OR 
Symptoms causing 
greater than 
minimal 
interference with 
usual social & 
functional activities 
≥ 10 cm in diameter 
OR ≥ 100 cm2 
surface area OR 
Ulceration OR 
Secondary infection 
OR Phlebitis OR 
Sterile abscess OR 
Drainage OR 
Symptoms causing 
inability to perform 
usual social & 
functional activities 
Potentially life-
threatening 
consequences (e.g., 
abscess, exfoliative 
dermatitis, necrosis 
involving dermis or 
deeper tissue) 
Injection site 
pruritus  
Itching localized to 
the injection site 
that is relieved 
spontaneously or 
in < 48 hours of 
treatment 
Itching beyond the 
injection site that is 
not generalized OR 
Itching localized to 
the injection site 
requiring ≥ 48 
hours treatment 
Generalized itching 
causing inability to 
perform usual social 
& functional 
activities 
NA 
 
 

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Systemic 
(General) 
Mild  
(Grade 1) 
Moderate 
(Grade 2) 
Severe  
(Grade 3) 
Potentially  
Life Threatening 
(Grade 4) 
Acute systemic 
allergic reaction 
Localized urticaria  
(wheals) with no 
medical intervention 
indicated  
Localized urticaria 
with medical 
intervention indicated 
OR Mild angioedema 
with no medical 
intervention indicated 
Generalized urticaria  
OR Angioedema with 
medical intervention 
indicated OR 
Symptomatic mild 
bronchospasm  
Acute anaphylaxis OR  
Life-threatening 
bronchospasm OR 
laryngeal edema  
Fever 
37.7 – 38.6°C 
38.7 – 39.3°C 
39.4 – 40.5°C 
> 40.5°C 
Vomiting 
Transient or 
intermittent vomiting 
with no or minimal 
interference with oral 
intake 
Frequent episodes of 
vomiting with no or 
mild dehydration 
Persistent vomiting 
resulting in greater 
than milde 
dehydration OR 
Aggressive 
rehydration indicated 
(e.g., IV fluids)  
Life-threatening 
consequences (e.g., 
hypotensive shock) 
Irritability 
Crying more than 
usual but easily 
consoled 
Crying more than 
usual and somewhat 
difficult to console 
Continuous crying 
that is inconsolable 
for 4 hours or more 
 
Decreased 
activity 
Slightly subdued, but 
responds normally to 
stimuli 
Subdued and does 
not respond as 
readily as normal to 
stimuli 
Lethargic 
Obtunded 
Decreased 
appetite 
Loss of appetite 
without decreased 
oral intake 
Loss of appetite 
associated with 
decreased oral 
intake without 
significant weight 
loss 
Loss of appetite 
associated with 
significant weight 
loss 
Life-threatening 
consequences OR 
Aggressive 
intervention indicated 
[e.g., tube feeding or 
total parenteral 
nutrition (TPN)] 
Nausea 
Transient (< 24 
hours) or intermittent 
AND No or minimal 
interference with oral 
intake 
Persistent nausea 
resulting in 
decreased oral 
intake for 24 to 48 
hours 
Persistent nausea 
resulting in minimal 
oral intake for > 48 
hours OR 
Rehydration 
indicated (e.g., IV 
fluids) 
Life-threatening 
consequences (e.g., 
hypotensive shock) 
Myalgia 
(generalized) 
Muscle pain causing 
no or minimal 
interference with 
usual social & 
functional activities 
Muscle pain causing 
greater than minimal 
interference with 
usual social & 
functional activities 
Muscle pain causing 
inability to perform 
usual social & 
functional activities 
Disabling muscle pain 
causing inability to 
perform basic self-
care functions 
Headache  
Symptoms causing 
no or minimal 
interference with 
usual social & 
functional activities 
Symptoms causing 
greater than minimal 
interference with 
usual social & 
functional activities 
Symptoms causing 
inability to perform 
usual social & 
functional activities 
Symptoms causing 
inability to perform 
basic self-care 
functions OR 
Hospitalization 
indicated OR 
Headache with 
significant impairment 
of alertness or other 
neurologic function 

