Phase II, Randomized, Double-Blind, Placebo-Controlled Study of the Safety and Immunogenicity of the Recombinant Live Attenuated Tetravalent Dengue Virus Vaccine Admixture TV005 (TetraVax-DV TV005) in Healthy Adults, Adolescents, and Children in Dhaka, Bangladesh
Phase: PHASE2
Conditions: Dengue
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

Phase II, randomized, double-blind, placebo-
controlled study of the safety and immunogenicity of 
the recombinant live attenuated tetravalent dengue 
virus vaccine admixture TV005 (TetraVax-DV TV005) 
in healthy adults, adolescents, and children in Dhaka, 
Bangladesh 
Version 2.10 
16 June 2017 
NCT02678455 

1 
PR-15097  
 
V2.10: 16Jun17 
 
 
Phase II, randomized, double-blind, placebo-controlled study of the 
safety and immunogenicity of the recombinant live attenuated 
tetravalent dengue virus vaccine admixture TV005 (TetraVax-DV 
TV005) in healthy adults, adolescents, and children in Dhaka, 
Bangladesh 
 
 
CIR Protocol Number: 
 
 
UVM IRB Protocol Number: 
16-101 
ICDDR,B Protocol Number: 
PR-15097  
FDA IND Number: 
 
 
14268 
Ionizing Radiation:   
 
None 
Multi-institution: 
 
 
Yes (ICDDR,B & UVM) 
Project Assurance: 
FWA# 00000287 (Johns Hopkins Bloomberg 
School of Public Health 
 
FWA# 00000723 (University of Vermont) 
 
FWA# 00001468 (International Center for 
Diarrhoeal Disease Research, Bangladesh) 
 
Protocol Version: 
 
 
2.10 
Date:  
 
 
 
June 16, 2017 
 
 
Sponsored by: 
Office of Clinical Research Policy and Regulatory Operations (OCRPRO) 
Division of Clinical Research (DCR) 
National Institute of Allergy and Infectious Diseases (NIAID) 
National Institutes of Health (NIH) 
 
 
 

2 
PR-15097  
 
V2.10: 16Jun17 
 
 
Team Roster 
Principal Investigator: 
Rashidul Haque, MBBS, PhD 
Centre for Vaccine Sciences 
International Centre for Diarrhoeal Disease Research, 
Bangladesh 
Mohakhali, Dhaka -1212 
+880- 0173093859 (phone) 
+88 02 9827001-10 ext.3483 (office hours phone) 
 
Co-Principal Investigator: Firdausi Qadri, PhD 
 
 
 
 
Centre for Vaccine Sciences 
International Centre for Diarrhoeal Disease Research,         
Bangladesh 
Mohakhali, Dhaka, 1212 Bangladesh 
+88 02 9827001-10 ext.2431 (office hours phone) 
 
Co-Principal Investigator: Beth D. Kirkpatrick, MD 
 
 
 
 
Vaccine Testing Center 
University of Vermont College of Medicine 
Given Building 
89 Beaumont Avenue 
Burlington, VT 05405 
+1 802 656-5822 (phone) 
 
Co-Principal Investigator: Anna Durbin, MD 
 
 
 
 
Center for Immunization Research 
Johns Hopkins Bloomberg School of Public Health 
Johns Hopkins University 
Hampton House, 624 N. Broadway 
Baltimore, MD 21205 
+1 410 614-4736 (phone) 
 
Co-Investigator: 
 
Mohammad Shafiul Alam, PhD 
 
 
 
 
Parasitology Research Group 
International Centre for Diarrhoeal Disease Research, 
Bangladesh 
Mohakhali, Dhaka, 1212 Bangladesh 
+88 02 9827001-10 ext.3482 (office hours phone) 
 
Co-Investigator: 
 
Farhana Khanam, MBBS 
 
 
 
 
Centre for Vaccine Sciences 
The information contained herein is the property of the National Institute of Allergy and Infectious 
Diseases, National Institutes of Health and may not be reproduced, published, or disclosed to others 
without the written authorization of the National Institute of Allergy and Infectious Diseases. 

3 
PR-15097  
 
V2.10: 16Jun17 
 
International Centre for Diarrhoeal Disease Research, 
Bangladesh 
Mohakhali, Dhaka, 1212 Bangladesh 
+88 02 9827001-10 ext.2413 (office hours phone) 
 
Co-Investigator: 
 
Masud Alam, MBBS 
 
 
 
 
Centre for Vaccine Sciences 
International Centre for Diarrhoeal Disease Research, 
Bangladesh 
Mohakhali, Dhaka, 1212 Bangladesh 
+88 02 9827001-10 ext.3482 (office hours phone) 
 
Co-Investigator:  
 
Benjamin Lee, MD 
 
 
 
 
Vaccine Testing Center 
University of Vermont College of Medicine 
Given Building 
Burlington, VT 05405 
+1 802 656-7748 (phone) 
 
Co-Investigator:  
 
Marya Carmolli 
 
 
 
 
Vaccine Testing Center 
University of Vermont College of Medicine 
Given Building 
Burlington, VT 05405 
+1 802 656-9296 (phone) 
 
Co-Investigator:  
 
Mary Claire Walsh 
 
 
 
 
Vaccine Testing Center 
University of Vermont College of Medicine 
Given Building 
Burlington, VT 05405 
+1 802 656-7764 (phone) 
 
Scientific Investigators: 
Stephen S. Whitehead, PhD  
 
Laboratory of Infectious Diseases 
National Institute of Allergy and Infectious Diseases, 
National Institutes of Health 
Room 3W10A 6, Building 33 
33 North Drive 
Bethesda, MD  20892, USA 
Phone: 301-496-7692 
 
Sponsor Medical Monitor: Marc J. Teitelbaum, MD, MS  
Clinical Monitoring Research Program (CMRP) 
Support to Regulatory Compliance and Human Subjects 
Protection Program / NIAID / NIH 
Leidos Biomedical Research, Inc. 
Frederick National Laboratory for Cancer Research 

4 
PR-15097  
 
V2.10: 16Jun17 
 
5705 Industry Lane, Room 249  
Frederick, Maryland  21704 
Phone (301) 228-4707 
Mobile (301) 917-4842 
Marc.Teitelbaum@nih.gov 
 
Shirley Jankelevich, MD 
Clinical Monitoring Research Program (CMRP) 
Frederick National Laboratory for Cancer Research  
Leidos Biomedical Research, Inc.  
Support to: Regulatory Compliance & Human Subjects 
Protection Program / NIAID / NIH 
5705 Industry Lane, Room 248  
Frederick, Maryland  21704 
Phone 301-846-7322 
Fax 301-846-6224 
shirley.jankelevich@nih.gov 
 
Clinical Study Site:  
International Centre for Diarrhoeal Disease Research, 
 
 
 
 
Bangladesh 
GPO Box 128 
Mohakhali, Dhaka, 1212 Bangladesh 
 
Research Laboratory:  
International Centre for Diarrhoeal Disease Research, 
Bangladesh 
GPO Box 128 
Mohakhali, Dhaka, 1212 Bangladesh 
 
Research Laboratory:  
Vaccine Testing Center 
University of Vermont College of Medicine 
Given Building 
89 Beaumont Avenue 
Burlington, VT 05405 
 
Clinical Laboratory:  
International Centre for Diarrhoeal Disease Research, 
Bangladesh 
GPO Box 128 
Mohakhali, Dhaka, 1212 Bangladesh 
   
Institutional Review Board: Ethical Review Committee  
(ICDDR,B) 
International Centre for Diarrhoeal Disease Research, 
Bangladesh 
GPO Box 128 
Mohakhali, Dhaka, 1212 Bangladesh 
 
Institutional Review Board: Research Review Committee  
(ICDDR,B) 
International Centre for Diarrhoeal Disease Research, 
Bangladesh GPO Box 128 
Mohakhali, Dhaka, 1212 Bangladesh 
 

5 
PR-15097  
 
V2.10: 16Jun17 
 
Institutional Review Board: Research Protections Office 
(UVM) 
 
 
Institutional Review Board 
 
 
 
 
University of Vermont 
 
 
 
 
213 Waterman Building 
 
 
 
 
85 South Prospect Street 
 
 
 
 
Burlington, VT 05405 
 
 
 
 
802-656-5040 (phone) 

6 
PR-15097  
 
V2.10: 16Jun17 
 
Table of Contents 
 
Team Roster ...................................................................................................................... 2 
Table of Contents .............................................................................................................. 6 
List of Tables .................................................................................................................... 9 
List of Figures ................................................................................................................. 10 
Protocol Summary: ......................................................................................................... 13 
1 Description of the Research Project ............................................................................ 14 
1.1 Primary Objectives .............................................................................................. 14 
1.2 Secondary Objectives .......................................................................................... 14 
1.3 Exploratory Objectives ........................................................................................ 14 
2 Introduction ................................................................................................................. 15 
2.1 Background - Dengue .......................................................................................... 15 
2.2 Background - Dengue Vaccines .......................................................................... 17 
2.3 Vaccine Description ............................................................................................. 17 
2.3.1 rDEN1Δ30 Vaccine ....................................................................................... 17 
2.3.1.1 Final Container rDEN1∆30 .................................................................... 18 
2.3.1.2 Composition rDEN1∆30......................................................................... 18 
2.3.1.3 Investigational Product Label rDEN1∆30 .............................................. 18 
2.3.2 rDEN2/4Δ30 (ME) Vaccine .......................................................................... 18 
2.3.2.1 Final Container rDEN2/4Δ30(ME) ........................................................ 19 
2.3.2.2 Composition rDEN2/4Δ30(ME) ............................................................. 19 
2.3.2.3 Investigational Product Label rDEN2/4Δ30(ME) .................................. 19 
2.3.3 rDEN3Δ30/31 Vaccine ................................................................................. 19 
2.3.3.1 Final Container of rDEN3∆30/31 ........................................................... 20 
2.3.3.2 Composition rDEN3∆30/31 ................................................................... 20 
2.3.3.3 Investigational Product Label rDEN3∆30/31 ......................................... 20 
2.3.4 rDEN4Δ30 Vaccine ....................................................................................... 20 
2.3.4.1 Final Container rDEN4∆30 .................................................................... 21 
2.3.4.2 Composition rDEN4∆30......................................................................... 21 
2.3.4.3 Investigational Product Label rDEN4∆30 .............................................. 21 
2.4 Rationale .............................................................................................................. 21 
2.4.1 Animal Studies .............................................................................................. 21 
2.4.2 Clinical Experience with Recombinant Live Attenuated Dengue Vaccine 
Candidates .............................................................................................................. 23 
2.4.2.1 Clinical Experience with LATV DEN1, DEN2, DEN3, and DEN4 
Monovalent Vaccines ......................................................................................... 23 
2.4.2.2 Summary of Clinical, Virologic, and Serologic Response to Monovalent 
Vaccines .............................................................................................................. 25 
2.4.2.3 Clinical, Virologic, and Serologic Response to a Tetravalent Admixture 
TV005 ................................................................................................................. 28 
2.4.3 Clinical Experience with TV005 in an Endemic Population ........................ 29 
2.4.4 Mosquito Transmissibility of Monovalent Vaccine Candidates ................... 33 
3 Study Design................................................................................................................ 34 
3.1 Overall Study Design ........................................................................................... 34 
3.2 Dosing Strategy.................................................................................................... 35 
3.3 Sample Size and Placebo Ratio ........................................................................... 36 
3.4 Duration of Participation ..................................................................................... 36 
3.5 Estimated Duration of the Study .......................................................................... 36 

7 
PR-15097  
 
V2.10: 16Jun17 
 
3.6 Treatment Assignment ......................................................................................... 36 
3.7 Blinding ............................................................................................................... 37 
4 Selection and Enrolment of Participants ..................................................................... 37 
4.1 Inclusion Criteria ................................................................................................. 37 
4.2 Exclusion Criteria ................................................................................................ 38 
4.3 Elimination Criteria During the Study ................................................................. 40 
4.4 Contraindications to Vaccination......................................................................... 40 
4.5 Participant Withdrawal and Termination Criteria ............................................... 41 
5 Vaccine Preparation ..................................................................................................... 42 
5.1 Pre-Vaccine Preparation ...................................................................................... 43 
5.2 Vaccine Storage ................................................................................................... 43 
5.3 Vaccine Accountability ........................................................................................ 44 
5.4 Storage Disposition of Used/Unused Supplies .................................................... 44 
6 Study Procedures ......................................................................................................... 44 
6.1 Recruitment and General Screening .................................................................... 44 
6.2 Screening Procedures ........................................................................................... 45 
6.3 Detailed Study Procedures ................................................................................... 46 
6.3.1 Immunization Procedure ............................................................................... 49 
6.3.2 Visit 1, Study Day 0, Administration of Vaccine or Placebo ....................... 49 
6.3.3 Visit 2, Study Day 7 (+/- 1 day) .................................................................... 50 
6.3.4 Visit 3, Study Day 14 (+/- 1 day) .................................................................. 51 
6.3.5 Visit 4, Study Day 28 (+ 3 days) ................................................................... 52 
6.3.6 Visit 5, Study Day 56 (+/- 7 days) ................................................................ 53 
6.3.7 Visit 6, Study Day 180 (+/- 14 days) ............................................................ 53 
6.3.8 Visit 7, Study Day 360 (+/- 14 days) ............................................................ 54 
6.3.9 Visit 8, Study Day 720 (+/- 14 days) ............................................................ 54 
6.3.10 Visit 9, Study Day 1080 (+/- 14 days) ........................................................ 55 
6.3.11 Unscheduled Visits ...................................................................................... 58 
6.3.11.1 Close-out of Solicited Laboratory Adverse Events .............................. 58 
6.3.11.2 Fever Surveillance ................................................................................ 58 
6.3.11.3 Definition of Suspected Dengue ........................................................... 58 
6.3.11.4 Definition of Laboratory Confirmed Dengue ....................................... 59 
6.4 Solicited and Unsolicited Adverse Events ........................................................... 59 
6.4.1 Solicited Local Injection Site Adverse Events .............................................. 60 
6.4.2 Solicited General Adverse Events ................................................................. 60 
6.5 Clinical Laboratory Testing ................................................................................. 60 
6.6 Medical History and Concomitant Medications .................................................. 62 
6.7 Immunology Testing ............................................................................................ 62 
6.7.1 Antibody Testing ........................................................................................... 62 
6.7.2 Other Exploratory Immunological Assays .................................................... 63 
7 Adverse Event Monitoring .......................................................................................... 63 
7.1 Definitions ........................................................................................................... 63 
7.1.1 Adverse Event ............................................................................................... 63 
7.1.2 Serious Adverse Event (SAE) ....................................................................... 64 
7.1.3 Unexpected Adverse Events (UAEs) ............................................................ 65 
7.1.4 Serious and Unexpected Suspected Adverse Reaction (SUSAR) ................. 65 
7.1.5 Unanticipated Problem that is not an Adverse Event (UPnonAE) ............... 65 
7.1.6 Pre-existing Conditions, Worsening of Pre-existing Conditions .................. 65 
7.2 Assessment of Adverse Events ............................................................................ 66 
7.2.1 Identification of Adverse Events ................................................................... 66 

8 
PR-15097  
 
V2.10: 16Jun17 
 
7.2.2 Protocol Specific Adverse Event Definitions ............................................... 67 
7.2.3 Determination of Severity ............................................................................. 68 
7.2.4 Relationship of Adverse Events to Investigational Product Vaccine ............ 70 
7.2.5 Adverse Event Action Taken ........................................................................ 71 
7.2.6 Adverse Event Outcome ............................................................................... 71 
7.2.7 Adverse Event Seriousness ........................................................................... 72 
7.3 Adverse Event Reporting ..................................................................................... 72 
7.3.1 Non-Serious Adverse Events ........................................................................ 72 
7.3.2 Serious Adverse Events ................................................................................. 72 
7.4 Sponsor’s Reporting Responsibilities .................................................................. 73 
7.5 Stopping Criteria .................................................................................................. 73 
7.6 Pausing Criteria.................................................................................................... 74 
8 Data Collection and Monitoring .................................................................................. 74 
8.1 Source Documentation and Data Collection ........................................................ 74 
8.2 Study Documentation .......................................................................................... 75 
8.3 Retention of Specimens ....................................................................................... 75 
8.4 Retention of Records ........................................................................................... 76 
8.5 Protocol Compliance............................................................................................ 77 
8.6 Investigator’s Brochure ........................................................................................ 77 
8.7 Study Monitoring ................................................................................................. 77 
9 Statistical Considerations ............................................................................................ 78 
9.1 General Design .................................................................................................... 78 
9.2 Study Endpoint Analysis ..................................................................................... 78 
10 Statistical methods ..................................................................................................... 79 
10.1 Estimated Sample Size....................................................................................... 79 
10.2 Cohort Evaluations ............................................................................................ 79 
10.3 Safety ................................................................................................................. 80 
10.3.1 Adverse Events ............................................................................................ 80 
10.3.2 Suspected and Confirmed Dengue .............................................................. 80 
10.4 Immunogenicity ................................................................................................. 80 
10.5 Viremia .............................................................................................................. 81 
10.6 CMI Exploratory Objectives .............................................................................. 81 
11 Monitoring Plan ......................................................................................................... 82 
11.1 DSMBs: NIAID Intramural and ERC Data and Safety Monitoring Boards...... 82 
11.2 Sponsor Medical Monitor .................................................................................. 83 
11.3 Safety Review and Communications Plan ......................................................... 83 
12 Protection of Human Subjects ................................................................................... 83 
12.1 Institutional Review Boards............................................................................... 83 
12.2 Informed Consent .............................................................................................. 84 
12.3 Risks .................................................................................................................. 85 
12.3.1 Venipuncture ............................................................................................... 85 
12.3.2 Immunization .............................................................................................. 85 
12.4 Benefits .............................................................................................................. 86 
12.5 Compensation .................................................................................................... 86 
12.6 Confidentiality ................................................................................................... 86 
12.7 Biohazard Containment ..................................................................................... 87 
13 Study Specific Policies and Procedures ..................................................................... 87 
13.1 Collaborations .................................................................................................... 87 
13.2 Study Sponsor and Funding ............................................................................... 87 
14 Facilities ..................................................................................................................... 89 

9 
PR-15097  
 
V2.10: 16Jun17 
 
Literature Cited ............................................................................................................... 89 
    
 
List of Tables 
 
Table 1: List of Abbreviations ........................................................................................ 11 
Table 2: Serum antibody responses induced by TV005 when given as a single 
subcutaneous dose .................................................................................................. 22 
Table 3: Monovalent components of TetraVax-DV ....................................................... 23 
Table 4: Clinical response to monovalent DENV vaccine candidates for use in study 
admixtures .............................................................................................................. 26 
Table 5: Magnitude, onset, and duration of viremia in participants inoculated with 
monovalent DENV vaccine candidates for use in tetravalent vaccine admixtures 27 
Table 6: Serum antibody responses induced by each monovalent DENV candidate 
vaccine given as a single subcutaneous dose .......................................................... 27 
Table 7: Magnitude, onset, and duration of viremia in participants inoculated with 
TV005, dose 1 (CIR 268 and CIR 279) .................................................................. 30 
Table 8: Serum antibody responses induced by TV005 when given as a single 
subcutaneous dose .................................................................................................. 30 
Table 9: Geometric mean antibody titer to each serotype following a single dose of 
TV005 ..................................................................................................................... 30 
Table 10: TV005 induce a broad neutralizing antibody response to all 4 serotypes ...... 31 
Table 11: Geometric mean antibody titer to each serotype following a second dose of 
TV005 (CIR 279).................................................................................................... 31 
Table 12: Percent and cumulative seropositivity rates to multiple DENV following the 
first and second dose of TV005 (CIR 279). ............................................................ 32 
Table 13: Clinical response to TV005 vaccine candidate in an endemic setting 
(Thailand) ............................................................................................................... 32 
Table 14: Dosing Strategy .............................................................................................. 35 
Table 15: Study specific procedures ............................................................................... 47 
Table 16: Approximate Blood volumes per study visit per cohort ................................. 56 
Table 17: Laboratory assays ........................................................................................... 62 
Table 18: Solicited adverse events: study participants > 5 years of age ........................ 64 
Table 19: Solicited adverse events: study participants < 5 years of age ........................ 64 
Table 20: Severity definitions ......................................................................................... 69 
Table 21: Assessment of solicited adverse events .......................................................... 69 
Table 22: Assessment of solicited laboratory AEs ......................................................... 70 
 
 
 
 
 
 
 
 
 
 
 
 

10 
PR-15097  
 
V2.10: 16Jun17 
 
List of Figures 
 
Figure 1: Label for Final Vial rDEN1Δ30-1545 (Enlarged Sample) ............................. 18 
Figure 2: Label for Final Vial rDEN2/4∆30(ME)-1495, 7163 (Enlarged Sample) ........ 19 
Figure 3: Label for Final Vial rDEN3∆30/31 (Enlarged Sample) .................................. 20 
Figure 4: Label for Final Vial rDEN4∆30-7132, 7163, 8308 (Enlarged Sample) ......... 21 
 
 
 

11 
PR-15097  
 
V2.10: 16Jun17 
 
 
 
Table 1: List of Abbreviations  
Abbreviation 
Definition  
AE 
Adverse event 
ALT 
alanine transaminase 
ANC 
Absolute neutrophil count 
AST 
aspartate aminotransferase 
CBC 
complete blood count 
CIR 
Center for Immunization Research 
CMI 
Cell mediated immunity 
CRF 
Case report form 
CRL 
Charles River Laboratories 
DENV  
Dengue viruses (serotypes DEN1, DEN2, DEN3, and DEN4) 
DF 
Dengue fever 
DGDA 
Directorate General of Drug Administration  
DHF 
Dengue haemorrhagic fever 
DSMB 
Data Safety Monitoring Board 
DSMP  
Data safety monitoring plan 
DSS 
Dengue shock syndrome 
EPI 
Expanded Program on Immunization 
ERC 
Ethical Review Committee (icddr,b regulatory body) 
FA 
Field Assistant 
FDA 
Food and Drug Administration 
FRA 
Field Research Assistant 
GCP 
Good Clinical Practice 
HBsAg 
Hepatitis B surface antigen 
HBV 
Hepatitis B virus 
HCV 
Hepatitis C virus 
HLA 
Human leukocyte antigen 
icddr,b 
International Centre for Diarrhoeal Disease Research, Bangladesh 
ICH 
International Conference on Harmonization 
IND 
Investigational new drug 
IRB 
Institutional Review Board 
JHSPH 
Johns Hopkins Bloomberg School of Public Health 
LID 
Laboratory of Infectious Diseases 
MID50 
50% Mosquito infectious dose 
NIAID 
National Institute of Allergy and Infectious Diseases 
NIH 
National Institutes of Health 
NS 
Non-structural 
OCRPRO 
Office of Clinical Research Policy and Regulatory Operations 
PBMC 
Peripheral blood mononuclear cell 
PFU 
Plaque-forming units 
PI 
Principal Investigator 
PRNT 
Plaque reducing neutralizing antibody titer assay 
PT/PTT 
prothrombin time/ partial thromboplastin time 
RCHSPB 
Regulatory Compliance and Human Subjects Protection Branch 
ROP 
Retro-orbital pain 

12 
PR-15097  
 
V2.10: 16Jun17 
 
RRC 
Research Review Committee (icddr,b regulatory body) 
SAE 
Serious adverse event 
SCID 
Severe combined immunodeficiency 
SE 
Standard error 
SERF 
Safety expedited report form 
SOP 
Standard Operating Procedure 
SRCP 
Safety Review and Communication Plan 
SSP 
Study Specific Procedure 
SUSAR 
Serious and unexpected adverse reaction 
TV005  
Tetravalent 005 
UAE 
Unexpected adverse event 
UP 
Unanticipated problem 
UTR 
untranslated region 
WHO  
World Health Organization 
Wt 
Wild type 
 
  
 
 
 
 
 
 

13 
PR-15097  
 
V2.10: 16Jun17 
 
Protocol Summary: 
Principal Investigator: Dr. Rashidul Haque 
Research Protocol Title: Phase II, randomized, double-blind, placebo-controlled study of the safety and 
immunogenicity of the recombinant live attenuated tetravalent dengue virus vaccine admixture TV005 
(TetraVax-DV T005) in healthy adults, adolescents, and children in Dhaka, Bangladesh 
Proposed start date:  January 2016                                               Estimated end date: February 2020  
Background (brief): 
a. Burden:  
Dengue viruses (DENV) are now the leading arboviral infection globally, with over 2 billion persons at risk of 
infection with continually expanded regions affected by dengue (1).  The first dengue fever outbreak in 
Bangladesh occurred in 2000, and the burden of dengue disease in this area has sustained since then (2).   
b. Knowledge gap:  
A dengue vaccine has not yet been tested on the Indian subcontinent, despite very large populations of persons 
at risk for infection. In Dhaka, an extremely densely populated city of 12-20 million inhabitants, antibody 
testing demonstrates that 80% of the population (9.6-16 million persons) has had exposure to dengue.  
c. Relevance: 
Dengue is an emerging and reemerging arboviral disease of great global public health importance. 
Since the outbreak in 2000, dengue fever has had a continued presence throughout Bangladesh. In recent time 
several live attenuated vaccines for dengue have been developed and tested in different parts of the world. 
Understanding the safety and immunogenicity of a live attenuated vaccine has never been assessed in 
Bangladeshi population. Successful completion of this study will give us safety and immunogenicity data of 
such vaccine in Bangladesh.  
Objectives:  
Primary Objectives 
• 
Evaluation of the safety of TV005 live-attenuated dengue vaccine in healthy adults, followed by 
adolescents, and children in a dengue-endemic area of Dhaka (Mirpur), Bangladesh 
• 
Evaluation of vaccine immunogenicity based on neutralizing antibody level to DEN1, DEN2, DEN3, and 
DEN4 at days 14, 28, 56, and day 180. 
Secondary Objectives 
• 
To measure the frequency, quantity, and duration of viremia after vaccination (both vaccine and natural 
dengue virus) measured at day 7 and 14. 
• 
To determine the number of vaccinees with recoverable vaccine virus or wild type (wt) dengue virus 
after vaccination. 
• 
To determine the effect of pre-existing dengue antibodies on recovery of vaccine virus and or 
frequencies of seroconversion in each age group. 
• 
To determine the durability of neutralizing antibody response at 1 year, 2 years, and 3 years after 
vaccination with TV005. 
Methods: 
A randomized, step-wise, double-blind, placebo- controlled study design will be employed with 4 age de-
escalation cohorts. 192 study participants (48 in each age cohort) will be enroled. Study participants will be 
screened, and enroled to receive either TV005 vaccine or placebo.  There will be 8 follow-up visits to collect 
information on vaccine safety and immunogenicity.  Evaluation of vaccine safety will be determined by the 
frequency of solicited and unsolicited adverse events.  Evaluation of immunogenicity will be measured by 
Plaque Reducing Neutralizing Antibody Titer (PRNT) assay.   
Outcome measures/variables: 
The main outcome variables are the following: 
• 
Proportion of study participants with solicited or unsolicited Adverse Events. 
• 
Proportion of study participants who are seropositive or seroconvert following vaccination. 
• 
Proportion of study participants with vaccine virus recovered following vaccination. 
• 
Proportion of study participants with recoverable vaccine virus or wild type virus after vaccination 
 
 

14 
PR-15097  
 
V2.10: 16Jun17 
 
 
 
1 Description of the Research Project 
 
 
Specific Objectives: 
 
1.1 Primary Objectives 
The primary objectives are to determine the safety, immunogenicity, and durability of 
the TV005 tetravalent live attenuated dengue vaccine in healthy adults followed by 
assessment in adolescents and children residing in an endemic area in Mirpur, Dhaka 
Bangladesh. 
1 Determine the safety of the TV005 tetravalent live- attenuated dengue vaccine as 
assessed by the frequency of vaccine-related adverse events (AEs), graded by 
severity. The study will begin in adults and age de-escalate by cohort based upon the 
interpretation of safety and immunogenicity data for the adult cohort, provided that 
stopping criteria are not met following analysis of safety data through Study Day 28 
by the Data and Safety Monitoring Boards (DSMBs). 
2 Determine the immunogenicity of the TV005 tetravalent live attenuated dengue 
virus (DENV) vaccine, as assessed by neutralizing antibody titers to DENV-1, 
DENV-2, DENV-3, and DENV-4 at 28, 56, and 180 days after each vaccination. 
Monovalent, bivalent, trivalent, and tetravalent seroconversion rates will be 
determined. 
 