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Systemic 
(General) 
Mild  
(Grade 1) 
Moderate 
(Grade 2) 
Severe  
(Grade 3) 
Potentially  
Life Threatening 
(Grade 4) 
Chills  
Symptoms causing 
no or minimal 
interference with 
usual social & 
functional activities 
Symptoms causing 
greater than minimal 
interference with 
usual social & 
functional activities 
Symptoms causing 
inability to perform 
usual social & 
functional activities 
NA 
Fatigue  
Symptoms causing 
no or minimal 
interference with 
usual social & 
functional activities 
Symptoms causing 
greater than minimal 
interference with 
usual social & 
functional activities 
Symptoms causing 
inability to perform 
usual social & 
functional activities 
Incapacitating 
symptoms of fatigue 
or malaise causing 
inability to perform 
basic self-care 
functions 
 
 
Chemistry 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially  
Life Threatening 
(Grade 4)** 
Creatinine  
1.1-1.3 ULN 
>1.3-1.8 ULN 
>1.8 to <3.5 ULN 
≥3.5 ULN 
Albumin – 
Hypoalbuminemia G/L 
NA 
20 to <30 
<20 
-- 
ALT 
1.25 to <2.5 ULN 
2.5 to <5.0 ULN 
5.0 to <10.0 ULN 
≥10 ULN 
Bilirubin 
1.1 to <1.6 ULN 
1.6 to <2.6 ULN 
2.6 to <5.0 ULN 
≥5.0 ULN 
Hematology 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially  
Life Threatening 
(Grade 4) 
Hemoglobin - g/dL 
 
 
 
 
36 to 56 days of age  
(male and female) 
8.5 to 9.6 
7.0 to <8.5 
6.0 to <7.0 
<6.0 
57 days of age to  
< 13 years of age  
(male and female) 
9.5 to 10.4 
8.5 to <9.5 
6.5 to <8.5 
<6.5 
≥ 13 years of age  
(male only) 
10.0 to 10.9 
9.0 to <10.0 
7.0 to <9.0 
<7.0 
≥ 13 years of age  
(female only) 
9.5 to 10.4 
8.5 to <9.5 
6.5 to <8.5 
<6.5 
 
 
 
 
 
WBC Decrease - cell/mm3 
2,000 to 2,499 
1,500 to 1,999 
1,000 to 1,499 
< 1,000 
Platelets - cell/mm3 
100 to <124 X103 
50 to <100 X103 
25 to <50 X103 
<25 X103 
 
 

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14.3. APPENDIX III: Memory Aid 
Participant and Parental Assessments and Use of Memory Aid 
The Memory Aid prompts daily participant and parental assessment and recording in the Memory 
Aid until 7 days following dosing, beginning about 8 hours post-dose on the dosing day and 
thereafter each night. The participant/parent assesses and records observations of all signs and 
symptoms at the injection site, systemic signs and symptoms, and other AEs. 
The Memory Aid specifically prompts assessment of the solicited injection site reactions 
considered to be study endpoints (Section 5.5.6). The participant’s/parent’s assessment of 
reactogenicity will follow the Toxicity Grading Scale provided in Appendix II. The participant/parent 
takes and records oral (adults) or axillary (toddlers and infants) temperature (with thermometer 
provided by study staff) at each assessment time. The Memory Aid prompts that, if temperature 
exceeds 38 degrees Celsius, the participant/parent then repeats and records temperature 
measurements every 4 hours until temperature is lower than 38 degrees Celsius. 
Participants/parents will be provided with a standardized tool to measure diameter of palpable 
swelling and erythema (redness). The Memory Aid prompts the participant/parent to call site staff 
if redness or swelling widest diameter exceeds grade 1.  
The Memory Aid is not used as the source document for data entry into the electronic data capture 
system. Study staff will review the Memory Aid with the participant/parent to elicit as much 
information as possible about any reported symptoms. Based on this information, study staff will 
use their clinical judgment to assess the event, and its severity, and record the data on the source 
document that captures reactogenicity symptoms, which will be used for the purposes of data 
entry. The existence of any discrepancies between the participant/parent and staff assessment 
of event severity will be noted on the case report form. The Memory Aid will be retained with 
participant study records. 
On Days 3 and 7 after each study injection, study staff will review the Memory Aid information 
during a scheduled clinic visit (for adults, Day 7 only). The participant/parent will be instructed to 
bring the Memory Aid to any in-person clinic visits within 7 days after a vaccine administration.  
Any additional, unsolicited symptoms will be similarly discussed with the participant/parent, and 
clinic staff should evaluate the event(s) according to their clinical judgment and guidance provided 
in the protocol. This assessment should be described carefully in source documents and entered 
on the AE form. Any reported Concomitant Medications are recorded on the Concomitant 
Medications form. 
 