1.2 Secondary Objectives 
1 Assess the frequency, quantity, and duration of vaccine viremia following 
vaccination for both vaccine and wild-type dengue. 
2 Determine the number of vaccinees with recoverable DENV-1, DENV-2, DENV-3, 
and DENV-4 vaccine virus components or wild-type dengue virus during the first 14 
days following vaccination.  This is defined as recovery of vaccine virus from the 
blood or serum of a participant and/or by new seropositivity (Plaque Reducing 
Neutralizing Antibody Titer (PRNT)50 ≥ 1:10) in a previously seronegative study 
participant or seroconversion to DENV (> 4-fold rise in neutralizing antibody titers 
against DENV-1, DENV-2, DENV-3, or DENV-4) in those volunteers who were 
seropositive at baseline. 
3 Determine the effect of pre-existing DENV antibodies on recovery of vaccine virus 
from the blood or serum of a study participant and/or seroconversion frequencies to 
DENV among the different age groups. 
4 Determine the durability of neutralizing antibody response at 1 year, 2 years, and 3 
years after vaccination with TV005. 
 
1.3 Exploratory Objectives 
To evaluate the individual immune response to vaccination examining the following: 
1 New serological measures of immunogenicity and vaccine efficacy surround 
neutralization and enhancement. 

15 
PR-15097  
 
V2.10: 16Jun17 
 
2 Cell-mediated immunity including stimulation of T and B cells for measures of 
adaptive immune response post vaccination 
3 Evaluation of innate immune response post vaccination 
4 Other immunologic assays may be performed in response to the development of 
additional assay techniques and current literature and scientific knowledge over the 
course of the study.   
 
 
2 Introduction 
 
2.1 Background - Dengue 
The World Health Organization (WHO) estimates that dengue fever (DF) is the fastest 
emerging arboviral infection and is spread by Aedes mosquitos. Dengue has far-
reaching public health consequences throughout the tropical and sub-tropical regions of 
the world, primarily in urban and semi-urban areas. Globally, over 2.5 billion people--
over 40% of the world's population--are at risk for DF, and more than 75%, or 1.8 
billion, live in the Asia- Pacific region (3, 4). WHO estimates that there may be 50-100 
million dengue infections worldwide each year (1) and approximately one half million 
cases annually of the more severe disease, dengue haemorrhagic fever/dengue shock 
syndrome (DHF/DSS) (3).  An estimated 90% of those individuals with severe dengue 
infection requiring hospitalization are children less than 5 years of age. Infection with 
dengue virus (DENV) is the leading cause of hospitalization and death in children in at 
least 8 tropical Asian countries (4).  For these reasons, the WHO has made development 
of a dengue vaccine a top priority (Resolution WHA 46.31). 
 
Dengue virus was first documented in Bangladesh, known as East Pakistan at the time, 
in 1964, when the DENV-3 serotype was isolated from a patient with febrile illness.  
Since this first documented illness, sporadic suspected cases of dengue fever have 
occurred in Bangladesh between the 1960’s and the 1990’s (5).  In the summer of 2000, 
the first outbreak of dengue fever and dengue haemorrhagic fever occurred in the major 
cities of Bangladesh with over 5000 cases of dengue fever reported to the Ministry of 
Health.  While the majority of the cases were dengue fever, approximately 25% of cases 
were dengue haemorrhagic fever (2).  Since that outbreak, dengue fever has had a 
continued presence throughout Bangladesh.  One study has shown the overall 
seroprevalence of dengue in Dhaka to be approximately 80% (5).   
 
There are 4 serotypes of DENV (DENV-1, DENV-2, DENV-3, and DENV-4), each 
capable of causing dengue infection ranging from asymptomatic infection or mild and 
self-limited febrile illness, to a life-threatening disease. Infection with one dengue 
serotype induces long-lived homotypic immunity to the infecting serotype and short-
lived heterotypic immunity to the other serotypes (6).  Immunity appears mediated, at 
least in part, by neutralizing antibodies against the envelope (E) glycoprotein.   
 
Epidemiological studies have demonstrated that most cases of DHF/DSS occur in 
persons experiencing a second dengue infection with a serotype different from that 
which caused their first dengue infection (7).  For this reason, DHF/DSS occurs 
predominately in children or adults living in dengue-endemic regions with multiple 
DENV serotypes circulating simultaneously.   

16 
PR-15097  
 
V2.10: 16Jun17 
 
 
The goal of immunization is to induce a long-lived protective immune response against 
all 4 DENV serotypes.  This can be best and most economically achieved using a live 
attenuated tetravalent virus vaccine delivered in one or more doses.  Development of a 
live attenuated vaccine is a reasonable goal since it has already been achieved for other 
related mosquito-borne flaviviruses, including the yellow fever and Japanese 
encephalitis viruses which are also endemic to tropical regions of the world (3, 8). 
 
In humans, DENV infects predominately monocytes, dendritic cells, and lymphocytes 
and does not exhibit tropism for any particular organ, except perhaps the liver (9).  The 
majority of primary and secondary infections with DENV are asymptomatic.  Following 
an incubation period of approximately 1 week, self-limiting acute illness, (DF), occurs 
and is characterized by a febrile period of about 5 days accompanied by systemic 
symptoms such as headache, malaise, anorexia, arthralgia, and myalgia. Rash (including 
petechial haemorrhages), lymphadenopathy, leukopenia, thrombocytopenia, and viremia 
can accompany the fever.  Elevations of liver enzymes during DF are common, and the 
virus is known to infect human hepatocytes (10-12).  The virus does not establish 
persistent infection and is usually eliminated by the end of the second week.   
 
DHF/DSS occurs much less commonly than DF.  It develops at the time of 
defervescence and is characterized by an increased tendency to bleed into the skin or 
from mucous membranes and by a marked increase in vascular permeability resulting in 
haemoconcentration and shock.  This state of increased vascular permeability is short-
lived (a few days) and with proper management is fatal in only about 1% of patients (3).  
Because previous infection with one DENV serotype can increase the risk for DHF/DSS 
following infection with a different serotype, it is clear that a dengue vaccine will need 
to protect against each of the 4 DENV virus serotypes, namely DENV-1, DENV-2, 
DENV-3, and DENV-4. 
 
The DENV genome contains a single open reading frame encoding a polyprotein that is 
processed by proteases of both viral and cellular origin into 3 structural proteins, namely 
the capsid (C), membrane, (M) and envelope (E) proteins and at least 7 non-structural 
(NS) proteins.  Each end of the DENV genome consists of an untranslated region (UTR) 
that is predicted to be highly structured.  The E glycoprotein is on the surface of the 
virion, and immunity is mediated primarily by neutralizing antibodies to this protein.  
The E protein also uniquely defines each of the 4 DENV serotypes (DENV-1, DENV-2, 
DENV-3, and DENV-4).  The 3´ untranslated region (UTR) is highly conserved 
between the 4 DENV serotypes.   
 
Studies in mice, Rhesus monkeys, and mosquitoes were designed to evaluate the level 
of attenuation conferred independently by chimerization and the ∆30 (30 nucleotide 
deletion in the 3´UTR) mutation (13).  This attenuation strategy has been successfully 
used to design the TV005 tetravalent vaccine proposed to be tested in this clinical trial. 
The specific objectives of this study are to evaluate the safety, infectivity, and 
immunogenicity of the TV005 vaccine admixture in a dengue endemic area where 
volunteers have been variously exposed to DENV.  Since the greatest need for a dengue 
vaccine is among young children living in dengue endemic regions, the planned study is 
designed as an age de-escalation strategy. The TV005 admixtures will be comprised of 
four monovalent dengue vaccine candidates representing each of the four DENV 
serotypes: rDEN1∆30, rDEN2/4Δ30(ME), rDEN3∆30/31, and rDEN4∆30. 

17 
PR-15097  
 
V2.10: 16Jun17 
 
 
2.2 Background - Dengue Vaccines 
Numerous studies of wild-type (wt) DENV and live attenuated dengue vaccine 
candidates have been conducted over the past 60 years.  Various live attenuated dengue 
vaccine candidate viruses have been administered to nearly 1500 study participants in 
Phase 1 and Phase 2 clinical studies (14-24). The majority of dengue vaccines tested to 
date have been biologically derived by repeated passage in tissue culture.  Although 
some of these vaccine candidates have appeared promising in early clinical 
development, none has achieved the ideal balance between reactogenicity and 
immunogenicity, particularly when included as part of a tetravalent vaccine formulation 
(22, 23, 25). It has been difficult to achieve adequate antibody responses to all 4 
serotypes when the vaccine viruses are administered in the tetravalent formulation.   
 
Common adverse events (AEs) noted in these studies were signs and symptoms of mild 
DF, notably fever, headache, myalgias, rash, neutropenia, and elevated liver function 
tests.  In the published literature, all subjects who experienced an AE recovered 
completely and without sequelae. To date, no live attenuated dengue vaccine candidate 
has induced DHF or DSS.  The lack of a suitable licensed tetravalent live attenuated 
vaccine candidate forms the basis for testing the TV005 candidate vaccine described in 
this protocol. 
 
2.3 Vaccine Description 
The admixture of the candidate vaccine contains 4 live attenuated dengue viruses, each 
of a different serotype.  The description of each component is described below. The 
monovalent vaccine candidates may be stored at -80°C ± 15°C.  
 
The components of this vaccine candidate have collectively been administered to many 
volunteers without any significant adverse or permanent effect, however the formulation 
of the vaccine used in this trial is not a licensed product (24, 27, 35-40).  TV005 is 
currently being evaluated for safety and immunological effects which may result in 
licensure. 
 
2.3.1 rDEN1Δ30 Vaccine 
The vaccine candidate rDEN1∆30 is a live attenuated virus derived from the DEN1 
Western Pacific (WP) wt strain of DENV using recombinant DNA technology.  A full-
length cDNA clone of the DEN1 WP genome was constructed and a 30-nucleotide 
deletion was then created in the 3´UTR. Genome-length, capped, RNA transcripts were 
synthesized, purified, and transfected into qualified Vero cells.  Recovered virus was 
terminally diluted and then amplified by serial passaging in Vero cells.  The final 
amplification of virus was made in serum-free medium, and the titer was determined by 
plaque titration in Vero cells.  From initial transfection through final amplification, only 
serum-free medium was used for Vero cell culture and propagation of virus.  Reagents 
used during the transfection process and present initially in fluid harvested from the 
transfection were diluted more than 1012-fold as a result of the biological cloning 
(terminal dilution) and amplification of the virus.  The rDEN1Δ30-1545 seed virus was 
generated in the Laboratory of Infectious Diseases (LID), National Institute of Allergy 
and Infectious Diseases (NIAID), and the vaccine was manufactured at Charles River 
Laboratories (CRL) Biopharmaceutical Services facility in Malvern, PA.  The Final 
Drug Product, rDEN1Δ30-1545 (Lot DEN1#104A) was manufactured in qualified Vero 

18 
PR-15097  
 
V2.10: 16Jun17 
 
cells at CRL on 26 August 2004 based on a method developed by LID/NIAID/National 
Institutes of Health (NIH) and CRL.  
 
2.3.1.1 Final Container rDEN1∆30 
The Final Drug Product was dispensed as 0.6 mL aliquots of approximately 106.8  
plaque-forming units [PFU]/mL Live Recombinant Dengue Virus Type 1 rDEN1∆30-
1545 Vero Grown Virus Vaccine into 2.0 mL sterile cryovials and stored at -80°C ± 
15°C. 
 
2.3.1.2 Composition rDEN1∆30 
The Final Drug Product composition is a concentration of live attenuated rDEN1∆30-
1545 (Lot DEN1#104A) in Leibovitz L-15 Medium containing 1X SPG (sucrose, 0.218 
M; KH2PO4, 0.0038 M; K2HPO4, 0.0072 M; mono-sodium glutamate, 0.0054 M). The 
potency of rDEN1Δ30-1545 (Lot DEN1#104A) is 106.8 PFU/mL. 
 
2.3.1.3 Investigational Product Label rDEN1∆30 
 
0804- Vial # 
Lot DEN1#104A 
Live Recombinant Dengue Virus Type 1  
rDEN1Δ30-1545 
Vero Grown Virus Vaccine 
CAUTION: NEW DRUG LIMITED 
BY FEDERAL (USA) LAW 
TO INVESTIGATIONAL USE 
Store at -70°C or below 
Charles River Laboratories 
Malvern PA 
Figure 1: Label for Final Vial rDEN1Δ30-1545 (Enlarged Sample) 
 
*NOTE: Since manufacture of the Final Drug Product, evaluation of live 
attenuated dengue virus vaccines (developed at the LID/NIH/ NIAID) in both 
laboratory and clinical situations indicates that these viruses are stable and 
potent when stored at a temperature of -80°C ± 15°C. To maintain consistency in 
Investigational Product storage requirements, all vaccine monovalent candidates 
will be stored together in a dedicated, locked, and secure freezer at -80°C ± 
15°C, and the manufacturer’s stability data and confirmation that this storage 
parameter will be adequate for vaccine storage is documented in the 
Investigator’s Brochure.  
 
2.3.2 rDEN2/4Δ30 (ME) Vaccine 
The rDEN2/4∆30 (ME) virus is a live attenuated chimeric virus derived from 
rDEN4∆30.  A full-length cDNA copy of rDEN4∆30 was constructed in which the M 
and E coding sequences of DEN4 were replaced with those of DEN2 NGC prototype.  
Genome-length, capped, RNA transcripts were synthesized, purified, and transfected 
into qualified Vero cells.  Recovered virus was terminally diluted and then amplified by 
serial passaging in Vero cells.  The final amplification of virus was made in serum-free 
medium and the titer was determined by plaque titration in Vero cells.  The 
rDEN2/4Δ30(ME)-1495,7163 seed virus was generated in LID/NIAID and then 
manufactured at Charles River Laboratories Biopharmaceutical Services facility in 

19 
PR-15097  
 
V2.10: 16Jun17 
 
Malvern, PA.  The Final Drug Product, rDEN2/4Δ30(ME)-1495,7163 (Lot 
DEN2/4#106C) was manufactured in qualified Vero cells at CRL on 8 February 2007 
based on a method developed by LID/NIAID/NIH and CRL. 
 
2.3.2.1 Final Container rDEN2/4Δ30(ME) 
The Final Drug Product was dispensed as 0.6 mL aliquots of approximately 107.8 
PFU/mL Live Recombinant Dengue Virus Type 2 rDEN2/4Δ30(ME)-1495,7163 Vero 
Grown Virus Vaccine into 2.0 mL sterile cryovials (Corning #430659) and is stored at -
80°C ± 15°C. 
 
2.3.2.2 Composition rDEN2/4Δ30(ME) 
The Final Drug Product composition is a concentration of live attenuated 
rDEN2/4Δ30(ME)-1495,7163 (Lot DEN2/4#106C) in Leibovitz L-15 Medium 
containing 1X SPG. The potency of rDEN2/4Δ30(ME)-1495,7163 (Lot DEN2/4#106C) 
is 107.8 PFU/mL. 
 
2.3.2.3 Investigational Product Label rDEN2/4Δ30(ME) 
 
Live Recombinant Dengue Virus Type 2 
rDEN2/4∆30(ME)-1495,7163 
VERO Grown Virus Vaccine 
Lot DEN 2/4 #106C 
0207-vial # 
CAUTION: NEW DRUG LIMITED 
BY FEDERAL (USA) LAW 
TO INVESTIGATIONAL USE 
Store at -70±10oC 
Charles River Laboratories, Malvern, PA 
Figure 2: Label for Final Vial rDEN2/4∆30(ME)-1495, 7163 (Enlarged 
Sample) 
 
2.3.3 rDEN3Δ30/31 Vaccine 
rDEN3∆30/31 was constructed by further mutating the DEN3 3´ UTR of the DEN3 
Sleman/78 by combining the ∆30 deletion with an additional, 31 nucleotide deletion in 
the DEN3 3´UTR(23). Since the flavivirus 3´ UTR is non-coding, it is well suited for 
use as a genetic element to condition attenuation in DENV vaccine candidates.  The 
attenuation phenotype of the rDEN3∆30/31 virus vaccine is expected to be 
phenotypically stable since the ∆30 mutation was shown to be stable following 
administration of the DEN4∆30 virus vaccine to human study participants (24).  
Genome-length, capped, RNA transcripts were synthesized from the linearized 
p3Δ30/31 clone #28 using the AmpliCap SP6 Message Maker Kit (EpiCentre 
Technologies, Madison, WI; Lot AC650408) and purified using the RNeasy Mini Kit 
(Qiagen, Valencia, CA).   
 
Virus was recovered in qualified C6/36 cells (Sponsor’s Master File, MF #11371) 
transfected on 6 January 2006 with purified RNA transcripts using DOTAP liposomal 
transfection reagent (Roche, Indianapolis, IN).  An aliquot of the seed virus was 
submitted to Charles River Laboratories Biopharmaceutical Services (Malvern, PA), for 
production of a second lot of vaccine. The lot was amplified by 1 passage on qualified 

20 
PR-15097  
 
V2.10: 16Jun17 
 
Vero cells in serum free medium, medium containing buffer and essential amino acids 
and not containing fetal bovine serum, to produce the Final Drug Product, 
rDEN3Δ30/31Lot DEN3#113B. Final drug product was manufactured on 18 January 
2012. 
 
2.3.3.1 Final Container of rDEN3∆30/31 
The Final Drug Product was dispensed as 0.6 mL aliquots of approximately 107.7 
PFU/mL of Live Recombinant Dengue Virus Type 3 rDEN3Δ30/31 Vero Grown Virus 
Vaccine in L-15 medium containing 1X SPG (sucrose, 0.218 M; KH2PO4, 0.0038 M; 
K2HPO4, 0.0072 M; mono-sodium glutamate, 0.0054 M) into 2.0 mL sterile cryovials 
and is stored at -80°C ± 15°C. 
 
2.3.3.2 Composition rDEN3∆30/31 
The Final Drug Product composition is a concentration of live attenuated rDEN3Δ30/31 
in Leibovitz L-15 medium containing 1X SPG (sucrose, 0.218 M; KH2PO4, 0.0038 M; 
K2HPO4, 0.0072 M; mono-sodium glutamate, 0.0054 M).  The titer of rDEN3Δ30/31 
(Lot DEN3#113B) is 107.7 PFU/mL. 
 
2.3.3.3 Investigational Product Label rDEN3∆30/31 
 
Live Recombinant Dengue Virus Type 3 
rDEN3∆30/31 
VERO Grown Virus 
Date: 18JAN2012 
Vial#: XXXX 
Lot: DEN3#113B 
CAUTION: NEW DRUG LIMITEDBY 
FEDERAL (USA) LAW 
TO INVESTIGATIONAL USE 
Store at -70±10oC 
Charles River Laboratories, Malvern, PA      
 
Figure 3: Label for Final Vial rDEN3∆30/31 (Enlarged Sample) 
 
2.3.4 rDEN4Δ30 Vaccine 
The vaccine candidate rDEN4∆30 is a live attenuated virus derived from the DEN4 
Dominica/81 wt strain of DENV using recombinant DNA technology.  A full-length 
cDNA clone of the DEN4 Dominica/81 genome was constructed and a 30-nucleotide 
deletion was then created in the 3´ UTR.  Genome-length, capped, RNA transcripts 
were synthesized, purified, and transfected into qualified Vero cells.  Recovered virus 
was terminally diluted and then amplified by serial passaging in Vero cells.  The final 
amplification of virus was made in serum-free medium, and the titer was determined by 
plaque titration in Vero cells.  From initial transfection through final amplification, only 
serum-free medium was used for Vero cell culture and propagation of virus. The 
rDEN4Δ30-7132,7163,8308 seed virus was generated at LID/NIAID, and vaccine was 
manufactured at Charles River Laboratories Biopharmaceutical Services facility in 
Malvern, PA.  The Final Drug Product, rDEN4∆30-7132,7163,8308 (Lot DEN4#109A) 
was manufactured in qualified Vero cells at CRL on 18 July 2007 based on a method 
developed by LID/NIAID/NIH and CRL. 
  

21 
PR-15097  
 
V2.10: 16Jun17 
 
2.3.4.1 Final Container rDEN4∆30 
The Final Drug Product was dispensed as 0.6 mL aliquots of approximately 107.2 
PFU/mL Live Recombinant Dengue Virus Type 4 rDEN4∆30-7132,7163,8308 Vero 
Grown Virus Vaccine into 2.0 mL sterile cryovials (Corning #430659) and stored at -
80°C ± 15°C. 
 
2.3.4.2 Composition rDEN4∆30 
The Final Drug Product composition is a concentration of live, attenuated rDEN4∆30-
7132,7163,8308 (Lot DEN4#109A) in L-15 medium containing 1X SPG.  The potency 
of rDEN4∆30-7132,7163,8308 (Lot DEN4#109A) is 107.2 PFU/mL. 
 
 
2.3.4.3 Investigational Product Label rDEN4∆30 
 
 
 
 
 
 
 
 
 
 
Figure 4: Label for Final Vial rDEN4∆30-7132, 7163, 8308 (Enlarged 
Sample) 
 
2.4 Rationale 
 
2.4.1 Animal Studies 
Each of the component monovalent vaccine viruses of TV005 has been evaluated in 
juvenile rhesus monkeys and in the novel rodent model consisting of severe combined 
immunodeficiency (SCID) mice bearing intraperitoneal tumors of the human liver cell 
line HuH-7.  Replication of these candidate vaccine viruses in the SCID-HuH-7 mouse 
model was compared with that of their wt parent viruses.  The candidate vaccine viruses 
were attenuated in SCID-HuH-7 mice, with peak decreases in peak titers ranging from 
6-fold (rDEN4∆to to 200-fold (rDEN1∆30) lower than that of their wt parent DENV 
(13, 26, 28, 29).  Juvenile Rhesus monkeys were inoculated subcutaneously with 
monovalent doses (105 PFU) of rDEN1∆30, rDEN2/4Δ30(ME), rDEN3-3´D4∆30, 
rDEN3∆30/31, rDEN4∆30, or the wt parent DENV (10, 23, 25, 26). All monkeys 
inoculated with wt virus became viremic with the mean duration of viremia ranging 
from 2.8 days (DENV-1) to 5.5 days (DENV-2) (Table 2).  Fewer monkeys inoculated 
with the candidate vaccine viruses were viremic compared with the parent virus for each 
vaccine candidate except rDEN4∆30.  In addition, the mean number of days of viremia 
and the mean peak titer were also reduced for each candidate vaccine virus compared 
with its parent virus (Table 2). 
 