 

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14.4. APPENDIX IV: Literature Cited 
1. 
Tate, J.E., et al., 2008 estimate of worldwide rotavirus-associated mortality in children 
younger than 5 years before the introduction of universal rotavirus vaccination 
programmes: a systematic review and meta-analysis. The Lancet infectious diseases, 
2012. 12(2): p. 136-41. 
2. 
WHO, Rotavirus vaccines. Weekly Epidemiological Record, 2007. 82(32): p. 285-95. 
3. 
Jiang, B., J.R. Gentsch, and R.I. Glass, Inactivated rotavirus vaccines: a priority for 
accelerated vaccine development. Vaccine, 2008. 26(52): p. 6754-8. 
4. 
Diggle, L., Rotavirus diarrhoea and future prospects for prevention. Br J Nurs, 2007. 
16(16): p. 970-4. 
5. 
Santos, N. and Y. Hoshino, Global distribution of rotavirus serotypes/genotypes and its 
implication for the development and implementation of an effective rotavirus vaccine. 
Reviews in medical virology, 2005. 15(1): p. 29-56. 
6. 
Tshangela, A., et al., Rotavirus Surveillance in South Africa, 2011. Communicable 
Diseases Surveillance Bulletin, 2012. 10(3): p. 42-46. 
7. 
Mwenda, J.M., et al., Burden and epidemiology of rotavirus diarrhea in selected African 
countries: preliminary results from the African Rotavirus Surveillance Network. The 
Journal of infectious diseases, 2010. 202 Suppl: p. S5-S11. 
8. 
Estes, M.K., D.Y. Graham, and B.B. Mason, Proteolytic enhancement of rotavirus 
infectivity: molecular mechanisms. Journal of virology, 1981. 39(3): p. 879-88. 
9. 
Arias, C.F., M. Lizano, and S. Lopez, Synthesis in Escherichia coli and immunological 
characterization of a polypeptide containing the cleavage sites associated with trypsin 
enhancement of rotavirus SA11 infectivity. The Journal of general virology, 1987. 68 
(Pt 3): p. 633-42. 
10. 
Bellido, D., et al., Brucella spp. lumazine synthase as a bovine rotavirus antigen delivery 
system. Vaccine, 2009. 27(1): p. 136-45. 
11. 
Dunn, S.J., et al., Immunogenicity, antigenicity, and protection efficacy of baculovirus 
expressed VP4 trypsin cleavage products, VP5(1)* and VP8* from rhesus rotavirus. Arch 
Virol, 1995. 140(11): p. 1969-78. 
12. 
Gil, M.T., et al., Homotypic protection against rotavirus-induced diarrhea in infant mice 
breast-fed by dams immunized with the recombinant VP8* subunit of the VP4 capsid 
protein. Viral Immunol, 2000. 13(2): p. 187-200. 
13. 
Kovacs-Nolan, J., et al., Cloning and expression of human rotavirus spike protein, VP8*, 
in Escherichia coli. Biochem Biophys Res Commun, 2001. 282(5): p. 1183-8. 
14. 
Larralde, G., et al., Serotype-specific epitope(s) present on the VP8 subunit of rotavirus 
VP4 protein. Journal of virology, 1991. 65(6): p. 3213-8. 
15. 
Marelli, B., et al., Oral immunization with live Lactococcus lactis expressing rotavirus VP8 
subunit induces specific immune response in mice. J Virol Methods, 2011. 175(1): p. 28-
37. 
16. 
Padilla-Noriega, L., et al., Humoral immune responses to VP4 and its cleavage products 
VP5* and VP8* in infants vaccinated with rhesus rotavirus. Journal of clinical microbiology, 
1992. 30(6): p. 1392-7. 
17. 
Perez Filgueira, D.M., et al., Passive protection to bovine rotavirus (BRV) infection induced 
by a BRV VP8* produced in plants using a TMV-based vector. Arch Virol, 2004. 149(12): 
p. 2337-48. 

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21 June 2016 
 
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18. 
Tan, M., et al., Norovirus P particle, a novel platform for vaccine development and antibody 
production. Journal of virology, 2011. 85(2): p. 753-64. 
19. 
Matsui, S.M., et al., Passive protection against rotavirus-induced diarrhea by monoclonal 
antibodies to the heterotypic neutralization domain of VP7 and the VP8 fragment of VP4. 
Journal of clinical microbiology, 1989. 27(4): p. 780-2. 
20. 
Kirkwood, C.D., R.F. Bishop, and B.S. Coulson, Human rotavirus VP4 contains strain-
specific, serotype-specific and cross-reactive neutralization sites. Arch Virol, 1996. 141(3-
4): p. 587-600. 
21. 
Kovacs-Nolan, J., D. Yoo, and Y. Mine, Fine mapping of sequential neutralization epitopes 
on the subunit protein VP8 of human rotavirus. Biochem J, 2003. 376(Pt 1): p. 269-75. 
22. 
Favacho, A.R., et al., Cloning, expression, and purification of recombinant bovine rotavirus 
hemagglutinin, VP8*, in Escherichia coli. Protein Expr Purif, 2006. 46(2): p. 196-203. 
23. 
Lee, J., et al., Immunological response to recombinant VP8* subunit protein of bovine 
roravirus in pregnant cattle. The Journal of general virology, 1995. 76 (Pt 10): p. 2477-83. 
24. 
Andres, I., et al., Yeast expression of the VP8* fragment of the rotavirus spike protein and 
its use as immunogen in mice. Biotechnol Bioeng, 2006. 93(1): p. 89-98. 
25. 
Lentz, E.M., et al., VP8* antigen produced in tobacco transplastomic plants confers 
protection against bovine rotavirus infection in a suckling mouse model. J Biotechnol, 
2011. 156(2): p. 100-7. 
26. 
Istrate, C., et al., Parenteral administration of RF 8-2/6/7 rotavirus-like particles in a one-
dose regimen induce protective immunity in mice. Vaccine, 2008. 26(35): p. 4594-601. 
27. 
Ahlborg, N., et al., Linkage of exogenous T-cell epitopes to the 19-kilodalton region of 
Plasmodium yoelii merozoite surface protein 1 (MSP1(19)) can enhance protective 
immunity against malaria and modulate the immunoglobulin subclass response to 
MSP1(19). Infection and immunity, 2000. 68(4): p. 2102-9. 
28. 
Falugi, F., et al., Rationally designed strings of promiscuous CD4(+) T cell epitopes 
provide help to Haemophilus influenzae type b oligosaccharide: a model for new conjugate 
vaccines. Eur J Immunol, 2001. 31(12): p. 3816-24. 
29. 
Wen, X., et al., Construction and characterization of human rotavirus recombinant VP8* 
subunit parenteral vaccine candidates. Vaccine, 2012. 30(43): p. 6121-6. 
30. 
Wen, X., et al., Inclusion of a universal tetanus toxoid CD4(+) T cell epitope P2 significantly 
enhanced the immunogenicity of recombinant rotavirus DeltaVP8* subunit parenteral 
vaccines. Vaccine, 2014. 32(35): p. 4420-7. 
31. 
Baylor, N.W., W. Egan, and P. Richman, Aluminum salts in vaccines--US perspective. 
Vaccine, 2002. 20 Suppl 3: p. S18-23. 
32. 
Cadranel, S., et al., Factors affecting antibody response of newborns to repeated 
administrations of the rotavirus vaccine RIT 4237. Journal of pediatric gastroenterology 
and nutrition, 1987. 6(4): p. 525-8. 
33. 
Westerman, L.E., et al., Serum IgG mediates mucosal immunity against rotavirus 
infection. Proc Natl Acad Sci U S A, 2005. 102(20): p. 7268-73.