Live Recombinant Dengue Virus Type 4 
rDEN4∆30-7132,7163,8308 
VERO Grown Virus Vaccine 
Lot DEN 4 #109A 
0707-vial #     
CAUTION: NEW DRUG LIMITED 
BY FEDERAL (USA) LAW 
TO INVESTIGATIONAL USE 
Store at -70±10oC  
Charles River Laboratories, Malvern, PA 

22 
PR-15097  
 
V2.10: 16Jun17 
 
Table 2: Serum antibody responses induced by TV005 when given as a 
single subcutaneous dose 
Vaccine candidate 
Dose 
(log10 PFU) 
No. of 
monkeys 
No. 
Viremic 
Mean # of 
days 
viremic 
Mean peak 
serum titer 
(log10PFU/
mL) 
DENV-1 (wt) 
5 
4 
4 
2.8 
2.1 ± 0.1 
rDEN1∆30 
5 
4 
2 
0.5 
0.8 ± 0.1 
DENV-2 (wt) 
5 
4 
4 
5.5 
2.1 ± 0.15 
rDEN2/4Δ30(ME) 
5 
4 
1 
0.3 
0.7 ± 0.02 
DENV-3 (wt) 
5 
4 
4 
3.5 
1.8 ± 0.1 
rDEN3∆30/31 
5 
4 
0 
n/a 
n/a 
DENV-4 (wt) 
5 
4 
4 
3 
2.7 ± 0.09 
rDEN4∆30 
5 
6 
6 
2.5 
2.0 ± 0.15 
 
In early experiments, a tetravalent admixture of the live attenuated dengue vaccine 
candidates was evaluated for safety/toxicity, virus replication, and immunogenicity in 
Rhesus macaques (30), but contained a DENV-3 component different than that 
subsequently tested in humans. The admixture included rDEN1∆30, rDEN2/4Δ30(ME), 
rDEN3-3´D4∆30, and rDEN4∆30 (designated ‘TV2’). The animals received 105 PFU of 
each of the component viruses in the admixture in a single subcutaneous injection of 0.5 
mL. Monkeys were observed twice daily for mortality and morbidity, as well as for 
signs of neurologic, haemorrhagic, or dermal disease.  No signs consistent with these 
diseases were observed in any of the animals.  Multiple episodes of lymphopenia were 
seen in all monkeys inoculated with TV2, and in 3 of the 4 monkeys inoculated with 
placebo.  These results were seen throughout the study, including the samples taken on 
Day -8 and Day 0 prior to inoculation. Mild deviations from the normal reference range 
were noted on the clinical pathology profiles for most monkeys, but are not likely to be 
of statistical or biological significance.  The clinical profile tested included complete 
blood count (CBC)and differential, clotting profile (Prothrombin time/Partial 
thromboplastin time(PT/PTT)), clinical chemistry (Albumin, alanine transaminase (ALT), 
aspartate aminotransferase (AST), Calcium, Chloride, Cholesterol, Creatinine, Gamma 
glutamyltransferase (GGT), Globulin, Glucose, Lipase, Magnesium, Osmolality, 
Phosphorus, Potassium, Sodium, Total Bilirubin, Total Protein, and Triglyceride). Mild 
transient elevations in ALT and AST not related to the vaccine were seen. 
 
The DENV-4 component of the tetravalent vaccine was the only virus detected in any of 
the monkeys inoculated with the TV2 admixture.  The rDEN4∆30 vaccine virus was 
detected at low titer (101.0 to 101.3 PFU/mL) in 3 of the 4 monkeys that received TV2, 
and only on Study Day 2.  These findings confirm the attenuation (the absence of dengue 
vaccine virus detected in serum or dengue vaccine virus detected at a significantly lower 
level than seen in wild type virus (Table 2)) of each of the component vaccine viruses. 
 
Despite the low or undetectable levels of viremia observed in the animals, the 
tetravalent vaccine admixture elicited neutralizing antibody titers against the component 
vaccine viruses.  Inoculation of monkeys with TV2 resulted in 100% seroconversion to 
DENV-1, DENV-2, and DENV-4, and 50% to DENV-3 component.  Geometric mean 
titers for TV2 were ≥ 39, ≥69, ≥ 50, ≥ 70 for the DENV-1, DENV-2, DENV-3, and 
DENV-4 components respectively.   

23 
PR-15097  
 
V2.10: 16Jun17 
 
 
In summary, the TV2 tetravalent dengue virus vaccine formulation was safe and well-
tolerated in Rhesus monkeys and elicited antibody responses against the component 
viruses.   
 
2.4.2 Clinical Experience with Recombinant Live Attenuated Dengue Vaccine 
Candidates 
Each of the candidate live attenuated DENV vaccines included in TV005 has been 
evaluated in at least 1 Phase 1 clinical trial as a monovalent vaccine (Table 3). These 
trials were conducted at the Center for Immunization Research (CIR) at the Johns 
Hopkins Bloomberg School of Public Health (JHSPH) and at the University of Vermont 
Vaccine Testing Center.  The experience with each of these vaccine candidates is 
summarized below.  The absence of clinically apparent dengue-like illness is likely a 
result of the high degree of attenuation of the vaccine viruses.  Peak viremia titers 
observed in the studies described below were more than 1,000-fold lower than those 
observed with symptomatic wildtype DENV infection (31-33). 
 
Table 3: Monovalent components of TetraVax-DV 
Vaccine candidate 
Lot number 
IND number 
rDEN1∆30 
DEN1#104A 
IND 11677 
 
rDEN2/4Δ30(ME) 
DEN2/4#106C 
IND 11938 
rDEN3∆30/31 
M10377008 
DEN3#113B 
IND 13886 
 
rDEN4∆30 
DEN4#109A 
IND 8463 
 
 
2.4.2.1 Clinical Experience with LATV DEN1, DEN2, DEN3, and DEN4 
Monovalent Vaccines 
rDEN1∆30 was first evaluated at a dose of 103  PFU in 2 placebo-controlled Phase 1 
studies at the CIR/JHSPH(34).  In a single-dose-study, 28 healthy male and non-
pregnant female adult study participants, between the ages of 18 and 50, were enroled 
and were randomly assigned to receive vaccine or placebo. Twenty study participants 
received vaccine; eight study participants received placebo (vaccine diluent).   
 
In a 2-dose schedule comparison study of rDEN1∆30 lotDEN1#104A, 2 cohorts of 30 
flavivirus-naïve healthy male and non-pregnant female adult study participants between 
the ages of 18 and 50 were randomly assigned to receive their second dose of vaccine 
either 120 days or 180 days following their initial dose (35).  In each cohort, 25 study 
participants were randomly assigned to receive vaccine and 5 study participants were 
randomly assigned to receive placebo.  One study participant in this study was replaced 
after receiving vaccine and a second study participant was replaced after receiving 
placebo.  Thus far, a total of 71 study participants have received rDEN1∆30 at a dose of 
103 PFU.  The vaccine was found to be safe and immunogenic (Table 4, Table 5, and 
Table 6).  The candidate vaccine was further studied at a dose of 101 PFU to determine 
the 50% human infectious dose (HID50) of this candidate vaccine.  Fourteen out of 15 

24 
PR-15097  
 
V2.10: 16Jun17 
 
vaccinees (93%) were infected with 101 PFU of vaccine, indicating that the HID50 is 
well below 101 PFU. 
 
A total of 40 adult flavivirus-naïve study participants have received the 
rDEN2/4∆30(ME) candidate DENV-2 vaccine at a dose of 103 PFU.  rDEN2/4Δ30(ME) 
was first evaluated at a dose of 103 PFU in a Phase 1, placebo-controlled trial conducted 
at the CIR(36).  Twenty-eight healthy male and non-pregnant female adult study 
participants, between the ages of 18 and 50, were enroled; 20 study participants 
received vaccine, 8 study participants received placebo (vaccine diluent).  The vaccine 
was found to be safe and immunogenic (Table 4, Table 5 and Table 6).  
rDEN2/4Δ30(ME) was also evaluated in a 2-dose study at a dose level of 103 PFU, with 
the second dose given at 4 or 6 months post Dose 1.  A total of 50 study participants 
were enroled (25 in each cohort).  Twenty study participants received 103 PFU of 
rDEN2/4Δ30(ME) subcutaneously at Day 0 and again at 6 months.  Five study 
participants were enroled as placebo-controls and received vaccine diluent at time 0 and 
4 months.  Because no subject was infected with the second dose given at 6 months, 
study participants were not enroled in the cohort evaluating a second dose given at 4 
months.  The vaccine was found to be safe, well tolerated, and immunogenic (Tables 4, 
5, and 6).    
 
Eighteen adult flavivirus-naïve study participants were enroled in a trial evaluating the 
safety and immunogenicity of rDEN2/4∆30(ME) given as a single subcutaneous dose of 
101 PFU.   Fifteen study participants received vaccine and three received placebo.  A 
dose of 101 PFU was given in order to determine the HID50 of the candidate vaccine.  
Fifty-three percent of vaccinees were infected by a dose of 101 PFU, indicating that the 
HID50 of this candidate is approximately 101 PFU.   
 
rDEN3∆30/31 has been evaluated at a dose of 103 PFU given as a single subcutaneous 
dose in a Phase 1, placebo-controlled trial conducted at the CIR and at the University of 
Vermont(37).  Seventy male and non-pregnant female adult study participants between 
the ages of 18 and 50 were enroled. Fifty participants received vaccine and 20 received 
placebo (vaccine diluent).  The vaccine was found to be safe and immunogenic (Tables 
4, 5, and 6).  The candidate vaccine was further studied at a dose of 101 PFU to 
determine the HID50 of this candidate vaccine.  Eighteen out of 20 vaccinees (90%) 
were infected with 101 PFU of vaccine, indicating that the HID50 is well below 101 PFU. 
Because the clinical supply of Lot M10377008 of rDEN3∆30/31 was exhausted, a 
second clinical lot of rDEN3∆30/31, Lot DEN3#113B, was manufactured and has been 
used in all tetravalent studies initiated since 2012. Lot DEN3#113B will be used for this 
tetravalent study. 
 
rDEN4∆30, Lot 4-9 was evaluated in two clinical trials (27, 38).  In the first study, 20 
healthy male and non-pregnant female adult study participants received 105 PFU of the 
candidate vaccine as a single subcutaneous dose.  In the second study, 20 healthy male 
and non-pregnant female adult study participants received this vaccine at a dose of 103 
PFU, 102 PFU, or 101 PFU.  Twelve study participants received vaccine diluent as a 
placebo.  The vaccine was found to be safe and immunogenic (Tables 4, 5, and 6). The 
HID50 was determined to be less than 10 PFU. Five study participants who received 105 
PFU of the rDEN4∆30 candidate vaccine developed a transient elevation in serum ALT 

25 
PR-15097  
 
V2.10: 16Jun17 
 
levels.  Elevation in serum ALT was abrogated by decreasing the dose of the candidate 
vaccine (Table 4).  Thus far, a total of 80 study participants have received rDEN4∆30.  
  
Because the clinical supply of Lot# 4-9 of rDEN4∆30 was exhausted, a second clinical 
lot of rDEN4∆30, Lot#DEN4#109A, was manufactured.  Seventy healthy adult male 
and non-pregnant female study participants were enroled and received a single 
subcutaneous 103 dose of rDEN4∆30 (50 study participants) or placebo (20 study 
participants).  The vaccine has been well tolerated by all study participants.  The most 
common vaccine-related AE was asymptomatic, transient maculopapular rash in 20 of 
50 (40%) vaccinees (Table 4).  One vaccinee developed an elevated serum ALT level.  
Lot#DEN4109A will be used for this tetravalent vaccine study. 
 
2.4.2.2 Summary of Clinical, Virologic, and Serologic Response to 
Monovalent Vaccines 
All of the vaccines described above have been well tolerated and have been found to be 
both safe and strongly immunogenic when administered as monovalent vaccines.  The 
safety, infectivity, and immunogenicity profiles of the monovalent vaccines were found 
to be comparable.  (Table 4, Table 5, and Table 6)(37).  Vaccinees did not develop a 
dengue-like illness at any dose (dengue-like illness is defined in Section 6.3.11.3).  
Local reactogenicity was minimal in all study participants.  The most common AEs 
observed in all studies were transient neutropenia, rash, and headache (Table 4).  Fifty-
three of 231 vaccinees (23%) developed a transient neutropenia following vaccination.  
The neutropenia was graded as mild (1,000 – 1500/mm3) in 48 vaccinees (16%), as 
moderate (750 – 1499/m3) in 9 vaccinees (3%) and as severe (< 750/mm3) in 7 
vaccinees (2%).  The neutropenia was of short duration (generally 3 – 5 days).  All 
episodes of severe neutropenia were ≤ 4 days.  None of the neutropenic study 
participants developed clinical complications.  A total of 92 vaccinees (40%) developed 
a transient maculopapular rash over the trunk and proximal upper extremities following 
first vaccination.  The rash was characteristically non-pruritic and went unnoticed by the 
majority of affected study participants.  A total of 92 vaccinees (40%) complained of 
headache; however, this was comparable to the number of headaches reported in 
placebo recipients (36%). One of the 231 vaccinated study participants developed a 
fever, though this was determined to be not related to the vaccine. Other AEs 
experienced in the 28 day follow-up period following vaccination included arthralgia, 
myalgia, malaise, retro-orbital pain, and injection site reactions.  These occurred in 
fewer than 10% of vaccinated study participants. 
 
Vaccine virus was recovered from the blood of inoculated with each monovalent DENV 
candidate (Table 5).  The mean peak titers of the monovalent DENV candidate vaccine 
viruses were very low, ranging from 100.5 PFU/mL (recipients of 103 PFU of 
rDEN3∆30/31 and rDEN4∆30) to 101.6 PFU/mL (recipients of 105 PFU of rDEN4∆30) 
(Table 5).  

26 
PR-15097  
 
V2.10: 16Jun17 
 
Table 4: Clinical response to monovalent DENV vaccine candidates for use in study 
admixtures 
Vaccine candidate 
Dose 
(log10PF
U) 
No. of 
partici
pants 
% 
vire
mic  
No. of participants (%) with indicated clinical response 
Fever 
Rash 
Headache 
Neutrope
nia2 
↑ALT 
rDEN1∆30 
3 
71 
60 
    11 
(1.4) 
22 (31) 
29 (41) 
32 (45) 
   1 (1.4) 
 
 
 
 
 
 
 
 
 
rDEN2/4Δ30(ME) 
3 
40 
60 
0 (0) 
13 (32) 
12 (30) 
11 (28) 
 3 (8)2 
 
 
 
 
 
 
 
 
 
rDEN3∆30/31 
3 
50 
34 
0 (0) 
26 (52) 
18 (36) 
2 (4) 
 1 (2)1 
 
 
 
 
 
 
 
 
 
rDEN4∆30 (Lot 4-
9) 
3 
20 
35 
0 (0) 
11 (55) 
  7 (35) 
  5 (25) 
1 (5) 
rDEN4∆30 (Lot 
109A) 
3 
50 
26 
0 (0) 
20 (40) 
26 (52) 
3 (6) 
1 (2) 
 
 
 
 
 
 
 
 
 
Placebo4 
n/a5 
76 
0 
1 (1) 
2 (3) 
27 (36) 
6 (8) 
3 (4) 
1. 
Not related to vaccine. 
2. 
Peak ALT levels in these participants ranged from 1.3 to 1.7 x ULN (upper limit of normal).  
2/3 participants with elevated ALT had an ALT above the ULN on Day 0 prior to vaccination 
despite being normal at screening 
3. 
Participant recorded a fever on 1 occasion.  Fever lasted only 2 hours.  
4. 
Includes the placebo recipients enroled in the clinical trial of the monovalent candidates listed 
in this table. 
5. 
Not applicable. 

27 
PR-15097  
 
V2.10: 16Jun17 
 
Table 5: Magnitude, onset, and duration of viremia in participants inoculated with 
monovalent DENV vaccine candidates for use in tetravalent vaccine admixtures 
Vaccine candidate 
Dose 
(log10PFU
) 
N 
(%) with 
viremia 
Mean peak titer 
± SE (log10 
PFU/mL)1 
Mean day of onset 
of viremia ± SE 
Mean # of days of 
viremia ± SE 
rDEN1∆30 
3 
71 
60 
1.0 ± 0.08 
10.0 ± 0.3 
3.3 ± 0.3 
 
 
 
 
 
 
 
rDEN2/4Δ30(ME) 
3 
40 
60 
0.5 ± 0.03 
9.2 ± 0.6 
3.3 ± 0.6 
 
 
 
 
 
 
 
rDEN3∆30/31 
3 
50 
34 
0.6 ± 0.01 
  7.6 ± 0.64 
3.2 ± 0.5 
 
 
 
 
 
 
 
rDEN4∆30 (Lot 4-9) 
5 
20 
70 
1.6 ± 0.1 
10.5 ± 0.7 
  3.6 ± 0.45 
rDEN4∆30 (Lot 4-9) 
3 
20 
35 
0.5 ± 0.1 
9.1 ± 1.2 
1.6 ± 0.8 
rDEN4∆30 (Lot 109A) 
3 
50 
26 
0.7 ± 0.1 
10.8 ± 0.7 
2.2 ± 0.4 
1. Mean peak titer is calculated only for those participants who were viremic.  Lower limit of 
detection is 0.5 log10 PFU/mL. 
 
 
 
 
Table 6: Serum antibody responses induced by each monovalent DENV candidate 
vaccine given as a single subcutaneous dose 
 Vaccine candidate 
Dose 
(log10) 
No. 
vaccine
es 
%. 
infecte
d2 
Geometric mean titer, reciprocal (range)1 
% 
seroconverted
3 
Day 
0 
Day 28 
Day 42 
rDEN1∆30 
3 
70 
94 
<5 
170 (<5 - 6309) 
140 (<5 – 2753) 
934 
 
 
 
 
 
 
 
 
rDEN2/4Δ30(ME) 
3 
40 
100 
<10 
88 (11– 1043) 
104 (11 - 1377) 
100 
 
 
 
 
 
 
 
 
rDEN3∆30/31 
3 
50 
82 
<5 
84 (14-1715) 
79 (8-1341) 
81 
 
 
 
 
 
 
 
 
rDEN4∆30 (Lot 4-9) 
5 
20 
100 
<10 
567 (72 – 2455) 
399 (45 – 1230) 
100 
rDEN4∆30 (Lot 4-9) 
3 
20 
95 
<10 
139 (<10 – 
2365) 
129 (15 – 1222) 
95 
rDEN4∆30 (Lot 
109A) 
3 
50 
93 
<5 
112 (12 – 892) 
135 (22 - 748) 
93 
1.  Geometric mean titer calculated only for those participants who seroconverted. 
2.  Defined as either recovery of vaccine virus from the blood or by seroconversion. 
3.  Defined as a ≥4-fold rise in serum neutralizing titer compared to Day 0. 
4.  Vaccine virus was recovered from the blood of 1 subject who did not meet criteria for 
seroconversion. 
 
 
Each of the monovalent candidate vaccines induced seroconversion (as defined as a ≥ 4-fold 
rise in serum neutralizing antibody titer at Study Day 28 or 42 compared with Study Day 0) 
in a large majority of vaccinees (Table 6).  Seroconversion rates ranged from 81% 
(rDEN3∆30/31) to 100% (rDEN2/4Δ30(ME), rDEN4∆30) following a single dose.  
rDEN1∆30 induced sterilizing immunity to a second dose of vaccine administered at 4 or 6 

28 
PR-15097  
 
V2.10: 16Jun17 
 
months in all vaccinees (data not shown)(32). DEN2/4∆30 induced sterilizing immunity to a 
second dose of vaccine administered at 6 months (data not shown)(37). 
 
2.4.2.3 Clinical, Virologic, and Serologic Response to a Tetravalent Admixture 
TV005 
The tetravalent admixture TV005 is a combination of four live attenuated monovalent 
vaccines, each of a different serotype.  The TV005 is comprised of 103 of rDEN1∆30, 104 of 
rDEN2/4Δ30(ME), 103 of rDEN3∆30/31, and 103 of rDEN4∆30 live attenuated monovalent 
vaccines.   
 
Clinical experience with TV005 (US) 
TV005 has been evaluated in healthy flavivirus-naïve adult volunteers in two studies (CIR 
268 and CIR 279) at the Center for Immunization Research, Johns Hopkins University and 
the Vaccine Testing Center, University of Vermont. In CIR 268, 20 volunteers received 
TV005 and 8 volunteers received a placebo. The protocol was later amended to allow for a 
second dose of vaccine (or placebo) to be given 6 months later. A limited number of 
volunteers returned for a second dose. In CIR 279 volunteers received a subcutaneous dose of 
vaccine (40) or placebo (16) at time 0 and a second dose of the same 6 months later (39). 
 
Virologic response 
Virology data from both CIR 268 and CIR 279 is presented together. Following the first dose 
of TV005, 46/60 (77%) vaccinees had at least one vaccine virus recovered from the blood 
(Table 7) Twenty-four vaccinees had only 1 vaccine virus detected in the blood 
(DEN1∆30=7; DEN2/4∆30=1; DEN3∆30/31=10; DEN4∆30=6).  Seventeen vaccinees had 2 
vaccine viruses detected in the blood (DEN2/4∆30, DEN3∆30/31=5; DEN3∆30/31, 
DEN4∆30=3; DEN2/4∆30, DEN4∆30=4; DEN1∆30, DEN4∆30=3; DEN1∆30, 
DEN3∆30/31=2) and 5 vaccinees had 3 vaccine viruses detected in the blood (DEN1∆30, 
DEN3∆30/31, and DEN4∆30=2; DEN1∆30, DEN2/4∆30, DEN3∆30/31=2; 
DEN2/4∆30(ME), DEN3∆30/31, DEN4∆30=1).  Vaccine virus was recovered from the blood 
of only 1 vaccine-recipient following the second dose. DENV3∆30/31 was recovered from 
the subject only on Study Day 9 following dose 2 (titer = 0.5 log10 PFU/mL). This subject 
had a PRNT60 of 1:12 to DEN3 Slemen/78 on Study Day 180 prior to second dose.  
 
Serologic response 
Serologic endpoints differed in the 2 studies (CIR 268 and CIR 279). In CIR 268, 
seropositivity was defined as a PRNT60 of ≥ 1:10. In CIR 279 seropositivity was defined as a 
PRNT50 of ≥ 1:10. In addition, in CIR 268 samples were collected only at Study Day 28, 42 
and 180 post-vaccination and the peak titer post-each dose was the peak titer that occurred 
out to Study Day 42. In CIR 279, samples were collected at Study Day 28, 56, 90 and 180 
post-vaccination and the peak titer post-each dose was the peak titer that occurred out to 
Study Day 90. 
 
Following the first vaccination in CIR 268, 80% of vaccinees seroconverted to DENV-1, 
60% of vaccinees seroconverted to DENV-2, 80% of vaccinees seroconverted to DENV-3 
and 100% to DENV-4 (Table 8).  60% of vaccinees had a tetravalent response, 30% had a 
trivalent response, 0% had a bivalent response, and 10% had a monovalent response (Table 
12). 
 

29 
PR-15097  
 
V2.10: 16Jun17 
 
Following the first vaccination in CIR 279, 92% of vaccinees seroconverted to DENV-1, 
97% of vaccinees seroconverted to DENV-2, 97% of vaccinees seroconverted to DENV-3 
and 97% to DENV-4 (Table 8). The increase in seropositivity frequency to DENV-2 induced 
by TV005 in CIR 279 compared to TV003 in CIR 279 was statistically significant (p = 
0.006226, Fisher Exact test). Geometric mean peak titers following dose 1 are presented in 
Table 9. The higher seroconversion frequencies observed in CIR 279 are likely due to the 
additional day 90 post-vaccination time-point as we noted that antibody titers in some 
vaccinees continued to rise and some study participants had peak antibody titers at Study Day 
90 or sometimes even later. There was no significant boost in antibody titer following a 
second dose of vaccine (Table 9).  Titers had declined by Study Day 180 and did not increase 
appreciably after the second dose. The geometric mean antibody titers recorded post-dose 2 
of vaccine were much lower than those observed following dose one. Only one of three study 
participants who had developed a trivalent antibody response following the first dose of 
TV005 seroconverted to the missing fourth serotype following the second dose. 
 
2.4.3 Clinical Experience with TV005 in an Endemic Population 
TV005 is currently being evaluated in a Phase II safety and immunogenicity clinical trial in 
volunteers ages 12 months to 50 years in an endemic area in Thailand.  Safety data for 
vaccine related adverse events (AE)s has been completed for the adult cohort only.  In this 
cohort 30 study participants received TV005, and 24 study participants received placebo.  
These data show that in this population the vaccine is well tolerated.  Similar to studies 
conducted in the United States, mild rash was common following vaccination, occurring in 
approximately 40% of vaccinated study participants in the adult cohort (48% of all study 
participants).  Fatigue was also common following vaccination, occurring in approximately 
33% of vaccinated study participants in the adult cohort.  Rash and fatigue were the only two 
vaccine related AEs that were significantly different between vaccine and placebo recipients 
(Table 13).   
 
The data from the adolescent cohort are similar to the adult cohort. No safety concerns were 
noted by the DSMB, and the study has continued to age de-escalate to children ages 5 – 11 
years old (data not available).   
 
Conclusions:   
TV005 was well tolerated by study participants, and a single dose was highly immunogenic 
(tetravalent response in 90% of vaccines).  Very little boost in antibody titer was observed 
following a second dose of vaccine administered six months following initial vaccination.   

30 
PR-15097  
 
V2.10: 16Jun17 
 
Table 7: Magnitude, onset, and duration of viremia in participants inoculated 
with TV005, dose 1 (CIR 268 and CIR 279) 
 
Vaccine component 
No. 
participants 
vaccinated 
 
No. viremic 
(%) 
Maximum 
titer (log10 
PFU/mL) 
Mean peak 
titer1 ± SE 
Mean day of 
onset of 
viremia 
(range) 
Mean 
duration in 
days 
(range) 
DEN1∆30 
60 
17 (28) 
1.7 
0.62 ± 0.1 
10.9 (8-15) 
2.1 (1-5) 
DEN2/4∆30(ME) 
13 (22) 
0.5 
0.50 ± 0.0 
7.5 (6-10) 
1.0 (all 1) 
DEN3∆30/31 
23 (42) 
1.0 
0.60 ± 0.0 
9.3 (3-14) 
2.5 (1-7) 
DEN4∆30 
18 (30) 
1.5 
0.64 ± 0.1 
9.3 (6-16) 
1.9 (1-6) 
 
Total Viremic 
46 (77) 
 
 
 
 
 
 
 
 
Table 8: Serum antibody responses induced by TV005 when given as a single 
subcutaneous dose 
%  of vaccinees seropositive to specific serotype 
Admixture 
N 
DENV-1 
DENV-2 
DENV-3 
DENV-4 
TV005 (268)1 
20 
80 
60 
90 
100 
TV005 (279)2 
39 
92 
973 
97 
97 
1. Seropositive = 60 percent plaque reduction neutralization titer (PRNT60) of 
>1:10 
2. Seropositive = 50 percent plaque reduction neutralization titer (PRNT50) of 
>1:10 
3. Statistically significant (p = 0.006226, Fisher Exact test) 
 
 
Table 9: Geometric mean antibody titer to each serotype following a single dose 
of TV005 
Geometric Mean Peak Titer (range) 
Admixture 
N 
DENV-1 
DENV-2 
DENV-3 
DENV-4 
TV005 (268)1 
20 
40 (16-91) 
44 (10-149) 
35 (14-113) 
70 (14-338) 
TV005 (279)2 
39 
35 (10-190) 
91 (12-651) 
100 (17-343) 
205 (19-2190) 
1. Titer calculated using PRNT60 (per protocol) 
2. Titer calculated using PRNT50 (per protocol) 
 
 

31 
PR-15097  
 
V2.10: 16Jun17 
 
Table 10: TV005 induce a broad neutralizing antibody response to all 4 
serotypes 
% with multivalent response (cumulative) 
 
N 
Tetravalent 
Tri- 
Bi- 
Mono- 
None 
TV005 
(268)1 
20 
60  
30 (90) 
0 (90) 
10 (100) 
0 
TV005 
(279)2 
39 
90  
8 (98) 
0 (98) 
2 (100) 
0 
1. Based on PRNT60.  Seropositive response is defined as a PRNT60 ≥ 1:10 by Study 
Day 42. 
2. Based on PRNT50.  Seropositive response is defined as a PRNT50 ≥ 1:10 by Study 
Day 90. 
 
Table 11: Geometric mean antibody titer to each serotype following a second 
dose of TV005 (CIR 279) 
TV005 
N1 
DENV-1 
DENV-2 
DENV-3 
DENV-4 
Day 180 
33 
10 (<5-41) 
35 (<5-241) 27 (<5-68) 
40 (9-1582) 
Peak titer post-dose 2 
33 
16 (<5-54) 
55 (11-297) 
36 (10-312) 75 (26-951) 
 
 1. Only those participants who received both doses of vaccine were included in this analysis 
 
 

32 
PR-15097  
 
V2.10: 16Jun17 
 
 
Table 12: Percent and cumulative seropositivity rates to multiple DENV 
following the first and second dose of TV005 (CIR 279). 
 
 
% with multivalent response (cumulative) 
 
N1 
Tetravalent Trivalent 
Bivalent 
Monovalent None 
TV005, 
dose 1 
33 
91 
9 (100) 
0 (100) 
0 (100) 
0 
TV005, 
dose 2 
33 
94 
6 (100) 
0 
0 
0 
1. Only those participants who received both doses of vaccine were included 
 
 
 
Table 13: Clinical response to TV005 vaccine candidate in an endemic setting 
(Thailand) 
Vaccine 
Candida
te 
No. of 
partici
pants 
No. of participants (%) with indicated clinical response 
Any 
AE 
ALT 
↑ 
Arthr
algia 
Rash 
Fatigu
e 
Fev
er 
Head
ache 
Mya
lgia 
Neutrop
enia 
Naus
ea 
TV005 
30 
25 
(83) 
1 (3) 
2 (7) 
12 
(40) 
10 
(33) 
4 
(13) 
10 
(33) 
7 
(23) 
0 (0) 
4 
(13) 
Placebo 
24 
15 
(63) 
0 (0) 
1 (4) 
2 (8) 
0 (0) 
0 
(0) 
6 (25) 3 
(13) 
1 (4) 
2 (8) 
 
 

 
33 
PR-15097  
 
V2.10: 16Jun17 
 
 
2.4.4 Mosquito Transmissibility of Monovalent Vaccine Candidates 
For DENV to be transmitted from 1 person to another there is a 10–14 day incubation period 
within the mosquito.  Therefore, for these vaccine viruses to be transmitted to an in-house 
family member, the subject would have to be viremic with a peak virus titer greater than 105 
PFU/mL, would have to be bitten by a viable vector mosquito at the peak of viremia, the 
mosquito would have to live for an additional 10–14 days, and the same mosquito would then 
have to bite another family member.  The peak virus titers of all the live attenuated DENV 
vaccine viruses tested thus far were at least 1000-fold below the viremia level required for 
transmission to mosquitoes making risk of transmission minimal.  
 
Both the wt rDEN1 and rDEN1∆30 demonstrated a low infectivity (50% mosquito infectious 
dose [MID50] of >103 PFU/mL)(28).  This is consistent with previously reported studies that 
demonstrated a very low infectivity of DENV-1 for Aedes aegypti fed an infectious blood 
meal.  It is unlikely that the vaccine virus would be transmitted from an infected vaccinee to a 
mosquito since rDEN1∆30 is poorly infectious for mosquitoes and because of the low level 
of viremia demonstrated in humans (40).   
 
 
Transmissibility of rDEN2/4∆30(ME) to both Ae. aegypti and Toxorhynchites splendens 
mosquitoes is reduced compared with both rDEN4 and DEN2-NGC (10).  The minimum 
infectious dose of rDEN2/4∆30(ME) needed to infect 50% of Ae. aegypti mosquitoes 
(MID50) was ≥10-fold higher than that of either rDEN4 or DEN2-NGC, indicating impaired 
infectivity for the midgut.  rDEN2/4∆30(ME) was less infectious than either parent virus for 
T. splendens, indicating reduced ability of the chimeric virus to disseminate to the head of the 
mosquito.  It is unlikely that the vaccine virus would be transmitted from an infected vaccinee 
to a mosquito since rDEN2/4∆30(ME) is poorly infectious for mosquitoes and because of the 
low level of viremia demonstrated in humans (36).  rDEN2/4∆30(ME) should be poorly 
transmitted to mosquitoes, as previously observed for the attenuated rDEN4∆30 vaccine 
candidate, which was not transmitted from 10 infected vaccinees to over 300 feeding 
mosquitoes(27,41).  
 
The parent virus of the rDEN3-3´D4∆30 and rDEN3Δ30/31 virus vaccines, 
DEN3-Sleman/78, is very poorly transmitted to mosquitoes: ingestion by Ae. aegypti 
mosquitoes of 104.1 PFU of wt DEN3 Sleman/78 infected the midgut of only 4 of 28 (14%) 
mosquitoes tested and disseminated from the midgut in only 2 of 28 (7%) mosquitoes.  The 
required dose of wt DEN3 Sleman/78 is in excess of 105 PFU/mL in blood to allow for 
transmission to Ae. aegypti mosquitoes, the natural vector of DENV(42).   
 
The rDEN4∆30 virus construct has been extensively studied with respect to its 
transmissibility to humans by mosquitoes (41).  The rDEN4∆30 was restricted both in its 
ability to infect the midgut and to cause a disseminated infection in mosquitoes.  The ability 
of rDEN4∆30 to be transmitted from viremic study participants to Ae. albopictus mosquitoes 
was also evaluated.  Mosquitoes fed on study participants on Study Days 7, 8, and 9 post-
infection.  Vaccine virus was not recovered from any of the 352 mosquitoes that fed on the 
study participants  even though some of the study participants were viremic on each of the 
days tested(41).  The restricted capacity of rDEN4∆30 to disseminate from the midgut of the 
mosquito to its head is specified by the ∆30 mutation. 
 

 
34 
PR-15097  
 
V2.10: 16Jun17 
 
The restricted capacity of rDEN4∆30 to disseminate from the midgut of the mosquito to its 
head is specified by the ∆30 mutation. As with the other monovalent attenuated DENV 
vaccine viruses included in TV005, this mutation is engineered into rDEN3∆30/31.  Ingestion 
by Ae. aegypti mosquitoes of 104.1 PFU of wt DEN3 Sleman/78 infected the midgut of only 4 
of 28 (14%) mosquitoes tested and disseminated from the midgut in only 2 of 28 (7%) 
mosquitoes.  In addition, the low level of viremia in vaccinated study participants will 
preclude transmissibility to mosquitoes.  rDEN3∆30/31 should be poorly transmitted to 
mosquitoes, as previously observed for the attenuated rDEN4∆30 vaccine candidate, which 
was not transmitted from 10 infected vaccinees to over 300 feeding mosquitoes(27,41). 
 
Research Design and Methods 
 
 
3 Study Design 
3.1 Overall Study Design 
This is a placebo-controlled, double-blind study in normal healthy adult, adolescent, and 
child volunteers recruited from Mirpur, Dhaka.  The purpose of this study is to evaluate the 
safety, reactogenicity, and immunogenicity of 1 dose of the live attenuated tetravalent dengue 
vaccine TV005 when given as a subcutaneous injection.  The candidate vaccine, TV005, will 
contain 103 PFU of rDEN1∆30, 104 PFU of rDEN2/4∆30(ME), 103 PFU of rDEN3∆30/31 
and 103 PFU of rDEN4∆30. A placebo cohort is included in the study as a control to 
distinguish vaccine-associated versus non-vaccine-associated AEs.  Placebo for this study 
will be the vaccine diluent, 1X L-15.  Placebo is prepared from a specific safety tested lot of 
2X L-15 medium (BB-MF #12959) that is obtained from a NIAID contracted repository and 
mixed 1:1 with sterile water for injection. 
 
After providing written informed consent/assent, participants will undergo eligibility 
screening, including medical history, physical examination, haematology testing, liver 
function testing, and hepatitis C screening (for adults and adolescents only), and hepatitis B 
screening.  Pregnancy testing will be performed on female participants as outlined in Section 
4.1 #6. Screening will be performed within 30 days prior to Study Day 0.  See Section 6 for a 
detailed description of study visits and procedures. 
 
All clinically significant abnormalities will be reviewed with study participants and they will 
be referred to an appropriate local care provider for follow-up care.  Study participants will 
be determined to be eligible based on the inclusion and exclusion criteria found in Section 4 
of this protocol.  For participants who are eligible, the Study Day 0 visit will be scheduled for 
the participants to receive their vaccination.  Pregnancy testing will be repeated on female 
participants of reproductive age on the day of vaccination, and must be negative prior to 
vaccination.  Participants will be considered enroled in the study only after they are 
vaccinated (receive either vaccine or placebo).  After vaccination, participants will be 
observed in the clinic for at least 30 minutes in order to assess for any AEs. Study 
participants will return to the clinic on Study Days 7, and 14 post-vaccination for evaluation 
by a clinician and to have blood drawn for safety, clinical laboratory studies, and assays to 
assess for vaccine virus viremia.  They will also have a history-directed clinical evaluation 
performed at each specified visit (see Section 6 for detailed description of study procedures).  
They will return to the clinic on Study Days 28, 56, 180, 360, and 720 for further evaluation 
of immune responsiveness.  On Study Day 1080 (year 3) participants will return to the clinic 
for their final visit.  

 
35 
PR-15097  
 
V2.10: 16Jun17 
 
 
Investigators and or designee will identify suspected dengue and febrile illness cases during 
the three year duration of the study.  The following systems will be utilized: 
 
First line of surveillance 
Except for days with scheduled clinic visits, Field Research Assistants (FRAs) or designees 
will make daily home visits to study participants or parent/legally acceptable representative to 
collect fever and AE information up to day 14 (Day 0 – Day 14). If study participants can’t 
be reached at the home, FRAs or designees will call the study participants or parent/legal 
acceptable representative to get information over the phone.  
 
Second line of surveillance  
Study participants or parents/legally acceptable or authorized representative (LAR) will be 
instructed to contact the study Investigator and/or other appropriate study staff or come to the 
clinic in the event of febrile episodes during entire study period or in case of any untoward 
medical event.  
 
Third line of surveillance  
After Day 14, study staff will contact study participants or their parent/LAR weekly by phone 
or in person to check for fever and /or AEs through study day 180.  After study day 180, 
study staff will contact study participants monthly, by phone or in person, to remind study 
participants to contact the clinic if they have a febrile illness.  Study staff will also ask study 
participants if they have been hospitalized for a febrile illness since the last call.  If the study 
participant was hospitalized for a febrile illness, they will be instructed to come to the clinic 
for suspected Dengue follow-up.   
 
FRAs or designee will assess study participants for solicited local symptoms (see Table 20 
and Table 21) at the injection site and record this information on surveillance source 
document on the day of dengue/placebo vaccine administration.  FRAs or designee will 
collect and record temperature through Study Day 14, and AEs through Study Day 28, on the 
age-appropriate surveillance source document. 
 
3.2 Dosing Strategy 
 
 
          Table 14: Dosing Strategy 
Cohort 
Age (years) 
No. Vaccinee 
No. Placebo 
Total 
(01) Adult 
18-50 
36 
12 
48 
(02) Adolescent 
11-<18 
36 
12 
48 
(03) Children 
5-<11 
36 
12 
48 
(04) Young 
Children 
1-<5 
36 
12 
48 
 
Total: 
144 
48 
192 
 
Each cohort will be enroled in an age de-escalation manner. The first cohort (Cohort 01) of 
48 adults will receive one subcutaneous dose of TV005 vaccine or placebo in a 3:1 ratio.  
Following the Study Day 28, study participants in the adult cohort will have blinded safety 
data analysed separately evaluating TV005 and placebo.  If stopping criteria are not met 
following analysis of safety data through Study Day 28, enrolment will continue into the next 
age de-escalation portion of the study based upon the approval of the DSMBs.  The second 

 
36 
PR-15097  
 
V2.10: 16Jun17 
 
cohort (Cohort 02) of 48 adolescents will then be enroled into the study.  It is estimated that it 
will take approximately one – two months for DSMB approval to proceed to the next cohort 
after Study Day 28, including compiling the data report, DSMB review and approval. The 
sequential enrolment of the final 2 cohorts (Cohort 03 and Cohort 04) will proceed in a 
similar manner, with DSMB evaluation before each subsequent group. All study participants 
will be followed for approximately three years after receipt of the vaccine. 
 
3.3 Sample Size and Placebo Ratio 
192 study participants (144 vaccinees and 48 placebo recipients) will be enroled in the study. 
All cohorts will consist of 48 study participants (36 vaccine, 12 placebo).  All cohorts will 
use a 3:1 (TV005: placebo) ratio. The age de-escalation strategy was chosen for the following 
reasons:  
• This study is designed to build upon the previous Phase I and II studies of each 
monovalent candidate and the tetravalent admixtures, in the United States and 
Thailand, which used similar cohorts and the similar vaccine to placebo ratios (24, 27, 
35, 40). 
• Placebo recipients have been included to allow assessment of whether common AEs 
are vaccine-related.   
 
3.4 Duration of Participation 
Study participants will be followed for approximately three years from vaccination. 
Participants will be screened for eligibility up to 30 days prior to vaccination on Study Day 0. 
 
3.5 Estimated Duration of the Study 
The study will span a total of approximately 4 years, from the time of first study vaccination 
until the last enroled participant completes the last visit. This 4 year study duration includes 
four cohorts of an approximate 37 month duration (one month of screening and three years of 
follow-up) and allows for interim safety evaluations by the DSMBs after enrolment of each 
cohort has been completed and prior to enrolment of the subsequent cohort.  
 
3.6 Treatment Assignment 
All study participants will be randomly assigned to receive TV005 vaccine or placebo.  
Treatment assignment will be done using a random number generator to prepare the sequence 
in which study participants are assigned to receive TV005 or placebo.  Study participants will 
be randomized in blocks of 4 by the vaccine preparation study staff using a pre-generated 
randomization code list for all study participants. Each block of 4 will include 3 vaccine 
recipients for TV005 and 1 placebo recipient.  Study participants will be enroled sequentially, 
and a separate randomization list will be generated for each of the four cohorts.   
 
Clinical staff and study participants will remain blinded to treatment assignment until all 
participants in that cohort have completed their full three year follow-up period.  Study 
investigators will remain blinded to treatment assignment until study participants have 
completed follow-up out to their Day 180 visit.  Study participants and study staff may only be 
unblinded after these specified times, or if unblinding is deemed necessary for safety reasons 
(see Section 3.7). 
 
A master log of treatment assignments in sealed envelopes will be maintained in a record 
separate from other study records.  It will be kept in a locked room with limited access by a 
designated individual at the International Centre for Diarrhoeal Disease Research, 

 
37 
PR-15097  
 
V2.10: 16Jun17 
 
Bangladesh (icddr,b). A copy of the treatment assignments will also be provided to the NIH 
DSMB, and ERC DSMB if requested.  
 
3.7 Blinding 
This study will be conducted as a double-blind study to avoid biased assessment of AEs.  
Vaccine or placebo will be prepared and drawn up into syringes by laboratory personnel who 
are not involved with the clinical assessment of study participants.  Syringes are labelled 
according to Study Specific Procedures with treatment numbers and pre-printed participant 
numbers.  Because vaccine diluent is used as placebo, there will be no difference in the 
appearance of the syringes, and clinical staff will have no access to the master list of 
treatment assignments for study participants. 
 
The study participant  and clinical staff will not know the treatment group to which study 
participants are assigned.  In addition, other personnel assigned to monitor the study will not 
know the treatment assignment of the participant.  
 
If the need arises to unblind a specific study participant’s treatment assignment for 
emergency medical management, the Principal Investigator (PI) will contact the assigned 
designee at icddr,b who is responsible for maintaining the envelopes containing the unblinded 
information and can obtain the treatment assignment of the study participant in question if 
needed.  Only that specific study participant’s assignment will be unblinded. The Sponsor 
and the DSMBs will be notified within 2 business days.  Unblinding will be documented in 
the participant’s study chart. The research monitor and the Ethical Review Committee (ERC) 
at icddr,b will be notified of the unblinding.  
 
For each age cohort, clinical staff and study participants will remain blinded to treatment 
assignment until all participants in that cohort have completed their full three year follow-up 
period.  Other study investigators will remain blinded to treatment assignment until all 
participants in that cohort have completed their Day 180 follow-up visit. 
 
Routine unblinding for Investigators (not clinical staff) will occur when all participants have 
completed their Day 180 follow-up visit.  Once all of the data for each study participant in 
each cohort up to study day 180 has been entered into the CRFs and uploaded, the clinical 
designee will notify the site PI that the cohort is ready for unblinding.  The site PI will make a 
formal request for the treatment assignments in writing from the unblinded study staff.  The 
unblinded study staff will then send the treatment assignments to the site PI. Clinical staff and 
study participants will remain blinded to treatment assignment until each study participant in 
each cohort has reached their full three year follow-up period. 
 
 
4 Selection and Enrolment of Participants 
 
4.1 Inclusion Criteria 
All of the following criteria must be fulfilled for a participant to qualify for inclusion in this 
study: 
 
1. Healthy males or females (non-pregnant or lactating) between the ages of 12 months and 
50 years at the time of enrolment into the study. 
 

 
38 
PR-15097  
 
V2.10: 16Jun17 
 
2. Reside in Mirpur Wards 2, 3, or 5 (or other Wards within Mirpur as designated by the PI 
if needed to reach enrolment targets) at the time of screening. 
 
3. Study participants and/or their parents/guardians/LARs who the Investigator believes can 
and will comply with the requirements of the protocol (e.g., return for follow-up visits).  
 
4. Good general health as determined by physical examination, laboratory screening, and 
review of medical history. 
 
5. If the participant is > 18 years of age, an informed consent form signed and dated by the 
study participant (and by an independent witness if required by local regulations). If the 
study participant is a minor, an assent form (for participant’s age 11-17 years) and 
informed consent form signed and dated by the participant's properly identified parent(s) 
or legally acceptable representative.  If a participant is < 11 years of age, the participant’s 
parent(s) or legally acceptable representative will consent by signing the consent form for 
minor participants. 
 
6. If the participant is female, she must not be pregnant or planning to become pregnant up 
to 28 days post vaccination.  Female participants under the age of 18 will be enroled, if 
they have not yet reached menarche. Females 18 years of age or older, must be properly 
using a method of pregnancy prevention that is known to be highly effective per the PI. 
Reliable methods of contraception include: hormonal birth control, condoms with 
spermicide, diaphragm with spermicide, surgical sterilization, intrauterine device, and 
abstinence (> 6 months since last sexual encounter). For female study participants who 
are 18 years of age or older pregnancy testing will occur at screening and again before 
vaccination and they must have a negative pregnancy test prior to vaccination.  
Additionally, they will receive counselling on the importance of effective contraception 
during the 30 days prior to vaccination and continuing up to Day 28. Counselling will 
occur at their screening visit, on all clinic visit days up to Day 28.  Female participants 18 
years of age and older will be subject to pregnancy testing on the Study Day 28 and 56 
visits. If female participants under the age of 18 that have not yet reached menarche prior 
to enrolment, reach menarche during the study, they will continue to be enroled and 
followed-up through the entire three year follow-up period.  
 
4.2 Exclusion Criteria 
A participant will be excluded from enrolment if any of the following criteria are met:  
 
1. Pregnant or lactating female or female planning to become pregnant within 28 days of 
receiving an investigational product or planning to discontinue abstinence or 
contraceptive precautions within 28 days of receiving an investigational product. 
Menstruating females under the age of 18 will be excluded from being enroled. 
 
2. Acute or chronic, clinically significant pulmonary, cardiovascular, hepatic, renal, 
autoimmune, hematologic, or endocrine disease or functional defect, as determined by 
history, physical examination, or screening tests.  
 
3. History of any neurological, psychiatric, or behavioural disorder or seizures, with the 
exception of a single febrile seizure in childhood. 
 

 
39 
PR-15097  
 
V2.10: 16Jun17 
 
4. Self-reported or suspected congenital or acquired immunodeficiency; or receipt of 
immunosuppressive therapy such as anti-cancer chemotherapy or radiation therapy 
within the preceding 6 months, or long-term systemic corticosteroid therapy 
(prednisone or equivalent,  > 0.5 mg/kg/day or 20 mg/day,  for more than 2 
consecutive weeks within the past 3 months). Inhaled and topical steroids are allowed. 
 
5. Having a height-for-age z-score (HAZ) or weight-for-age z-score (WAZ) of <-3 for 
children under the age of two.  Severe malnutrition as observed by the study physician 
for all study participants ages 2 years old and older.  
 
6. Hepatitis C virus (HCV) infection by screening and confirmatory assays (screening in 
adults and adolescents only), or Hepatitis B virus (HBV) infection, by Hepatitis B 
surface antigen (HBsAg) screening (all participants) or, unwilling to allow HCV 
(adults and adolescents only) and HBV testing.  
 
7. Screening laboratory values of Grade 1 or above for absolute neutrophil count (ANC), 
ALT, or platelet count, as defined in this protocol. 
 
8. For children under 5 years of age, screening laboratory values of Grade 1 or above for 
Haemoglobin. 
 
9. History of allergic reaction likely to be exacerbated by any component of the vaccine; 
any history of a severe allergic reaction or anaphylaxis. 
 
10. Current alcohol abuse or drug addiction that might interfere with the ability to comply 
with trial procedures. 
 
11. Any other condition that in the opinion of the Investigator would jeopardize the safety 
or rights of a participant participating in the trial or would render the participant 
unable to comply with the protocol. 
 
12. Planned administration of any vaccine  from 30 days prior to receipt of the study 
vaccine and ending 30 days after; with the exceptions of standard infant and child 
Expanded Program on Immunization (EPI) inactivated vaccines and the inactivated 
influenza vaccine or the inactivated rabies vaccine (without administration of 
immunoglobulin) administered to adults or children. 
 
13. Use of any investigational or non-registered drug or vaccine other than the study 
vaccine within 30 days preceding receipt of study vaccine/placebo or planned use at 
any time during the study period or history of having received any investigational 
dengue vaccine at any previous time. 
 
14. Current participation in any clinical or investigational study. 
 
15. Administration of immunoglobulins and/or blood products within 90 days preceding 
the dose or planned administration at any time during the study period, which might 
interfere with assessment of the immune response. 
 
16. A planned or anticipated move to a location that will prohibit full participation in the 
trial for the three year duration. 

 
40 
PR-15097  
 
V2.10: 16Jun17 
 
 
17. Potential adult volunteers or parents of potential child volunteers, who do not have 
easy access to a fixed or mobile telephone. 
18. Any participant that resides in a household with an individual previously enroled in an 
older age cohort in this study.  Only one participant from each household will be 
enroled. 
 
19. Any participant identified as a site employee of the Investigator or study clinic, with 
direct involvement in the proposed study or other studies under the direction of that 
Investigator or study clinic, as well as any immediate family member (such as 
husband, wife and their children, adopted or natural) of the clinic employees or the 
Investigator. 
 
4.3 Elimination Criteria During the Study 
The following criteria should be checked at each visit subsequent to the first visit. If any 
become applicable during the study, it will not require withdrawal of the participant from the 
study but may determine a participant’s evaluability in the per-protocol (PP) analysis. The 
participant will continue to be evaluated for safety purposes at the regularly scheduled visits. 
See Section 10.2 for definition of study cohorts to be evaluated.  
 
1. Use of any investigational or non-registered product (drug or vaccine) other than the 
study vaccine/placebo up to Day 56.  
 
2. Chronic administration (defined as more than 14 days) of immunosuppressants or other 
immune-modifying drugs up to Day 56. For corticosteroids, this will mean prednisone, or 
equivalent, ≥0.5 mg/kg/day. Inhaled and topical steroids are allowed. 
 
3. Administration of a licensed vaccine during the period starting from 30 days before dose 
of vaccine/placebo and ending 30 days after, with the exception of standard infant and 
childhood EPI “inactivated” vaccinations. 
 
4. Administration of immunoglobulins and/or any blood products up to Day 56.  
 
5. Pregnancy within the 28 days after investigational product administration.   
 
4.4 Contraindications to Vaccination 
The following conditions constitute absolute contraindications to administration of the study 
vaccine; if any of these AEs occur during the study, the study participant may continue study 
procedures at the discretion of the Investigator. The participant must be followed until 
resolution of the event, as with any AE. 
 
• Any confirmed or suspected immunosuppressive or immunodeficient condition 
• Pregnancy 
The following conditions constitute temporary contraindications to administration of the 
study vaccine: 
• Acute disease at the time of vaccination (Acute disease is defined as the presence of a 
moderate or severe illness with or without fever. Vaccine can be administered to 
persons with a minor illness such as diarrhoea or mild upper respiratory infection.) 

 
41 
PR-15097  
 
V2.10: 16Jun17 
 
• Fever ≥ 38.0ºC 
If any of these temporary contraindications occurs at the time of scheduled vaccination, the 
study participant may be vaccinated at a later date, within the time window specified in the 
protocol or excluded from participation at the discretion of the Investigator.  
 
4.5 Participant Withdrawal and Termination Criteria 
A study participant will not be considered to have completed the trial if any of the following 
reasons apply.  However, any participant who has received vaccine or placebo will be 
encouraged to remain in the study for periodic safety evaluations for the duration of the study 
at the discretion of the Investigator. 
 
1. Research terminated by Sponsor or Investigator – applies if the entire study is 
terminated by the Sponsor or Investigator for any reason. 
 
2. Withdrawal of consent – applies to a participant who withdraws consent to participate in 
the study for any reason. 
 
3. Noncompliant with protocol – applies to a participant who does not or is not able to 
comply with protocol-specific visits or evaluations on a consistent basis such that 
adequate follow-up is not possible and the participant’s safety, and the integrity of the 
study data, would be compromised by continuing in the trial.  
 
4. Participant withdrawal – may occur if the Investigator believes that it is in the best 
interest of the participant to be withdrawn from the study. 
 
5. Other – category used when previous categories do not apply and requires an 
explanation.   
 
For study participants where the reason for early termination is voluntary withdrawal of 
consent, the Investigator should attempt to determine the reason for the subject’s decision. 
The study staff will attempt to contact all study participants that withdraw or are terminated 
to obtain further safety information and to inform them of any significant findings which may 
affect their safety or health, unless the participant specifies that they do not want to be 
contacted again. This information will be documented in the source document.  
 
Participants will be allowed to withdraw from the study at any time without prejudice or loss 
of benefits to which they are entitled. The Investigator or designee will document whether the 
decision to withdraw from the study was made by the participant or the Investigator and 
describe the reasons for withdrawal. Information relative to the withdrawal will be 
documented on the related study documents. 
 
With the exception of expenses resulting from research-related injury, medical costs incurred 
following a participant’s withdrawal from the study will become the responsibility of the 
participant or the participant’s parent/legal guardian. 
 
Any and all data and biologic samples collected from the participant prior to his or her 
withdrawal may be used by Investigators as indicated in the protocol, unless the participant 
specifies a wish that the samples are destroyed. 
 

 
42 
PR-15097  
 
V2.10: 16Jun17 
 
All IRB/ERCs will be notified of any study participant’s withdrawal at the time of the 
continuing review submission. 
 
The Sponsor will be notified of any participant’s withdrawal according to the Sponsor’s 
guidelines. 
 
Special Situations: 
 
Pregnancy:   
If a female study participant who receives investigational product becomes pregnant up to 28 
days after receiving the investigational product, it may pose potential risks or contribute 
towards an AE for the participant or fetus. If study participant becomes pregnant within 28 
days of having received study product, the participant will not be included in the 
immunogenicity evaluations from her estimated date of conception.  She will, however, be 
encouraged to remain in the study for periodic safety evaluations and will be followed until 
completion of her pregnancy.  If any study participant tests positive for pregnancy within 56 
days after receiving the investigational product, the pregnant participant will be followed for 
the duration of the pregnancy, and the newborn will be assessed for any potential adverse 
outcomes.  The participant will be asked to sign a release of medical information form so that 
records can be obtained from her clinician or obstetrician regarding the outcome of the 
pregnancy.  All pregnancies occurring within 56 days after receiving investigational product 
will be reported to the Sponsor, the icddr,b ERC, UVM IRB, the Research Monitor, and to 
the DSMBs as soon as study staff becomes aware of the pregnancy, and as required by 
regulations. A Pregnancy Notification/Outcome Form will be completed by the study staff 
and forwarded to the Sponsor (see Section 7.3 for Adverse Event Reporting details). 
 
Lost to follow-up: 
A study participant who is not reachable by telephone or other means of communication and 
therefore not able to be located is considered lost to follow-up.  A participant may be 
considered lost to follow-up and withdrawn from the study once 3 attempts have been made 
to contact the participant, followed by non-response to a letter sent to the last known address 
requesting that the participant contact the clinical site.  
 
Incarceration: 
In the event that a study participant becomes incarcerated during the course of the study, 
every attempt will be made to contact the participant for determination of his/her safety. A 
participant may be terminated from the study if his/her period of incarceration will make 
him/her unavailable for scheduled visits. 
 
Immunosuppression: 
Study participants will be asked to report any history of immunosuppression at the time of 
enrolment as part of the exclusion criteria. Investigators will assess participants for 
immunosuppressive disease by clinical history, physical exam, and medical evaluation. Study 
participants potentially afflicted with any other self-reported immunosuppressive condition 
will not be enroled.  
 
5 Vaccine Preparation 
 

 
43 
PR-15097  
 
V2.10: 16Jun17 
 
5.1 Pre-Vaccine Preparation 
Monovalent vaccine virus for this protocol will be stored at a NIAID-contracted repository 
until requested by the clinical site.  Vials of frozen vaccine for administration will be 
formally requested to be transferred to the clinical site by the PI/designee after local 
Institutional Review Board (IRB) and Ethical Committee (EC) approvals for the study have 
been granted and the U.S. Food and Drug Administration (FDA) has been in receipt of the 
protocol for at least 30 days without issuing a clinical hold. Vaccine may be transferred to the 
study site prior to IRB approval and FDA review for the purpose of determining the titer of 
the vaccine only. The icddr,b team will be responsible for obtaining Directorate General of 
Drug Administration (DGDA) approval for import of the investigational product. The DGDA 
requires the approved protocol, Research Review Committee (RRC) and ERC approval 
letters, and an invoice with the drug name, consignee, and consigner to be submitted at the 
time of application for an import permit for the investigational product.    
 
After transfer to the clinical site, each monovalent vaccine will be stored in a locked and 
dedicated freezer at -80°C ± 15°C until time of use.  After the initial vaccine shipment is 
received by the clinical site, a pair of vials for each serotype of the vaccine will be thawed.  
To verify potency, virus titer will be determined by plaque titration of each undiluted 
monovalent vaccine and after combining into a representative tetravalent admixture held on 
ice for 4 hours.  Additional titration may occur at approximately 6 month intervals to ensure 
stability of the stored vaccine.  The vaccine is supplied as a concentrate that must be diluted 
to the proper dose prior to administration. 
 
Lab personnel who are not blinded to treatment assignment will be responsible for preparing 
the vaccine and placebo.  Prior to vaccination, the PI/designee will supply a prescription 
request form including necessary information and the number of doses to be administered to 
the study staff responsible for vaccine preparation. 
 
Admixture TV005 (103 of rDEN1∆30, 104 of rDEN2/4Δ30(ME), 103 of rDEN3∆30/31, and 
103 of rDEN4∆30) will be prepared according to the site’s Study specific Procedure (SSP).  
Study staff will prepare the correct dose of vaccine (or placebo) for each study participant in 
a biosafety hood using aseptic technique.  Vaccine will be diluted with 1X L-15 medium, 
which is prepared from a specific safety tested lot of 2X L-15 medium and sterile water for 
injection (1:1, v/v).  A Type II Master File for L-15 was created and submitted to the FDA 
(BB-MF #12959) on 27 February 2006.  The specific safety testing results for the current lot 
of 2X L-15 medium are available in the Master File.  The diluted vaccine (or placebo) will be 
drawn up to a volume of 0.5 mL in a 1 mL syringe and labeled according to the SSP.  The 
labeled syringes will be placed in a plastic syringe case and will be transported in an insulated 
cooler containing wet ice or an electronic insulated cooler to the clinic for administration.  
Vaccine must be used within 4 hours of being removed from the freezer.  Note that the 
electric coolers do not contain any wet ice, but rather work on airflow and can maintain the 
desired temperature for approximately 20 hours.  In addition, the admixture syringes will be 
kept in syringe cases within the cooler.  These syringe cases are transparent plastic casing 
which will enclose the syringes. 
 
5.2 Vaccine Storage 
Each monovalent vaccine candidate should remain frozen at -80°C ± 15°C until just prior to 
use on each scheduled vaccination day. Vaccine should never be refrozen for reuse in vaccine 
preparation.  Diluent components will be stored at 2°C to 8°C as per the manufacturer’s 

 
44 
PR-15097  
 
V2.10: 16Jun17 
 
recommendation.  Vaccine and diluent components should be opened from new containers 
for each use.  No component should be reused for future vaccine or placebo preparation. 
 
5.3 Vaccine Accountability 
Laboratory vaccine preparation personnel will maintain an accurate inventory and 
accountability record of vaccine for this study.  Partially used vials of vaccine or placebo 
components will not be refrozen or reused for future vaccinations. 
 
5.4 Storage Disposition of Used/Unused Supplies 
After laboratory personnel have diluted the vaccine and drawn up the syringes for 
administration, they will remove the label from the vaccine vial and place it in the vaccine 
preparation form.  In this manner, monitoring personnel will be able to verify the 
accountability of all vaccine vials used for the study.  In addition, the number of vaccine vials 
used will be accounted for in the study specific drug accountability log.  The diluent will be 
accounted for in the general accountability log, with identification of the specific study.  
 
An aliquot of undiluted (if available) and diluted vaccine will be titrated by laboratory 
personnel after vaccine has been prepared and delivered to the clinical staff.  This is done to 
confirm the potency of vaccine administered to the study participants. 
 
At least 1 aliquot (if available) of diluted vaccine will also be frozen and stored at-80°C ± 
15°C at the clinical site for future re-titration if needed.  After the syringes have been 
dispensed and aliquots removed for titration, any remaining vaccine will be destroyed by the 
laboratory personnel per SSP/SOP.  Any unused diluent/placebo from opened vials/bottles 
will also be destroyed. 
 
6 Study Procedures 
The following sections provide a detailed listing of the procedures and studies to be 
performed in this protocol at designated time points. 
 
6.1 Recruitment and General Screening 
Study participants will be recruited from Mirpur, Dhaka Wards, 2, 3, and 5 (or other Wards 
within Mirpur as designated by the PI if needed to reach enrolment targets).  Approximately 
100 participants per age group will be approached for recruitment from a household list.  If 
they agree, they will be taken to the clinic for consenting and screening procedures to be 
performed by a Medical Officer or their designee.  Approximately 100 potential participants 
will be approached in order to recruit 48 study participants in each age cohort. Only one 
study participant will be recruited per household. 
 
During the screening process, the study participant will read the consent form and will be 
encouraged to ask questions.  The participant may either sign the consent/assent form during 
the screening visit or return after further consideration.  The participant will have a medical 
history review, pregnancy prevention counselling (if applicable), physical exam, and/or lab 
specimens drawn during the general screening process after the informed consent/assent is 
signed.  
 
Approximately 400 adults, adolescents, and children will be screened in order to enrol 192 
healthy participants.  From the 192 enroled, 144 will receive the tetravalent dengue vaccine, 
and 48 will receive the placebo. Only participants who have signed the informed consent 

 
45 
PR-15097  
 
V2.10: 16Jun17 
 
and/or assent forms and have met the eligibility and inclusion/exclusion criteria will be 
enroled in the study. 
 
6.2 Screening Procedures 
All participants will undergo the following screening procedures within 30 days prior to 
vaccination: 
 
1. Explain the study and Informed Consent to the study participant. The consent form will 
be signed by all adult study participants.  An assent form will be signed by all children 
age 11-17 years of age, in addition to having the consent form signed by the parent(s) or 
legal guardian(s). A standard adult informed consent will be signed by the parent(s) or 
legal guardian(s) for participants less than 11 years of age. 
 
2. Ensure that the study participant has signed the Informed Consent/Assent, and receives a 
copy of the signed Informed Consent and assent forms if applicable. 
 
3. Check eligibility using inclusion and exclusion criteria for study participation. 
 
4. Collect and record demographic data and contact information. 
 
5. Elicit a complete medical history, including menstrual and contraceptive history and/or 
history of surgical sterilization for female study participants of child-bearing potential. 
Data will be recorded on appropriate study documents and if the study participant is 
enroled, the CRF will be uploaded at the time of enrolment (Day0). 
 
6. Perform a complete standard physical examination, including vital signs (height, weight, 
blood pressure, respiration, and pulse may be recorded depending on age). Data will be 
recorded in a source document and entered into CRF at the screening visit.  If participant 
is enroled, the CRF will be uploaded at the time of enrolment (Day 0). 
 
7. Obtain urine for rapid point-of-care β-HCG testing in females of child-bearing potential. 
A positive β-HCG will exclude the participant from the trial. 
 
8. Assign study identification number. 
 
9. Obtain whole blood for the following laboratory screening tests. See Table 16 for blood 
volumes to be drawn in each age cohort:  
• Complete blood count (CBC) with differential 
• Alanine transaminase (ALT) 
• Prothrombin time/partial thromboplastin time (PT/PTT) 
• Hepatitis B Virus (HBV) 
• Hepatitis C Virus (HCV) for adult and adolescent study participants only 
 
10. Any participant with a positive HBV or HCV test during screening will be referred to an 
appropriate medical provider who will provide further testing, counselling, and follow up 
care as needed. These participants would be excluded from enroling in the study.  
 
11. Counsel females of reproductive age or older to avoid becoming pregnant within 28 days 
of receiving a study vaccination series. 

 
46 
PR-15097  
 
V2.10: 16Jun17 
 
 
12. Counsel study participants regarding the procedures for capturing any AEs or serious 
adverse events (SAEs).  SAEs will be recorded throughout the entire study period. 
Participants will be instructed to contact the Investigator or designee immediately should 
they manifest any signs or symptoms they perceive as serious and for any hospitalizations 
for any reason (see Section 7.1.2, Serious Adverse Events). Participants will be given an 
identification card and a set of phone numbers to contact study staff 24 hours a day, 7 
days a week. 
 
13. Remind participant of anticipated study visit for dose 1, Study Day 0. 
 
6.3 Detailed Study Procedures 
Study procedures to be performed at each visit are listed below.  Photographs may be taken 
of the injection site. In addition, photographs may be taken of other areas of the skin before 
and/or after vaccination to record the characteristics of any rash that may develop.  
The volume of blood to be drawn over the full 156-week of vaccination phase in adults (age 
> 18 years of age) is approximately 257 mL. This amount is well within the Office for 
Human Research Protections (OHRP) and NIH guidelines (NIH Clinical Center Medical 
Administrative Policy M 95-9) for blood volumes in clinical research and should not 
compromise the health of study participants. Please see Table 16 for blood volumes to be 
drawn from each age cohort. These amounts do not include potential approximate 2 mL 
blood draws if participants are suspected to have dengue fever or additional amounts as 
needed to assess for resolution of laboratory AEs.  (See Section 6.3.11.3). 
 
On the day of vaccination, more study participants may be invited to the clinic than will be 
vaccinated.  These participants will be alternates and will be vaccinated only if other 
participants are not available for vaccination or are found to be ineligible on the day 
vaccination. Up to 48 participants will be assigned to each vaccination day. Up to five 
alternate participants will be identified for each vaccination day to replace any participants 
who are unable to attend the assigned first visit date. Those participants who are alternates 
will be informed that they are alternates when they are invited to the clinic.  
 

 
47 
PR-15097  
 
V2.10: 16Jun17 
 
 Table 15: Study specific procedures 
Study Day  
Screening 
(D-30 - D-5) 
Day 
0/Dosing 
Day 7 
Day 
14 
Day 
28 
Day 56 
Day 
180 
Day 
360 
Day 
720 
Day 
1080 
Study Activity 
Conduct household recruiting visits 
X 
  
  
  
  
  
  
 
 
 
Education 
X 
X 
  
  
  
  
  
 
 
 
Schedule screening visit 
X 
  
  
  
  
  
  
 
 
 
Assign subject ID 
X 
  
  
  
  
  
  
 
 
 
Review study procedures 
X 
  
  
  
  
  
  
 
 
 
Informed Consent/ Assent (child 11-
17) 
X 
X 
  
  
  
  
  
 
 
 
Check Inclusion/Exclusion 
X 
X 
  
  
  
  
  
 
 
 
Check elimination criteria 
  
  
X 
X 
X 
X 
X 
X 
X 
X 
Check contra-indications 
  
X 
  
  
  
  
  
 
 
 
Medical History 
X 
  
  
  
  
  
  
 
 
 
Interim Medical History 
  
X 
X 
X 
X 
X 
X 
X 
X 
X 
Standard Physical Exam 
X 
  
  
  
  
  
  
 
 
 
History-directed physical exam 
  
X 
X 
X 
X 
X 
X 
X 
X 
X 
Pre-vaccination temperature 
  
X 
  
  
  
  
  
 
 
 
Urine Pregnancy Testing*** 
X 
X 
  
  
 X 
 X 
 
 
 
Pregnancy prevention education*** 
X 
 X 
 X 
 X 
  
  
  
 
 
 
Blood Safety Tests: CBC/Diff, ALT 
X 
  
X 
X 
  
  
  
 
 
 
Blood Safety Tests: PT/PTT 
X 
  
  
X 
  
  
  
 
 
 
HBV, HCV* 
X 
  
  
  
  
  
  
 
 
 
Blood for Viremia 
  
  
X 
X** 
  
  
  
 
 
 
Blood for Serology 
  
X 
  
X** 
X 
X 
X 
X 
X 
X 
Blood for CMI 
  
X** 
X** 
  
X** 
X** 
X** 
X** 
X** 
X** 
Vaccination/ 30 minute observation 
  
X 
  
  
  
  
  
 
 
 
Record data from surveillance source 
document into CRF 
  
  
X 
X 
 X 
  
  
 
 
 

 
48 
PR-15097  
 
V2.10: 16Jun17 
 
Record concomitant medication 
  
X 
X 
X 
X 
  
  
 
 
 
Record Adverse Events 
  
X 
X 
X 
X 
  
  
 
 
 
Record Serious Adverse Events 
  
X 
X 
X 
X 
X 
X 
X 
X 
X 
Fever Surveillance/Evaluation of 
febrile episodes 
  
X 
X 
X 
X 
X 
X 
 
X 
 
X 
 
X 
Scheduling for follow-up visits 
  
X 
X 
X 
X 
X 
 X 
X 
X 
 
Reminder for next study visit 
X 
X 
X 
X 
X 
X 
 X 
X 
X 
 
Home visits for fever and AE surveillance on study days 0-14 except for scheduled clinic visits (0, 7, and 14).  After day 14 until 6 months, study 
participants will be contacted by phone or visited at home weekly.  After 6 months, study participants will be contacted by phone or in person 
monthly.  Information on AEs will be collected up to day 28. 
*HCV conducted on adults and adolescents only 
**Adult and adolescent age groups only 
***Pregnancy testing and prevention education if applicable (see Section 4.1#6) 
 
 

 
49 
PR-15097  
 
V2.10: 16Jun17 
 
6.3.1 Immunization Procedure 
All cohort study participants will receive vaccine or placebo on Study Day 0.  Monovalent 
vaccine candidates will be kept frozen at -80°C ± 15°C until just before use, whereupon they 
will be thawed, diluted, combined into a tetravalent admixture, and drawn up for 
administration (see Section 5 for vaccine preparation).  Vaccine or placebo will be kept on 
wet ice or in a dedicated, temperature-controlled electronic cooler from the time it is diluted 
until it is delivered to clinical staff for administration.  A vaccine volume of 0.5 mL will be 
delivered by subcutaneous injection in the deltoid region of the upper arm or the thigh in 
paediatric study participants with a needle of appropriate gauge and length after wiping the 
injection site with alcohol. 
 
6.3.2 Visit 1, Study Day 0, Administration of Vaccine or Placebo 
 
1. Verify that Informed Consent/Assent was obtained and that the consent/assent form 
was signed by the study participant and/or parent or guardian and by the study staff. 
 
2. Verify eligibility and that all inclusion and exclusion criteria have been met, and 
confirm that no contraindications to vaccination are present. If a study participant has 
any absolute contraindications to vaccination, the participant will be withdrawn from 
the study. If a participant has a temporary contraindication to vaccination (see Section 
4.4), the vaccination may be postponed until resolution of the temporary 
contraindication. 
 
3. Obtain vital signs and perform interim medical history and history-directed physical 
exam, concentrating on any acute complaints and any changes to medical history or 
exam findings since the screening visit. An examination of the thorax and skin will be 
performed to evaluate for dengue-like-rash. A pre-vaccination body temperature will 
be recorded. Any participant found to have a temperature of >38°C on the day of 
vaccination will not receive the vaccine on that day, as this is a temporary 
contraindication to vaccination.  
 
4. Obtain urine for rapid point-of-care β-HCG testing in females of reproductive age 
(see Section 4.1#6). A positive β-HCG will exclude the participant from the trial per 
protocol. Ensure the test is negative before proceeding. 
 
5. Perform pregnancy prevention counselling for females of reproductive age. 
 
6. Assign allocated study identification number and ensure study identification number 
is linked in enrolment log with the vaccine treatment number.  
 
7. Record all concomitant medications in CRF. 
 
8. Obtain whole blood for baseline serology (PRNT). These laboratory studies are drawn 
as baseline values and will not determine eligibility. 
 
9. Obtain whole blood for baseline Cell Mediated Immunity (CMI) testing for adult and 
adolescent study participants only. (See Table 16 for CMI blood volume) 
 
10. Administer dose of the vaccine or placebo as described in Section 6.3.1. 

 
50 
PR-15097  
 
V2.10: 16Jun17 
 
 
11. Observe for at least 30 minutes after vaccination and evaluate for immediate 
hypersensitivity.  Any AEs which occur after vaccination will be recorded by study 
staff into the CRF.  
 
12. Thermometers will be provided to study participants to monitor for febrile illness 
during the study period.  Surveillance source documents and small rulers will be used 
by FRAs or designee to record local and general solicited symptoms, including a 
temperature taken daily for 15 days (Day 0 through 14).  The observations will be 
recorded each day by FRAs or designee and will account for the previous 24 hours.  If 
multiple temperature measurements are taken in one 24 hour period, the maximum 
temperature will be recorded.   
a. FRAs or designee will record on the surveillance source document daily 
temperatures, and solicited and unsolicited symptoms (minimum Day 0-14) 
until the Study Day 14 visit. 
b. After Day 14, FRAs or designee will contact participants weekly by phone or 
home visit up to day 28, to continue to monitor and record solicited and 
unsolicited symptoms.  To monitor febrile illness, FRAs or designee will also 
contact participants weekly by phone or home visit up to Study Day 180 and 
then montly between Day 180 and three year follow-up. 
 
13. FRAs or designee will record on the source document concomitant medication until 
their next study visit (minimum Day 0-28) after vaccination. 
 
14. Record any AE and SAEs which have occurred from the time of screening. Serious 
adverse events will be recorded throughout the entire study period. Study participants 
will be instructed to contact the Investigator or designee immediately should they 
manifest any signs or symptoms they perceive as serious and for any hospitalizations 
for any reason (see Section 7.1.2, Serious Adverse Events). They will be given an 
identification card and a set of phone numbers to contact study staff 24 hours a day, 7 
days a week. 
 
15. Study staff will educate the study participants about the need to contact study staff 
immediately with any acute febrile illness consistent with suspected dengue.  
 
16. Ensure documentation of any febrile illness consistent with suspected dengue since 
last study visit.   
 
17. Study participants will be reminded of subsequent study visits, including the date of 
the next study visit. 
 
6.3.3 Visit 2, Study Day 7 (+/- 1 day) 
 
1. Confirm that study participants are still eligible according to elimination criteria.  (See 
Section 4.3). 
2. Obtain vital signs and perform interim medical history and history-directed physical 
exam, concentrating on any acute complaints. An examination of the thorax and skin 
will be performed to evaluate for dengue-like-rash. 
 

 
51 
PR-15097  
 
V2.10: 16Jun17 
 
3. Obtain whole blood for safety labs, to include CBC with differential and ALT 
according to blood volumes in Table 16.   
 
4. Obtain whole blood for viremia evaluation 
 
5. Obtain whole blood for CMI testing for adult and adolescent age cohorts only (Table 
16). 
 
6. MO to enter new data from the surveillance source document into the CRF. 
7. Record any unsolicited or solicited AEs since previous visit. 
 
8. Record concomitant medication since previous visit. 
 
9. Record SAEs during the entire study period (participants will be instructed to contact 
the Investigator immediately should they manifest any signs or symptoms perceived 
as serious or have any hospitalization for any reason, and/or any febrile illness 
consistent with suspected dengue). 
 
10. Ensure documentation of any febrile illness consistent with suspected dengue since 
last study visit.   
 
11. Perform pregnancy prevention counselling for females of reproductive age (see 
Section 4.1#6). 
 
12. Remind study participant of next scheduled study visit. 
 
6.3.4 Visit 3, Study Day 14 (+/- 1 day) 
 
1. Confirm that study participants are still eligible according to elimination criteria. (See 
Section 4.3). 
 
2. Obtain vital signs and perform interim medical history and history-directed physical 
exam, concentrating on any acute complaints. An examination of the thorax and skin will 
be performed to evaluate for dengue-like-rash. 
 
3. Obtain whole blood for safety labs, to include CBC with differential, PT/PTT, and ALT, 
according to blood volumes in Table 16 (for participants ≥ 11 years of age only). 
 
4. Obtain whole blood for viremia evaluation in the adult and adolescent cohorts only. 
 
5. Obtain whole blood for serology (PRNT) for adult and adolescent cohorts only. 
6. MO to enter new data from the surveillance source document into the CRF. 
7. Record any unsolicited or solicited AEs since previous visit. 
 
8. Record concomitant medication since previous visit. 
 

 
52 
PR-15097  
 
V2.10: 16Jun17 
 
9. Record SAEs during the entire study period (participants will be instructed to contact the 
Investigator immediately should they manifest any signs or symptoms perceived as 
serious or have any hospitalization for any reason, and/or any febrile illness consistent 
with suspected dengue). 
 
10. Ensure documentation of any febrile illness consistent with suspected dengue since last 
study visit.   
 
11. Perform pregnancy prevention counselling for females of reproductive age (see Section 
4.1#6). 
 
12. Remind participant of next scheduled study visit. 
 
6.3.5 Visit 4, Study Day 28 (+ 3 days) 
 
1.   Confirm that study participants are still eligible according to elimination criteria. 
(See Section 4.3). 
 
2. Obtain vital signs and perform interim medical history and history-directed physical 
exam, concentrating on any acute complaints. An examination of the thorax and skin 
will be performed to evaluate for dengue-like-rash. 
 
3. Per clinician discretion, obtain whole blood for safety labs as needed to assess for 
resolution of any laboratory AEs that were new or ongoing at Study Day 14.  
 
4. Obtain whole blood for serology (PRNT) according to blood volumes in Table 16 
 
5. Obtain whole blood for CMI testing (for adult and adolescent age groups only), and 
for safety labs as needed to assess for resolution of any laboratory AEs.  
 
6. Obtain urine from females of reproductive age for rapid β-HCG pregnancy testing.     
 
7. MO to enter new data from the surveillance source document into the CRF. 
 
8. Record any unsolicited or solicited AEs since previous visit. 
 
9. Record concomitant medication since previous visit. 
 
10. Record SAEs during the entire study period (participants will be instructed to contact 
the Investigator immediately should they manifest any signs or symptoms perceived 
as serious or have any hospitalization for any reason, and/or any febrile illness 
consistent with suspected dengue). 
 
11. Ensure documentation of any febrile illness consistent with suspected dengue since 
last study visit.   
 
12. Remind participant of next scheduled study visit. 
 
 

 
53 
PR-15097  
 
V2.10: 16Jun17 
 
6.3.6 Visit 5, Study Day 56 (+/- 7 days)  
 
1.  Confirm that study participants are still eligible according to elimination criteria. (See 
Section 4.3). 
 
2. Obtain vital signs and perform interim medical history and history-directed physical 
exam, concentrating on any acute complaints. An examination of the thorax and skin 
will be performed to evaluate for dengue-like-rash. 
 
3. Per clinician discretion, obtain whole blood for safety labs as needed to assess for 
resolution of any laboratory AEs that were ongoing at study day 28.  
 
4. Obtain whole blood for serology (PRNT) according to blood volumes in Table 16. 
 
5. Obtain whole blood for CMI testing (for adult and adolescent age groups only). 
 
6. Obtain urine from females of reproductive age for rapid β-HCG pregnancy testing.     
 
7. Record SAEs during the entire study period (participants will be instructed to contact 
the Investigator immediately should they manifest any signs or symptoms perceived 
as serious or have any hospitalization for any reason, and/or any febrile illness 
consistent with suspected dengue). 
 
8. Record concomitant medication in the case of new or changed significant or chronic 
conditions, for medications taken for AEs that were ongoing at Study Day 28, or for 
those medications taken for an SAE.  
 
9. Ensure documentation of any febrile illness consistent with suspected dengue since 
last study visit.   
 
10. Remind participant of next scheduled study visit. 
 
6.3.7 Visit 6, Study Day 180 (+/- 14 days) 
 
1.  Confirm that study participants are still eligible according to elimination criteria. (See 
Section 4.3) 
 
2. Obtain vital signs and perform interim medical history and history-directed physical 
exam, concentrating on any acute complaints. An examination of the thorax and skin 
will be performed to evaluate for dengue-like-rash. 
 
3. Per clinician discretion, obtain whole blood for safety labs as needed to assess for 
resolution of any laboratory AEs that were ongoing at study day 28.  
 
4. Obtain whole blood for serology (PRNT) according to blood volumes in Table 16. 
 
5. Obtain whole blood for CMI testing for adult and adolescent age groups only.  
 
6. Record SAEs during the entire study period (participants will be instructed to contact 
the Investigator immediately should they manifest any signs or symptoms perceived 

 
54 
PR-15097  
 
V2.10: 16Jun17 
 
as serious or have any hospitalization for any reason, and/or any febrile illness 
consistent with suspected dengue).  
 
7. Record concomitant medication in the case of new or changed significant or chronic 
conditions, for medications taken for AEs that were ongoing at Study Day 28, or for 
those medications taken for an SAE.  
 
8. Ensure documentation of any febrile illness consistent with suspected dengue since 
last study visit.   
 
9. Remind participant of next scheduled study visit. 
 
6.3.8 Visit 7, Study Day 360 (+/- 14 days) 
 
1. Confirm that study participants are still eligible according to elimination criteria. (See 
Section 4.3) 
2. Obtain vital signs and perform interim medical history and history-directed physical 
exam, concentrating on any acute complaints.  An examination of the thorax and skin 
will be performed to evaluate for dengue-like rash. 
3. Per clinician discretion, obtain whole blood for safety labs as needed to assess for 
resolution of any laboratory AEs that were ongoing at study day 28.  
4. Obtain whole blood for serology (PRNT) according to blood volumes in Table 16. 
5. Obtain whole blood for CMI testing for adult and adolescent age groups only. 
6. Record SAEs during the entire study period (participants will be instructed to contact 
the Investigator immediately should they manifest any signs or symptoms perceived 
as serious or have any hospitalization for any reason, and/or any febrile illness 
consistent with suspected dengue). 
7. Record concomitant medication in the case of new or changed significant or chronic 
conditions, for medications taken for AEs that wer ongoing at Study Day 28, or for 
those medications taken for an SAE. 
8. Ensure documentation of any febrile illness consistent with suspected dengue since 
last study visit. 
9. Remind participant of next scheduled study visit. 
 
6.3.9 Visit 8, Study Day 720 (+/- 14 days) 
 
1. Confirm that study participants are still eligible according to elimination criteria. (See 
Section 4.3) 
2. Obtain vital signs and perform interim medical history and history-directed physical 
exam, concentrating on any acute complaints.  An examination of the thorax and skin 
will be performed to evaluate for dengue-like rash. 
3. Per clinician discretion, obtain whole blood for safety labs as needed to assess for 
resolution of any laboratory AEs that were ongoing at study day 28.  
4. Obtain whole blood for serology (PRNT) according to blood volumes in Table 16. 
5. Obtain whole blood for CMI testing for adult and adolescent age groups only. 
6. Record SAEs during the entire study period (participants will be instructed to contact 
the Investigator immediately should they manifest any signs or symptoms perceived 
as serious or have any hospitalization for any reason, and/or any febrile illness 
consistent with suspected dengue). 

 
55 
PR-15097  
 
V2.10: 16Jun17 
 
7. Record concomitant medication in the case of new or changed significant or chronic 
conditions, for medications taken for AEs that wer ongoing at Study Day 28, or for 
those medications taken for an SAE. 
8. Ensure documentation of any febrile illness consistent with suspected dengue since 
last study visit. 
9. Remind participant of next scheduled study visit. 
 
6.3.10 Visit 9, Study Day 1080 (+/- 14 days) 
 
1. Confirm that study participants are still eligible according to elimination criteria. (See 
Section 4.3) 
2. Obtain vital signs and perform interim medical history and history-directed physical 
exam, concentrating on any acute complaints.  An examination of the thorax and skin 
will be performed to evaluate for dengue-like rash. 
3. Per clinician discretion, obtain whole blood for safety labs as needed to assess for 
resolution of any laboratory AEs that were ongoing at study day 28.  
4. Obtain whole blood for serology (PRNT) according to blood volumes in Table 16. 
5. Obtain whole blood for CMI testing for adult and adolescent age groups only. 
6. Ensure any SAE during the entire study period were captured and followed to 
resolution.  Active surveillance of serious adverse events will end following this final 
study visit. 
7. Record concomitant medication in the case of new or changed significant or chronic 
conditions, for medications taken for AEs that wer ongoing at Study Day 28, or for 
those medications taken for an SAE. 
8. Ensure documentation of any febrile illness consistent with suspected dengue since 
last study visit.  Surveillance of suspected dengue will end following this final study 
visit. 
9. Perform study conclusion form and document study conclusion in CRF. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
56 
PR-15097  
 
V2.10: 16Jun17 
 
Table 16: Approximate Blood volumes per study visit per cohort 
Adult Ages  
18-50 
Screen 
Visit 1 
Day 0 
Visit 2 
Day 7 
Visit 3 
Day 
14 
Visit 4 
Day 
28 
Visit 5 
Day 56 
Visit  
6 
Day 
180 
Visit  
7 
Day 
360 
Visit  
8 
Day 
720 
Visit  
9 
Day 
1080 
Test 
CBC/Diff 
1 ml 
  
1 ml 
1 ml 
  
  
  
 
 
 
ALT/HBV/HCV 
4 ml 
  
1 ml 
1ml 
  
  
  
 
 
 
PT/PTT  
2 ml 
  
  
2 ml 
  
  
  
 
 
 
Viremia  
  
  
2 ml 
2 ml 
  
  
  
 
 
 
Serology  
  
10 ml 
  
10 ml 
10 ml 
10 ml 
10 ml 
10 ml 
10 ml 
10 ml 
CMI  
  
20 ml 
20 ml 
  
20 ml 
20 ml 
20 ml 
20 ml 
20 ml 
20 ml 
Total 
7 mL 
30 mL 
24 mL 
16 mL 
30 mL 
30 mL 
30 
mL 
30 
mL 
30 
mL 
30 
mL 
Adolescent 
ages 11-<18 
Screen 
Visit 1 
Day 0 
Visit 2 
Day 7 
Visit 3 
Day 
14 
Visit 4 
Day 
28 
Visit 5 
Day 56 
Visit 
6 
Day 
180 
Visit 
7 
Day 
360 
Visit 
8 
Day 
720 
Visit 
9 
Day 
1080 
Test 
CBC/Diff  
1 ml 
  
1 ml 
1 ml 
  
  
  
 
 
 
ALT/HBV /HCV 
4 ml 
  
1 ml 
1ml 
  
  
  
 
 
 
PT/PTT  
2 ml 
  
  
2 ml 
  
  
  
 
 
 
Viremia  
  
  
2 ml 
2 ml 
  
  
  
 
 
 
Serology  
  
5 ml 
  
5 ml 
5 ml 
5 ml 
5 ml 
5 ml 
5 ml 
5 ml 
CMI  
  
8 ml 
8 ml 
  
8 ml 
8 ml 
8 ml 
8 ml 
8 ml 
8 ml 
Total 
7 mL 
13 mL 
12 mL 
11 mL 
13 mL 
13 mL 
13 
mL 
13 
mL 
13 
mL 
13 
mL 
Ages 5-<11 
Screen 
Visit 1 
Day 0 
Visit 2 
Day 7 
Visit 3 
Day 
14 
Visit 4 
Day 
28 
Visit 5 
Day 56 
Visit 
6 
Day 
180 
Visit 
7 
Day 
360 
Visit 
8 
Day 
720 
Visit 
9 
Day 
1080 
Test 
CBC/Diff  
1 ml 
  
1 ml 
1 ml 
  
  
  
 
 
 
ALT/HBV 
1 ml 
  
1 ml 
1ml 
  
  
  
 
 
 
PT/PTT  
2 ml 
  
  
2 ml 
  
  
  
 
 
 
Viremia  
  
  
2 ml 
  
  
  
 
 
 
Serology  
  
3 ml 
  
  
3 ml 
3 ml 
3 ml 
3 ml 
3 ml 
3 ml 
Total 
4 mL 
3 mL 
4 mL 
4 mL 
3 mL 
3 mL 
3 mL 
3 mL 
3 mL 
3 mL 
Ages 1-<5 
Screen 
Visit 1 
Day 0 
Visit 2 
Day 7 
Visit 3 
Day 
14 
Visit 4 
Day 
28 
Visit 5 
Day 56 
Visit 
6 
Day 
180 
Visit 
7 
Day 
360 
Visit 
8 
Day 
720 
Visit 
9 
Day 
1080 
Test 
CBC/Diff  
1 ml 
  
1 ml 
1 ml 
  
  
  
 
 
 
ALT/HBV 
1 ml 
  
1 ml 
1 ml 
  
  
  
 
 
 
PT/PTT  
2 ml 
  
  
2 ml 
  
  
  
 
 
 
Viremia  
  
  
2 ml 
  
  
  
 
 
 
Serology 
  
3 ml 
  
  
3 ml 
3 ml 
3 ml 
3 ml 
3 ml 
3 ml 
Total 
4 mL 
3 mL 
4 mL 
4 mL 
3 mL 
3 mL 
3 mL 
3 mL 
3 mL 
3 mL 

 
57 
PR-15097  
 
V2.10: 16Jun17 
 
NB:  The table is colour coordinated accordingly:  Orange = Safety procedures; Green = 
Study procedures; Blue = Exploratory procedures. 

 
58 
PR-15097  
 
V2.10: 16Jun17 
 
6.3.11 Unscheduled Visits 
 
6.3.11.1 Close-out of Solicited Laboratory Adverse Events 
In the event of a grade 1 or higher and unresolved laboratory AE, study participants may be 
asked to come to the clinic at a time specified by the medical officer or designee to assess 
resolution of the AE. Additional blood may be drawn at that time as determined to be 
clinically necessary.   
 
6.3.11.2 Fever Surveillance 
 
First line of surveillance 
Field research assistants (FRAs) or designee will make home visits to study participants or 
parent/legal acceptable representative to collect fever and AE information once per day up to 
day 14.   
 
Second line of surveillance  
Study participants or parents/LAR will be instructed to contact the Study Investigator and/or 
other appropriate study staff or come to the clinic in the event of febrile episodes during 
entire study period.   
 
Third line of surveillance  
After Day 14, study staff will contact study participants or their parent/legal acceptable 
representative weekly by phone or in person to check on fever and /or SAEs, or follow-up 
and resolution of ongoing AEs through Study Day 180.  After Study Day 180, study staff will 
contact study participants monthly by phone or in person to remind them to contact the clinic 
if they have a febrile illness.  Study staff will also ask study participants if they have been 
hospitalized for a febrile illness since the last call.  If the study participant was hospitalized 
for a febrile illness, they will be instructed to come to the clinic for suspected Dengue follow-
up.  
 
6.3.11.3 Definition of Suspected Dengue 
Study participants will be instructed to contact the Investigator or study coordinator if they 
develop fever equivalent to an oral temperature ≥38ºC at any time during the study period 
following the first vaccination visit.  
 
The Investigator will instruct the participant to monitor fever and any associated symptoms 
for an additional day.  
 
If 2 days of fever occur, the study participant will be instructed to report to the Investigator 
for an unscheduled visit to evaluate the participant for suspected dengue as defined below.  
The case definition of “suspected dengue” used in this study protocol includes the following 
criteria:  
• The study participant has a fever (equivalent to an oral temperature ≥38ºC) measured 
at least twice daily or is feverish (feels as if they have a fever but is unable to measure 
temperature) on two successive days 
• No alternative diagnosis by a qualified physician can be made with reasonable 
certainty 
 

 
59 
PR-15097  
 
V2.10: 16Jun17 
 
If dengue is suspected, a blood sample (approximately 2 mL of whole blood) will be 
collected from the study participant and analysed for dengue non-structural 1(NS1) antigen, 
IgM antibody, and viremia. PCR will also be conducted in order to determine if suspected 
dengue is vaccine related. 
 
The Investigator will record any symptoms associated with the fever during the suspected 
dengue evaluation on related study documentation.  The participant will be followed by the 
medical officer or appropriate designee until the fever abates. 
 
If a study participant has a febrile illness determined to not be due to a Dengue virus infection 
or related to vaccine/placebo administration, further fever work up may be performed at the 
discretion of the medical officer. The volunteer will be followed until fever abates. 
 
6.3.11.4 Definition of Laboratory Confirmed Dengue 
The case definition of laboratory confirmed dengue is as follows. All conditions must apply: 
1. The study participant has a fever (equivalent to an oral temperature ≥38ºC) 
measured at least twice daily or is feverish (feels as if they have a fever but is 
unable to measure temperature) on two successive days. 
2. No alternative diagnosis by a qualified physician can be made with reasonable 
certainty. 
3. DENV is detected by NS1/IgM criterion, PCR, or viremia assay. 
Additional testing may be completed at the discretion of the Investigator, if required to 
confirm a clinical diagnosis. 
 
6.4 Solicited and Unsolicited Adverse Events 
FRAs or designee will make home visits or calls to study participants once per day from study Day 0 
through study day 14 after vaccination, and weekly thereafter through Study Day 180.  After Study 
Day 180, study staff will contact study participants monthly by phone or in person until the end of 
the study.  They will record temperature and AEs on age-specific surveillance source documents as 
detailed below. Temperature will be recorded daily from Study Day 0 through Study Day 14. AEs 
will be collected from the time of informed consent through Study Day 28. Solicited local and 
general AEs, and a note of whether or not any unsolicited AEs occurred will be recorded on the age-
specific surveillance SD. Adverse events will then be entered into the appropriate CRF by MO, or 
designee, at the next clinic visit. Solicited AEs are described in Table 18, Table 19, and  
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
60 
PR-15097  
 
V2.10: 16Jun17 
 
 
Table 22.  Unsolicited AEs are AEs that are not described in Tables 18, 19, and 22.  Please 
see Section 7 for recording and reporting obligations for AEs.  
 
 
Field staff will instruct study participants how to use thermometers, to take their temperatures 
if they feel they have elevated temperatures, for dengue surveillance for the duration of the 
study.   
 
FRAs or designee will measure local reactogenicity at the vaccination site using rulers, and 
will record on the surveillance source documents.   
 
6.4.1 Solicited Local Injection Site Adverse Events 
AEs will be collected from the time of informed consent through Study Day 28. For all study 
participants, the solicited local symptoms at the injection site listed in Tables 18 and 19 
below will be assessed at the day of dengue/placebo vaccine administration and then every 
day for 15 days (from Day 0 through Day 14) post-vaccination, and weekly until Study day 
180, and monthly between Study day 180 and three year follow-up, by FRAs or designee at 
home visits or calls using surveillance source documents and rulers.  
 
6.4.2 Solicited General Adverse Events 
AEs will be collected from the time of informed consent through Study Day 28. For all study 
participants, the age-specific solicited general symptoms listed in Table 18 and Table 19 will 
be assessed at the day of dengue/placebo vaccine administration then every day for 15 days 
(from Day 0 through Day 14) post-vaccination, and weekly until Study day 180, and monthly 
between Study day 180 and three year follow-up by FRAs or designee at home visits or calls 
using surveillance source documents. 
 
For all age groups, oral or axillary temperature will be recorded once daily.  If additional 
temperature measurements are performed at other times of day, the highest temperature will 
be recorded. 
Fever is defined as an oral temperature ≥38°C, axillary temperature ≥38°C, tympanic 
temperature on oral setting ≥38°C. 
 
See Section 7 for full details of AE monitoring and grading. 
 
6.5 Clinical Laboratory Testing 
Using standard techniques, the icddr,b clinical laboratories will perform the following tests: 
• Complete blood count plus white blood cell differential.  
• PT/PTT 
• ALT 
• Hepatitis B and C (for adults and adolescents only) screening 
Laboratory assays will be performed according to Table 17. Urine β-HCG testing will be 
performed at the clinical trial site using a U.S. FDA-approved pregnancy test kit.  Collected 
scheduled samples for quantitation of vaccine virus titers obtained during the first 14 days 
after dosing of the candidate vaccine will be stored at-80°C ± 15°C at icddr,b research 
laboratories, and tested by isolation in Vero cells at the icddr,b research laboratories. Testing 
of serum samples for suspected dengue will be performed by a rapid NS1 ELISA followed by 
IgM ELISA at the icddr,b research laboratory. Multiplex PCR will also be run.  Serum 
collected for neutralizing antibody (NAb) plaque reduction neutralization antibody assays 

 
61 
PR-15097  
 
V2.10: 16Jun17 
 
(PRNT assays) will be stored at -20°C or below and assayed at the icddr,b research 
laboratory. Peripheral Blood Mononuclear Cells (PBMCs) will be isolated and stored in 
liquid nitrogen at the icddr,b research laboratory and shipped to the University of Vermont 
for cellular immune studies as detailed below in Table 17.  
 
 

 
62 
PR-15097  
 
V2.10: 16Jun17 
 
 
 Table 17: Laboratory assays 
Marker 
Assay Method 
Assay Type 
Testkit/ 
Manufacturer 
Assay cut-off and 
unit 
Laboratory 
CBC/diff, ALT 
PT/PTT* 
Automated assay 
Quantitative 
Commercial 
Refer to table of 
normal lab values 
Local (icddr,b 
clinical labs) 
Vaccine DENV 
isolation 
Vero cell isolation 
Qualitative and 
quantitative 
In-house 
Positive/negative 
icddr,b research 
labs 
NS1 rapid test for 
suspected Dengue 
Automated assay 
Qualitative 
Commercial 
Positive/negative 
Icddr,b research 
labs 
NSI/IgM  ELISA 
for suspected 
Dengue 
Automated assay  
Qualitative 
Commercial 
Positive/negative 
Icddr,b research 
labs 
Multiplex PCR 
for suspected 
Dengue 
pcr 
Qualitative 
In-house 
Positive/negative 
TBD 
Neutralizing Ab 
to DENV 
PRNT  
Quantitative 
In house (in Vero 
cells) 
The titer giving 50% 
reduction in viral 
infection   
icddr,b research 
labs 
Anti-HBV, HCV 
Automated assay 
Qualitative 
Commercial 
Positive/negative 
Local (icddr,b 
clinical labs) 
Human chorionic 
gonadotropin 
Urinalysis 
Qualitative  
Commercial 
As specified by the 
manufacturer 
On-site Mirpur 
clinic 
 
 
 
6.6 Medical History and Concomitant Medications 
A complete medical history will be collected during screening. Any changes reported in 
medical history during each 28-day post vaccination visit will be assessed as possible 
AEs. After Study Day 28 following vaccination, the medical history will be updated for any 
new or changed significant or chronic conditions.  Study staff will collect current medications 
as part of the medical history, including over the counter medications and herbal 
supplements, at the time of enrolment.  All changes or updates to medications will be 
collected through Study Day 28 following vaccination.  After Study Day 28, concomitant 
medications will be collected in the case of new or changed significant or chronic conditions.  
Medications taken for AEs that occur prior to Study Day 28 and continue past study day 28, 
and medications taken for SAEs will be recorded throughout the trial.  
 
6.7 Immunology Testing 
 
6.7.1 Antibody Testing 
Serum antibody levels to DENV-1, DENV-2, DENV-3, and DENV-4 will be measured by 
plaque reduction neutralizing antibody assay using standard laboratory protocols.  The 50% 
plaque reduction neutralization titer (PRNT50) is defined as the highest dilution of antibody 
that reduces the number of foci or plaques by 50%, compared to the plaque titer of a mixture 
of virus with serum from the same participant prior to vaccination.    
 

 
63 
PR-15097  
 
V2.10: 16Jun17 
 
6.7.2 Other Exploratory Immunological Assays 
Additional immunologic assays may be performed on PBMCs to evaluate the individual 
immune response post-vaccination including: 
• New serological measures of immunogenicity and vaccine efficacy surrounding 
neutralization and enhancement. 
• Cell-mediated immunity including stimulation of T and B cells for measures of 
adaptive immune response post-vaccination. 
• Evaluation of innate immune response post-vaccination. 
• Other immunologic assays in response to the development of additional assay 
techniques and current literature and scientific knowledge over the course of the 
study. 
• PBMC phenotyping, to include human leukocyte antigen (HLA) typing of samples, 
may be performed. 
 
7 Adverse Event Monitoring 
 
7.1 Definitions 
 
7.1.1 Adverse Event 
An adverse event (AE) is defined as any untoward or unfavourable medical occurrence in a 
human subject, including any abnormal sign (e.g. abnormal physical exam or laboratory 
finding), symptom, or disease, temporally associated with the subject’s participation in the 
research, whether or not considered related to the research. All AEs occurring from the time 
of consent to the day 28 follow-up period will be evaluated for severity, action taken, 
seriousness, outcome and relationship to the investigational vaccine and to research 
participation as described in Section 7.2 in this protocol. The relationship to the 
investigational product and research participation will be determined by the Investigator.  
 
If a diagnosis is clinically evident, the diagnosis rather than the individual signs and 
symptoms or lab abnormalities will be recorded as the AE.  AEs will be collected through the 
28-day period following vaccination, to include capture of all solicited local and general AEs 
recorded on the surveillance source document. Any AEs identified in the 28-day post-
vaccination period will be followed until resolution.  
 
AEs are categorized as solicited AEs and unsolicited AEs.  Solicited AEs include local 
reactogenicity, systemic reactogenicity, and Laboratory AEs.  Solicited AEs are those events 
which the clinician is specifically evaluating during the 28 day post-vaccination period, as 
listed in Table 18, Table 19, and  

 
64 
PR-15097  
 
V2.10: 16Jun17 
 
Table 22.  
 
• reported as outlined to ethical review boards and IND sponsor at continuing review, and 
DSMB before each meeting outlined in Section11.1. 
 
 
 
          Table 18: Solicited adverse events: study participants > 5 years of age 
Systemic Reactogenicity 
Local Reactogenicity 
Fever 
Injection site pain 
Headache 
Injection site erythema 
Retro-orbital pain 
Injection site swelling 
Photophobia 
Injection site pruritus 
Nausea 
 
Fatigue 
 
Myalgia 
 
Arthralgia 
 
Diffuse rash on trunk 
 
 
 
Table 19: Solicited adverse events: study participants < 5 years of age 
Systemic Reactogenicity 
Local Reactogenicity 
Fever 
Injection site pain 
Irritability/fussiness 
Injection site erythema 
Drowsiness 
Injection site swelling 
Loss of Appetite 
Injection site pruritus 
Vomiting 
 
Decrease in activity (fatigue) 
 
Headache 
 
Diffuse rash on trunk 
 
 
 
7.1.2 Serious Adverse Event (SAE) 
  SAEs will be collected from the time of informed consent until study participants three year 
follow-up period has been completed.  An SAE is defined as involving one or more of the 
following outcomes: 
• Death  
• Life threatening event, defined as an event that places a subject at immediate risk of 
death at the time of the event and does not refer to an event that hypothetically might 
have caused death were it more severe 
• Inpatient hospitalization or prolongation of existing hospitalization, defined as at least 
an overnight stay in the hospital or emergency ward for treatment that would have 
been inappropriate if administered in the outpatient setting 
At each contact with the participant, information regarding AEs will be elicited by 
appropriate questioning and examinations and will be: 
 
• immediately documented in the subject’s medical record/source document, 
 
• recorded on the AE CRF and 
 

 
65 
PR-15097  
 
V2.10: 16Jun17 
 
• Congenital anomaly or birth defect 
• Persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions. 
• Other medically important event* 
 
*Medical and scientific judgment should be exercised in deciding events that may not be 
immediately life threatening or result in death or hospitalization but may jeopardize the 
subject or may require intervention to prevent one of the outcomes listed above.  
 
Each AE will be classified by the Investigator/clinically qualified designee as “serious” or 
“non-serious”.  An AE needs to meet only one or more of the above criteria to be considered 
serious. 
 
7.1.3 Unexpected Adverse Events (UAEs)  
An adverse event is considered unexpected if it is not listed in the Investigator’s Brochure or 
is not listed at the specificity or severity that has been observed.    It is the responsibility of 
the IND Sponsor to make this determination. 
 
7.1.4 Serious and Unexpected Suspected Adverse Reaction (SUSAR) 
A SUSAR is a Suspected Adverse Reaction that is both Serious and Unexpected. SUSARs 
are reported only if there is evidence to suggest a causal relationship between the drug and 
the adverse event, such as: 
1. A single occurrence of an event that is uncommon and known to be strongly associated 
with drug exposure; 
2. One or more occurrences of an event that is not commonly associated with drug exposure, 
but is otherwise uncommon in the population exposed to the drug; 
3. An aggregate analysis of specific events observed in a clinical trial that indicates those 
events occur more frequently in the drug treatment group than in a concurrent or 
historical control group. 
7.1.5 Unanticipated Problem that is not an Adverse Event (UPnonAE) 
An unanticipated problem that does not fit the definition of an adverse event, but which may, 
in the opinion of the investigator, involve risk to the subject, affect others in the research 
study, or significantly impact the integrity of research data. Such events would be 
considered a non-serious UP.  For example, we will report occurrences of breaches of 
confidentiality, accidental destruction of study records, or unaccounted-for study drug. 
 
Adverse Reaction (AR) 
An adverse event that is caused by an investigational agent (drug or biologic). 
 
Suspected Adverse Reaction (SAR) 
An adverse event for which there is a reasonable possibility that the investigational agent 
caused the adverse event.  ‘Reasonable possibility’ means that there is evidence to suggest a 
causal relationship between the drug and the adverse event.  A suspected adverse reaction 
implies a lesser degree of certainty about causality than adverse reaction which implies a 
high degree of causality. 
 
7.1.6 Pre-existing Conditions, Worsening of Pre-existing Conditions 
Stable chronic conditions which are present prior to enrolment and do not worsen are not 
considered AEs and will be accounted for in the study participant’s medical history.  

 
66 
PR-15097  
 
V2.10: 16Jun17 
 
Exacerbation or worsening of pre-existing conditions are defined as AEs and are evaluated 
using the same criteria described in Section 7.2 in this protocol.  Any SAE that occurs prior 
to enrolment for eligible participants will be recorded using the NIAID SAE report form (the 
Safety Expedited Report Form or SERF), with the study ID as the identifier.   
 
Protocol Deviation:  
Any change, divergence, or departure from the IRB approved study procedures in a research 
protocol. Protocol deviations are designated as serious or non-serious and further 
characterized as 
 
1. 
Those that occur because a member of the research team deviates from the protocol. 
2. 
Those that are identified before they occur, but cannot be prevented. 
3. 
Those that are discovered after they occur 
 
Serious Protocol Deviation:  
A deviation that meets the definition of a Serious Adverse Event or compromises the safety, 
welfare or rights of subjects or others. 
 
Non-compliance:  
The failure to comply with applicable NIH HRPP policies, IRB requirements, or regulatory 
requirements for the protection of human subjects. Non-compliance is further characterized 
as: 
1. 
Serious: Non-compliance that 
a. 
Increases risks, or causes harm, to participants 
b. 
Decreases potential benefits to participants 
c. 
Compromises the integrity of the NIH-HRPP 
d. 
Invalidates the study data 
2. 
Continuing: Non-compliance that is recurring 
3. 
Minor: Non-compliance that, is neither serious nor continuing. 
 
7.2 Assessment of Adverse Events 
 
7.2.1 Identification of Adverse Events 
Assessment of safety will include clinical observations and monitoring of haematological, 
blood chemistry, and immunologic parameters.  Safety will be evaluated by monitoring of the 
study participants for local and systemic adverse reactions during the course of the trial.  
Participants will be closely monitored for 30 minutes following immunization.  Additionally, 
participants will return to the clinic on Study Days 7, 14, 28, 56, 180, 360, 720, and 1080 
following vaccination, at a minimum and may be asked to return more often if warranted. 
Study staff record temperatures and AEs on the surveillance source document. Any AEs 
which occur from Day 14 to Day 28 post-vaccination will be captured by questioning of the 
participant by study staff at the Day 28 visit or by weekly phone calls or home visits. At each 
visit through Study Day 28 following vaccination, study staff will query the participant about 
any AEs noted on the surveillance source document and perform a history directed physical 
exam. A study clinician will be available to study participants by telephone 24 hours a day 
during the entire three year study evaluation period, and all participants will be provided with 
a telephone contact card during the initial enrolment visit. 
 
For all enroled study participants, all AEs occurring from the time the informed consent is 
signed through day 28 will be documented, recorded, and reported.  All solicited and 

 
67 
PR-15097  
 
V2.10: 16Jun17 
 
unsolicited AEs will be recorded into source documents during the period after the study 
participant receives the study vaccine, through and including post-vaccination Study Day 28, 
and will be entered into the CRF up to the Day 28 visit.  In order to ensure maximum 
participant safety, a study participant may be asked to return to clinic for an unscheduled visit 
for assessment of resolution of an adverse event at the discretion of the clinician. 
 
All SAEs and SUSARs will be reported following SAE reporting guidelines outlined in 
Section 7.3.2 of this protocol. SAE severity grading classifications are listed in Table 20.   
 
7.2.2 Protocol Specific Adverse Event Definitions 
 
Solicited general adverse events in study participants ≥ 5 years of age 
Fever 
 
Diffuse Rash on Trunk 
(Dengue Vaccine-like Rash): 
Oral temperature ≥38°C, axillary temperature ≥38°C, 
tympanic temperature on oral setting ≥38°C 
Macular/ maculo-papular rash, typically found on the trunk 
and proximal extremities most frequently seen 10-16 days 
after vaccination. 
Headache: 
A pain located in the head, over the eyes, at the temples, or 
at the base of the skull. 
Retro-orbital pain (ROP): 
Bilateral pain situated behind the orbits of the eye. 
Photophobia:  
An abnormal sensitivity to or intolerance of light. 
Nausea:  
Discomfort in the stomach with an urge to vomit. 
Fatigue:  
Excessive tiredness following minimal exertion. 
Myalgia:  
Pain in the muscles, found in > 2 muscle groups. 
Arthralgia:  
Pain in a joint, found in > 2 joints. 
 
Solicited general adverse events in children < 5 years of age 
 
Fever 
 
Diffuse Rash on Trunk 
(Dengue Vaccine-like 
Rash): 
Oral temperature ≥38°C, axillary temperature ≥38°C, 
tympanic temperature on oral setting ≥38°C 
Macular/ maculo-papular rash, typically found on the trunk 
and proximal extremities most frequently seen 10-16 days after 
vaccination. 
Irritability/fussiness:  
Excessive irritability, manifested by frequent or inconsolable 
crying. 
Drowsiness:  
Fatigue and sleepiness evident more than is normal for the 
child.  
Loss of appetite:  
Noticeable decreased desire to eat normally. 
Vomiting:  
Abnormal frequency of vomiting. 
Decreased activity: 
Noticeably diminished playing behaviour, lack of interest in 
normal activities. 

 
68 
PR-15097  
 
V2.10: 16Jun17 
 
Headache (child): 
Holding head in hands, sensitivity to loud noise or bright light. 
 
7.2.3 Determination of Severity 
The Investigator/designee will assess and grade AE severity using the classifications outlined 
in Table 18, Table 19, Table 20 and  
Table 22. 
 
 

 
69 
PR-15097  
 
V2.10: 16Jun17 
 
 
 
          Table 20: Severity definitions 
Severity  
Defined 
Grade 0 (Normal) 
No symptoms 
Grade 1 (Mild) 
Event that is easily tolerated, may require 1 dose of medication/treatment 
Grade 2 (Moderate) 
Event that interferes with daily activity or requires more than 1 dose of 
medication/treatment 
Grade 3 (Severe) 
Event that prevents daily activity and requires medical intervention 
Grade 4 (Life Threatening)  
Event which places the subject at immediate risk of death 
Grade 5 (Death) 
Event which results in death  
 
Table 21: Assessment of solicited adverse events 
 
 
 
 
Local Reactogenicity 
Grade 
Severity 
Injection Site Pain 
Injection Site Pruritis 
0 
1 
2 
3 
Absent 
Event that is easily tolerated 
Event that interferes with daily activity 
Event that prevents daily activity 
Injection Site Erythema (redness) 
Injection Site Swelling 
(Record greatest surface diameter 
in mm) 
0 
1 
2 
3 
absent 
<5 mm 
5 mm to 20 mm 
>20 mm 
Systemic Reactogenicity 
Grade 
Severity 
Fever (oral or equivalent) 
0 
1 
2 
3 
<38 °C 
38° C–38.5°C 
38.6°C – 39.1°C 
≥39.2°C 
Dengue Vaccine Like Rash 
0 
1 
2 
3 
Normal 
Rash is present but asymptomatic 
Rash is symptomatic (pruritus/pain) but does not interfere with function 
Rash is symptomatic and interferes with function 
Headache 
Retro-orbital pain (ROP) 
Photophobia 
Nausea 
Fatigue 
Myalgia 
Arthralgia 
Irritability/fussiness 
Drowsiness 
Loss of appetite 
Vomiting 
Decrease in activity (fatigue) 
 
0 
 
1 
 
 
2 
 
 
3 
 
Normal 
 
Event that is easily tolerated, may require 1 dose of 
medication/treatment 
 
Event that interferes with daily activity or requires more than 1 dose of 
medication/treatment 
 
Event that prevents daily activity and requires medical intervention 

 
70 
PR-15097  
 
V2.10: 16Jun17 
 
         Table 22: Assessment of solicited laboratory AEs (43) 
 
7.2.4 Relationship of Adverse Events to Investigational Product Vaccine 
The Investigator is obligated to assess the relationship between the vaccine/placebo and the 
occurrence of each AE/SAE. The Investigator will use clinical judgment to determine the 
relationship. Alternative causes, such as natural history of the underlying diseases, 
concomitant therapy, other risk factors as well as the temporal relationship of the event to the 
vaccine/placebo will be considered and investigated. The Investigator will also consult the 
Laboratory AEs 
Grade 
Severity 
Haemoglobin (females) 
(Ages 5 and older) 
1 
2 
3 
9.5 - 10.7 gm/dL 
8.0 - 9.4 gm/dL 
<7.9 gm/dL 
Haemoglobin (males) 
(Ages 5 and older) 
1 
2 
3 
11 – 12.5 gm/dL 
9.0 – 10.9 gm/dL 
<8.9 gm/dL 
Haemoglobin (Children <5) 
 
1 
2 
3 
 
10.0 – 11.4 gm/dL 
8.5 – 9.9 gm/dL 
<8.4 gm/dL 
 
Neutropenia (reduced ANC) 
1 
2 
3 
750 - 999/mm3 
500 – 749/mm3 
<500 mm3 
Leukocytosis (Increased WBCs) 
(Ages 11 and older) 
1 
2 
3 
11,500 - 13,000/mm3 
13,001 - 15,000/mm3 
>15,000 or <1,000/mm3 
Leukocytosis (Increased WBCs) 
(Ages 4 to <11 years) 
1 
2 
3 
15,000 – 16,500/mm3 
16,501 – 18,500/mm3 
>18,500 or <1,000/mm3 
Leukocytosis (Increased WBCs) 
(Ages 1 year to <4 years) 
1 
2 
3 
18,000 – 19,500/mm3 
19,501 – 21,500/mm3 
>21,500 or <1,000/mm3 
Thrombocytopenia  
(Decreased Platelets) 
 
1 
2 
3 
 
≥100,000 – 120,000/mm3 
≥75,000 - 99,999/mm3 
<74,999/mm3 
 
PT 
1 
2 
3 
>1.0 - 1.25 x ULN 
>1.25 - 1.5 x ULN 
>1.5 x ULN 
 
PTT 
 
1 
2 
3 
 
>1.0 - 1.66 x ULN 
>1.66 - 2.33 x ULN 
>2.33 x ULN 
 
ALT/SGPT 
 
1 
2 
3 
>1.25 - 2.5 x ULN 
>2.5 - 5.0 x ULN 
>5.0 x ULN 

 
71 
PR-15097  
 
V2.10: 16Jun17 
 
Investigator Brochure in the determination of relatedness between the vaccine and adverse 
events. 
 
There may be situations when a SAE has occurred and the Investigator has minimal 
information to include in the initial report to the IRBs, ECs, and sponsor. However, it is very 
important that the Investigator always makes an assessment of causality for every event prior 
to transmission of the SAE Report Form, specifically referred to as the Safety Expedited 
Report Form (SERF). The Investigator may change his/her opinion of causality in light of 
follow-up information, amending the SERF accordingly. The causality assessment is one of 
the criteria used when determining regulatory reporting requirements. 
 
All solicited local (injection site) reactions will be considered causally related to vaccination. 
Causality of all other AEs should be assessed by the Investigator using the following 
question, “Is there a reasonable possibility that the AE may have been caused by the 
vaccine/placebo?” 
 
No: 
The AE is not causally related to administration of the study vaccine(s). There 
are other, more likely causes and administration of the study vaccine(s) is not 
suspected to have contributed to the AE. 
Yes: 
There is a reasonable possibility that the vaccine(s) contributed to the AE.  
 
Non-serious and serious AEs will be evaluated as two distinct events. If an event meets the 
criteria to be determined “serious” (see Section 7.1.2 for definition of serious adverse event), 
the Investigator will make every attempt to consider ALL contributing factors.  
 
 
7.2.5 Adverse Event Action Taken 
The Investigator/designee will assess the action taken by the study participant or the study 
staff in relation to the AE using the following classifications:   
 
Action 
1 = None 
2 = Remedial therapy (≥ 1 dose of medication required) 
3 = Discontinued study  
4 = Hospitalization 
5 = Other 
 
7.2.6 Adverse Event Outcome 
The Investigator/designee will assess the outcome of the AE, either at resolution or at the end 
of the study period, using the following classifications: 
Outcome  
1 = Resolved 
2 = Recovered with sequelae 
3= Continuing 
4 = Death 
5 = Unknown 
 

 
72 
PR-15097  
 
V2.10: 16Jun17 
 
7.2.7 Adverse Event Seriousness 
The Investigator/designee will categorize all AEs as either serious or non-serious, using the 
criteria defined in Section 7 of this protocol.   
 
Any events defined as serious will also be reported following SAE reporting guidelines 
outlined in Section 7.3 of this protocol. 
 
7.3 Adverse Event Reporting 
 
7.3.1 Non-Serious Adverse Events 
Non-serious AEs will be followed to resolution or until the study ends, and are reported to the 
Sponsor as requested, to the IRBs/ECs according to IRB/EC policies (to include annual 
Continuing Review Reports), to the DSMBs as required, and to the U.S. FDA by the sponsor 
as required for the annual report.  AE data will be submitted to the IND Sponsor when 
requested for periodic safety assessments, review of IND annual reports, review of IND 
safety reports, and preparation of final study reports. 
 
AEs meeting the stopping criteria outlined in Section 7.5 of this protocol will be reported to 
the Sponsor, the IRBs/ECs, and the DSMBs following the SAE reporting guidelines.  
 
7.3.2 Serious Adverse Events 
All SAEs (regardless of relationship and whether or not they are also UPs) must be reported on 
the Safety Expedited Report Form (SERF) and sent to the Clinical Safety Office (CSO) by fax 
or e-mail attachment. Deaths and immediately life threatening SAEs must be reported to the 
CSO within 1 business day after the site becomes aware of the event. All other SAEs must be 
reported within 3 business days of site awareness. 
 
E-mail: rchspsafety@mail.nih.gov 
 
SAEs that have not resolved by the end of the follow-up period are followed until final 
outcome is known. If it is not possible to obtain a final outcome for an SAE (e.g., the subject is 
lost to follow-up), the reason a final outcome could not be obtained will be recorded by the 
investigator on the AE CRF and the SERF. 
 
icddr,b ERC reporting 
All SAEs/SUSARs, UAEs, and UPs/UPs involving risk to subject or others are also reported 
to the icddr,b ERC as per guidelines as below: 
The PI is responsible for ensuring timely submission of Serious Adverse Events (SAEs) 
within 24 hours of knowledge of their occurrence, including measures taken to 
mitigate/manage the event. In the event any participant experiences multiple SAEs, each 
event will be reported separately. If detailed information is not available at the time of 
submission of the initial report, follow-up report(s) will be submitted as soon as information 
is available. Final outcome of the event should be reported within 72 hours of knowledge of 
outcome. 
CSO CONTACT INFORMATION: 
Clinical Safety Office 
5705 Industry Lane 
Frederick, MD 21704 
Phone 001-301-846-5301 
Fax 001-301-846-6224 

 
73 
PR-15097  
 
V2.10: 16Jun17 
 
 
UVM IRB 
All SAEs/SUSARs, UAEs, and UPs/UPs involving risk to study participants or others will be 
reported to the UVM IRB as per guidelines below:  
• UVM IRB requests that the above events be reported in the annual Continuing 
Review Report. 
 
Unanticipated Problems (UP) Reporting: 
Unanticipated Problems that are also AEs must be reported to the CSO and sent by fax or e-
mail attachment no later than 7 calendar days of site awareness of the event. UPs that are not 
AEs are not reported to the CSO. 
 
Report UPs that are also AEs to the CSO on a SERF or a local IRB UP form. 
 
7.4 Sponsor’s Reporting Responsibilities 
Serious, unexpected, suspected adverse reactions (SUSARs) as defined in 21 CFR 312.32 and 
determined by the IND Sponsor will be reported to the U.S. FDA and to all participating 
Investigators as IND Safety Reports. The IND Sponsor will also submit an IND Annual 
Report  of the progress of the investigation to the U.S. FDA as defined in 21 CFR 312.33.  
 
7.5 Stopping Criteria  
Halting the study requires immediate discontinuation of study agent administered for all 
subjects and suspension of enrolment until a decision is made whether or not to continue 
enrolment and study agent administration. The IRBs/ECs, the IND Sponsor (OCRPRO), 
CSO, either DSMB, or the FDA (CBER) may stop the study at any time following review of 
any safety concerns. 
 
The halting rules are outlined below: 
 
• Two or more study participants experience the same or similar SAEs that are 
unexpected and are possibly, probably, or definitely related to the study agent 
   OR 
 
•  any safety issue that the PI and/or the CSO determines should halt the study 
 
• If a stopping criteria is met, a description of the adverse event(s) or safety issue must 
be reported by the PI within one business day to the Sponsor, CSO, IRBs/ECs, and 
DSMBs by fax or email. The IND Sponsor will promptly report to CBER that a 
stopping criterion has been met.  
The IND Sponsor, in collaboration with the PI, IRBs/ECs, and DSMBs, will determine if it is 
safe to resume the study. The IND Sponsor will notify the Site Investigators of this decision. 
The conditions for resumption of the study will be defined in this notification. The Site 
Investigators will notify their local IRBs/ECs of the decision. 

 
74 
PR-15097  
 
V2.10: 16Jun17 
 
7.6 Pausing Criteria 
Pausing is the suspension of administration of study agent to a single study participant until a 
decision is made whether or not to resume administration of the study agent.  The pausing 
criteria for a single study participant include any of the following: 
 
A study participant experiences an SAE that is possibly, probably, or definitely related to a 
study agent 
 
A study participant experiences a Grade 3 or greater AE that is possible, probably, or 
definitely related to a study agent.  Any safety issue that Site Investigator determines should 
pause administration of study agent to a single subject.  
 
Any safety issue that the Site Investigator determines should pause administration of a study 
agent to a single subject.   
The IND Sponsor, in collaboration with the PI, may also pause an individual study participant 
or entire group if a safety concern is identified. 
If a pausing requirement is met, a description of the AE(s) or safety issue must be reported to 
the CSO, the IRBs/ECs, and the DSMBs by the Site Investigator by email within one 
business day. 
The IND Sponsor in collaboration with the PI, will determine if it is safe to resume 
administration of the investigational product to the study participant/group. The CSO or 
designee will notify the Site Investigators of this decision. The Site Investigators will notify 
their local IRB of the decision to resume administration of the study agent prior to 
resumption. 
A subject who does not resume study agent will continue to be followed for safety. 
 
8 Data Collection and Monitoring 
 
8.1 Source Documentation and Data Collection 
Complete source documentation (laboratory test reports, hospital or medical records, medical 
progress notes, etc.) is required for every study participant for the duration of the study.  The 
participant’s study record must record his/her participation in the clinical trial and, after 
unblinding, the randomization treatment received (with doses and frequency) and 
concomitant medications or other interventions administered, as well as any adverse reactions 
experienced during the trial.  
 
Study data will be maintained in paper CRFs that are uploaded into DataFax and collected 
directly from subjects during study visits and telephone calls, or will be abstracted from 
subjects’ medical records. Source documents include all recordings of observations or 
notations of clinical activities and all reports and records necessary to confirm the data 
abstracted for this study. Certain CRFs will serve as Source Documents. Data entry into paper 
CRFs and DataFax will be performed by authorized individuals.  
 
 
Corrections to the paper source documents must be made by striking through the incorrect 
entry with a single line (taking care not to obliterate or render the original entry illegible) and 

 
75 
PR-15097  
 
V2.10: 16Jun17 
 
entering the correct information adjacent to the incorrect entry.  Corrections must be initialled 
and dated by the person making the correction whenever possible.  Source documentation 
should support the data collected on the CRFs, and must be signed and dated by the person 
recording and/or reviewing the data.   
 
The Investigator is responsible for the accuracy, completeness and timeliness of the data 
reported to the Sponsor in the DataFax System.  Data reported in the DataFax system should 
be consistent with source documents. All discrepancies should be explained. Source 
documentation will be made available for review or audit by the Sponsor or designee, the 
Sponsor’s monitors, and any applicable Federal authorities. 
 
8.2 Study Documentation 
Study-related documentation will be completed as required by the IRBs/ECs, the Sponsor, 
and regulatory authorities.  Continuing review documentation will be submitted by the 
Investigator to the IRBs/ECs by the anniversary date of initial review as specified by each 
IRB/EC. An annual report will be submitted by the Sponsor to the FDA according to 
regulations.  These reports will provide a brief description of the progress of the investigation 
as outlined in 21 Code of Federal Regulations 312.33, and will include any revisions of the 
protocol if not previously submitted. In addition, annual Continuing Review Reports will be 
submitted to both the UVM and icddr,b IRBs, per regulations. 
 
The PI and designated icddr,b laboratory personnel will maintain adequate records to account 
for the disposition of the investigational product, including dates of receipt and quantity, 
current inventory, and dispensation to study participants.  If the study is terminated, 
suspended, or completed, the designated personnel at icddr,b will destroy all remaining 
investigational product according to the Sponsor’s recommendation. 
 
Investigators will maintain study charts for each volunteer. The study chart will contain the 
original signed consent form, any clinical and/or laboratory data, and other pertinent 
paperwork (i.e., source documents). The study chart will be identified by the unique study 
number assigned to the volunteer. Since the study charts will have documentation with both 
personal and unique identifiers, the chart is one possible link between study data and a 
personal identifier. 
  
8.3 Retention of Specimens 
All eligible specimens collected as part of this trial will be stored for future research as part 
of the NIAID and UVM IRB-approved biosample repositories for vaccine research. 
Participants must provide consent for future use of their samples for research purposes.  The 
informed consent document will clearly include a section in which the participant may 
consent to allow any future use of retained blood specimens.  Study specimens will be stored 
at icddr,b in accordance with their guidelines for retention of specimens.    
 
If a study participant becomes pregnant, incarcerated, or lost to follow-up, all specimens 
collected from those study participants will be tested as outlined in the protocol and will be 
stored for future use, but only if the participant provided consent. 
 
Participants may decide at any time not to allow their samples to be stored for future 
research.  In this case, the Principal Investigator will ensure that all known remaining samples 
are destroyed and will report the disposition of the samples to the participant and to the IRB. 

 
76 
PR-15097  
 
V2.10: 16Jun17 
 
The decision will not affect the participant’s participation in this protocol or in any other 
protocol sponsored by the NIH.  
 
Samples that are retained by consent of the participant may be used to learn more about 
dengue or other flavivirus infections and other diseases.  Samples will not be sold or used to 
make commercial products.  Investigators within or outside of NIH who wish to study these 
samples and/or data must first obtain IRB approval.  Any clinical information about the 
sample (with or without patient identifiers) similarly requires prior IRB approval.  
 
All samples stored in the repository will be labelled with the participant’s study ID number. 
This number cannot identify study participants, but is linkable to other research databases 
(e.g., from questionnaires, clinical assessments, logbooks, etc.) generated only by the primary 
TV005 study.  The repository database will contain only the study subject ID numbers.  A 
master log linking the study subject ID numbers to the names of the study participants will be 
maintained securely with limited access to authorized research team members.  At the 
completion of the protocol (termination), samples and data will either be destroyed, or after 
IRB approval, transferred to another existing protocol or a repository.  
 
Any loss or unanticipated destruction of samples (for example, due to freezer malfunction) or 
data (for example, misplacing a printout of data with identifiers) that compromises the 
scientific integrity of the study will be reported to the IRB. 
 
Access to research samples will be limited using either a locked room or a locked freezer.  
Samples and data will be stored using codes assigned by the Investigators or their designee.  
Data will be kept in password-protected computers.  Only Investigators or their designee will 
have access to the samples and data. 
 
8.4 Retention of Records 
The PI is responsible for retaining all essential documents listed in the International 
Conference on Harmonization (ICH) Good Clinical Practice (GCP) Guideline.  Trial-related 
documents will be maintained by the Investigator in a secure storage facility for a period of 5 
years. Records will also be maintained in compliance with IRB and Bangladesh national, 
state, and federal medical records retention standards. The Sponsor is required to inform the 
Investigator when documents no longer need to be retained. All trial-related documents will 
be stored in a way that ensures that confidentiality will be strictly maintained to the extent 
provided by Bangladesh national, federal, state, and local law.  
 
It is the PI’s responsibility to retain copies of source documents until receipt of written 
notification to the contrary from the Sponsor.  All study files, source documents, and trial 
master files will be maintained and stored in a secure manner in the icddr,b in the office of 
the Clinical Research Coordinator (or equivalent). No study documents should be destroyed 
without prior written agreement between OCRPRO/DCR/NIAID, the PI, and the clinical 
research staff.  If the PI wishes to assign the study records to another party and/or move the 
records to another location, the PI or designee must provide written notification of such intent 
to OCRPRO/DCR/NIAID with the name of the person who will accept responsibility for the 
transferred records and/or their new location. NIAID must be notified in writing and written 
permission must be received by the site prior to destruction or relocation of research records. 
 

 
77 
PR-15097  
 
V2.10: 16Jun17 
 
8.5 Protocol Compliance 
The PI will conduct the trial in exact compliance with the written protocol. The Investigator 
will not implement any deviation from or change to the protocol without agreement, prior 
review, and documented approval by the Sponsor and the IRBs/ECs that granted original 
approval for the study.  The DSMBs will be notified of all protocol revisions (other than 
administrative) and will review any changes to the protocol that involve DSMB oversight or 
involve changes to the data and safety monitoring plan (DSMP) of the study.   
 
The Investigator may implement a deviation or change in the protocol in order to eliminate an 
immediate hazard to study participants without prior IRB/EC or Sponsor approval or when 
the change involves only logistical or administrative aspects of the trial such as a telephone 
number change. In the event of a medical emergency, the PI shall perform any medical 
procedures deemed medically appropriate.   
 
As soon as possible, the implemented deviation or change, the reasons for the change, and, if 
appropriate, the proposed protocol amendment(s) should be submitted to the Sponsor, 
IRBs/ECs, DSMBs, and to the regulatory authorities.  
 
8.6 Investigator’s Brochure 
Investigators will receive the current version of the Investigator’s Brochure, which 
comprehensively describes all the available preclinical experience with the experimental 
vaccine.  If relevant new information becomes available during the course of the trial, the 
Investigators will receive a revised brochure or an amendment to the current version. 
 
8.7 Study Monitoring 
Per ICH-GCP 5.18 and FDA 21 CFR 312.50 clinical protocols are required to be adequately 
monitored by the Sponsor. Monitoring will be conducted according to the “NIAID Intramural 
Clinical Monitoring Guidelines.”  Monitors under contract to the OCRPRO/DCR/NIAID will 
visit the clinical research site to monitor aspects of the study in accordance with the 
appropriate regulations and the approved protocol.  
 
A specific protocol monitoring plan will be discussed with the Principal Investigator and 
study staff prior to participant enrolment. The plan will outline the frequency of monitoring 
visits based on such factors as study enrolment, data collection status, and regulatory 
obligations.  
 
The objectives of a monitoring visit will be: 1) to verify the existence of signed informed 
consent documents and documentation of the ICF process for each monitored subject; 2) to 
verify the prompt and accurate recording of all monitored data points, and prompt reporting 
of all SAEs; 3) to compare DataFax abstracts with individual subjects’ records and source 
documents (subjects’ charts, laboratory analyses and test results, physicians’ progress notes, 
nurses’ notes, and any other relevant original subject information); and 4) to help ensure 
investigators are in compliance with the protocol. Monitors also will inspect the clinical site 
regulatory files to ensure compliance with regulatory requirements (Office for Human 
Research Protections-OHRP, FDA) and applicable guidelines (ICH-GCP).  During the 
monitoring visit, the Investigator and/or the designee and other study personnel should be 
available to discuss the study.  The investigator and/or designee will make study documents 
(e.g., consent forms, DataFax data abstracts and pertinent hospital or clinical records) readily 
available for inspection by the local IRB, FDA, the site monitors, and the NIAID staff for 
confirmation of the study data.  

 
78 
PR-15097  
 
V2.10: 16Jun17 
 
The Sponsor will retain original copies of the Form FDA 1572 and copies of other study 
documents as deemed necessary.  
 
 
 
9 Statistical Considerations 
 
9.1 General Design 
The goal of this Phase 2 vaccine trial is to determine the safety and immunogenicity of 
TV005, a live attenuated tetravalent dengue vaccine candidate in healthy human participants 
in a dengue endemic region.  The purpose of this Phase 2 trial is to evaluate the safety and 
reactogenicity, immune response to the 4 DENV serotypes, and persistence of antibody 
following subcutaneous administration of 1 dose of TV005 vaccine.  With 36 study 
participants vaccinated in each age cohort, there is 80% power to detect an AE that occurs 
with a frequency of 1 in 20 participants.  
 
9.2 Study Endpoint Analysis 
 
Primary Objectives 
1. To determine the safety of one dose of TV005, as assessed by summarizing the 
frequency of immediate, systemic, local, and laboratory AEs following vaccination.   
Adverse events will be displayed in tabular format with line listings of individual 
clinical and laboratory events classified as immediate, systemic, or local events.  
Adverse events will be summarized by severity and relationship to vaccine by 
individuals and by each group. 
 
2. To determine the immunogenicity of 1 dose of TV005, as assessed by determination 
of the serum plaque reduction neutralization titer 50% (PRNT50) toDENV-1, DENV-
2, DENV-3, and DENV-4 viruses for each participant at Study Day 0, 14, 28, 56, and 
180 post vaccination.  Monovalent, bivalent, trivalent, and tetravalent seropositivity 
and seroconversion frequencies will be determined at these time points. 
Secondary Objectives 
1. To assess the frequency, quantity, and duration of viremia of each monovalent 
component of the vaccine after vaccination.  The mean peak viremia, mean day of 
onset of viremia, and mean duration of viremia of each monovalent component within 
each dose cohort will be calculated. 
 
2. To determine the number of vaccinees infected with the vaccine viruses DENV-1, 
DENV-2, DENV-3, and DENV-4 or wild-type dengue virus.  Infection is defined as 
recovery of vaccine virus or wild-type dengue virus from the blood or serum of a 
participant and/or by seropositivity or seroconversion to DENV.   
a. Seropositivity will be defined as PRNT50 ≥ 1:10 on or before Day 56 post-
vaccination  
b. Seroconversion will be defined as ≥ 4-fold rise in DENV-1, DENV-2, 
DENV-3, or DENV-4 neutralizing antibody titers on or before Day 56 
compared with the pre-vaccination antibody titer.  
3. To assess the duration of the antibody response in recipients of the tetravalent 
vaccine.  The PRNT50 to DENV-1, DENV-2, DENV-3, and DENV-4 will be 
determined for all specimens collected at Study Day 180 post vaccination. 

 
79 
PR-15097  
 
V2.10: 16Jun17 
 
4. To determine the durability of neutralizing antibody at 1 year, 2 years, and 3 years 
after vaccination with TV005 
Exploratory Objectives 
Exploratory objectives may be performed as indicated below and in Section 10.6 (CMI 
Exploratory Objectives) in addition to additional CMI assays, as available. 
 
1. To evaluate the phenotype of PBMCs at primary infection with the TV005 vaccine 
2. To evaluate the cellular immune response to primary infection with the TV005 
vaccine 
3. To evaluate the innate immune response to primary infection with the TV005 vaccine 
4. To evaluate B and T cell memory responses following primary and secondary 
infections with the TV005 vaccine 
 
 
10 Statistical methods 
 
10.1 Estimated Sample Size 
192 study participants (144 vaccinees and 48 placebo recipients) will be enroled in the study. 
Each cohort will consist of 48 study participants (36 TV005 vaccine, 12 placebo). 
 
All cohorts will be enroled using 3:1 vaccine to placebo ratio, which was chosen for the 
following reasons:  
1. The study is designed to build upon the previous Phase 1 studies of each monovalent 
candidate and the tetravalent admixtures, which used similar cohorts and vaccine to 
placebo ratio. 
2. Placebo recipients have been included to assess whether common AEs are vaccine-
related. 
3. With 36 study participants vaccinated in each age cohort, there is 80% power to detect an 
AE that occurs with a frequency of 1 in 20 participants.  
 
10.2 Cohort Evaluations 
 
Total vaccinated cohort  
The Total Vaccinated Cohort (TVC) will include all vaccinated study participants. . Thus, the 
TVC for analysis of safety will include all study participants with vaccine administration 
documented, and the TVC for analysis of immunogenicity will include vaccinated 
participants for whom data concerning immunogenicity endpoint measures are available, i.e., 
at least one evaluable post-vaccination NAb PRNT assay result.  
 
Per protocol analysis (PP) of safety 
The PP cohort for analysis of safety will include study participants: 
1. Who have received the study vaccine/placebo according to their random assignment 
2. With sufficient data to perform an analysis of safety (safety follow-up) 
3. For whom administration site of study vaccine/placebo is known 
4. Who have not received a vaccine not specified or forbidden in the protocol 
5. For whom the randomization code has not been broken 
Per protocol cohort (PP) for analysis of immunogenicity 

 
80 
PR-15097  
 
V2.10: 16Jun17 
 
The PP cohort for analysis of immunogenicity will include all evaluable study participants 
(i.e. those meeting all eligibility criteria, complying with the procedures and study visit 
intervals defined in the protocol, and with no elimination criteria during the study) for whom 
data concerning immunogenicity endpoint measures are available. 
10.3 Safety 
The primary safety endpoint is the frequency of vaccine-related AEs, as classified by both 
severity and seriousness, through active and passive surveillance.  Separate assessments of 
systemic, local, and laboratory reactions will be performed. 
 
The primary analysis for safety will be based on the TVC. If the percentage of enroled 
participants excluded from the ATP cohort for analysis of safety is more than 5%, a second 
analysis based on this ATP cohort will be performed to complement the TVC analysis. 
 
10.3.1 Adverse Events 
The following parameters will be evaluated for each age cohort and for all cohorts combined: 
 
1. The proportion of enroled study participants with at least one AE (solicited or 
unsolicited), with at least one general AE (solicited or unsolicited) and with any AE 
during the solicited follow-up period will be estimated with exact 95% confidence 
interval (CI) after vaccination and overall. 
2. The proportion of study participants reporting each individual solicited local, general, and 
laboratory AE during the follow-up period will also be estimated with exact 95% CI.   
3. The proportion of study participants with at least one report of an unsolicited AE reported 
up to 29 days (days 0-28) after vaccination will be estimated with exact 95% CI. 
4. Similar tabulations will be done for grade 3 AEs and for AEs with relationship to 
vaccination. 
5. Differences in the frequencies of AEs between vaccines and placebos will be determined 
by Chi squared analysis and likelihood ratios.  
6. The proportion of AEs resulting in a medically-attended visit will be tabulated. 
7. Serious adverse events and participant withdrawal due to AE(s) will be described in 
detail. 
8. The percentage of study participants in each group who received a concomitant 
medication to treat study-related AEs will be tabulated with exact 95% CI.  
9. For fever, temperatures will be summarized in 0.5oC increments. 
 
10.3.2 Suspected and Confirmed Dengue  
The percentage of study participants with suspected or confirmed dengue will be tabulated 
with exact 95% CI for each age cohort throughout the entire study. Cases will be summarized 
in terms of clinical and laboratory findings. 
 
10.4 Immunogenicity 
Immunogenicity will be measured by serum PRNT50 to DENV-1, DENV-2, DENV-3, and 
DENV-4 viruses for each participant at Study Day 0, 14, 28, 56, 180, 360, 720, and 1080 post 
vaccination.  
 
Definitions: 
• Seropositivity to each serotype will be defined as a PRNT50 of ≥ 1:10 on or before 
Study Day 56 (28 for younger cohorts).  

 
81 
PR-15097  
 
V2.10: 16Jun17 
 
• Seroconversion to each serotype will be defined as a ≥ 4-fold in serum neutralizing 
antibody titer on or before Study Day 56 compared with Study Day 0.  
 
The primary analysis will be based on the PP cohort for analysis of immunogenicity. If more 
than 5% of enroled study participants are excluded from this PP cohort, second analysis 
based on the TVC will be performed to complement the PP analysis. At each blood sampling 
time point, the following parameters will be tabulated. Each of the four age cohorts will be 
analysed individually. 
 
1. The proportion of study participants who are seropositive per each serotype after 
vaccination will be estimated with 95% CI.   
2. The proportion of study participant who have seroconverted per each serotype after 
vaccination will be estimated with 95% CI.   
3. Differences in seropositivity or seroconversion between study participants with and 
without pre-existing DENVantibodies will be determined by Chi Square analysis and 
likelihood ratios.   
4. The proportion of study participants with monovalent, bivalent, trivalent, and tetravalent 
seropositivity and seroconversion will be estimated with 95% CI.   
5. An estimate of neutralizing antibody titer for study participants will be determined by 
geometric mean titer (GMT) for each serotype with 95% CI.   
6. The proportion of study participants of each seroptype who are seropositive or 
seroconvert at day 180 will be estimated with 95% CI to determine the durability of 
TV005. 
 
10.5 Viremia 
The proportion of study participants with vaccine viremia (any serotype and each serotype 
individually) will be estimated with 95% CI. The mean peak titer will be calculated for each 
individual serotype. The mean duration of viremia with range will be calculated for each 
individual serotype. Differences in mean peak titer and duration of viremia between each 
individual serotype will be measured by Mann-Whitney U analysis. 
 
The proportion of study participants with recoverable DENV-1, DENV-2, DENV-3, and 
DENV-4 vaccine virus components or wild-type dengue virus during the first 14 days 
following vaccination will be estimated with 95% CI.  This is defined by the recovery of 
vaccine virus from the blood or serum of a study participant and/ or by new seropositivity 
(PRNT50 ≥ 1:10) in a previously seronegative study participant or seroconversion (> 4-fold 
rise in neutralizing antibody titers against DENV-1, DENV-2, DENV-3, or DENV-4) in 
study participants who were seropositive at baseline. 
   
Differences in the recovery of vaccine virus from blood or serum post vaccination between 
study participants with and without pre-existing DENV antibodies will be determined by Chi 
Square analysis and likelihood ratio.   
 
10.6 CMI Exploratory Objectives 
At study time points Day 0, Day 7, Day 28, Day 56, Day 180, Day 360, Day 720, and Day 
1080 the individual immune response to vaccination will be evaluated.  Descriptive statistics 
including mean, standard deviation, range, median 1st, and 3rd quartiles may be tabulated. In 
addition to descriptive tables, box and whisker plots of the frequencies may be displayed if 
the data is available. 

 
82 
PR-15097  
 
V2.10: 16Jun17 
 
Because the CMI endpoints are exploratory, all collected specimens may not be analyzed at 
each time point. If additional CMI assay techniques become available during the course of 
the study, additional analyses may be performed.  
 
 
11 Monitoring Plan  
 
11.1 DSMBs: NIAID Intramural and ERC Data and Safety Monitoring Boards 
 
Both the Sponsor (NIAID) and the local ethical committee (ERC) require the formation of a 
DSMB to review study participant safety.  We have outlined the process by which each 
Board will oversee safety and work together as needed.   
 
The ERC DSMB reflects the disciplines and medical specialties necessary to interpret the 
data from the clinical trial and may include expertise in clinical aspects of disease/patient 
population being studied, biostatistics, and clinical trials conduct and methodology.  The 
ERC selects the DSMB chair and determines the terms of membership.  The ERC DSMB is 
constituted to review and evaluate the accumulated study data for participant safety, study 
conduct, and progress, and when appropriate, efficacy and makes recommendations to the 
ERC concerning the continuation, modification, or termination of the trial.  The ERC DSMB 
will meet once before the study opens to make recommendations as to whether the study can 
proceed.  The ERC DSMB will also meet after all safety data has been collected up to Day 28 
for each cohort.  The ERC DSMB will conclude each review with their recommendations to 
the ERC chair and the Investigator as to whether the study should continue without change, 
be modified, or terminated.   
 
The NIAID Intramural DSMB includes independent experts that do not have direct 
involvement in the conduct of the study and have no significant conflicts of interests as 
defined by NIAID policy. The Board will review the study prior to initiation and once for 
each age cohort after safety data is obtained for each cohort’s initial 28 day participation 
in the trial.  The Board may convene additional reviews as necessary. The Board will 
review the study data to evaluate the safety, efficacy, study progress, and conduct of the 
study. All SAEs, all UPs, and all IND Safety Reports will be reported by the PI to the 
DSMB at the same time they are submitted to the IRB or IND Sponsor. The PI will notify 
the DSMB of any cases of intentional or unintentional unblinding as soon as possible. The 
PI will notify the Board at the time pausing or halting criteria are met and obtain a 
recommendation concerning continuation, modification, or termination of the study. The 
PI will submit the written DSMB summary reports with recommendations to the IRB(s). 
 
For each DSMB, designated unblinded icddr,b  staff will provide the Executive Secretary 
with blinding codes in a sealed envelope, to be opened in the event that the DSMB requires 
the information in order to make its recommendations.  Prior to each DSMB review, the PI 
will submit the Sponsor’s Medical Monitor’s written recommendations and cumulative safety 
data in a format acceptable, by unblinded cohort if requested.  The Boards will review the 
study data to evaluate the safety, efficacy, study progress, and conduct of the study.   
 
Reports of SAEs will be submitted by the PI to the Boards at the same time they are reported 
to the Sponsor and IRB.  All Unanticipated Problems will be submitted to the DSMBs at the 
same time they are submitted to the IRB.  Safety Reports will be submitted to the DSMBs by 
the Investigator after their receipt. The PI will notify the DSMBs of any cases of intentional 

 
83 
PR-15097  
 
V2.10: 16Jun17 
 
or unintentional unblinding as soon as possible. The PI will notify the Boards at the time 
stopping or pausing criteria are met and will obtain a recommendation concerning 
continuation, modification, or termination of the study. The PI will submit the written 
DSMBs’ recommendations to the IRB of record.  DSMBs’ Policy Document, which serves as 
the charter document, will be provided to the University of Vermont IRB at the time of 
submission of the full protocol packet for review. 
 
The NIAID and ERC DSMB will review safety data simultaneously through enrolment of the 
youngest cohort (age de-escalation phase of the study).  Both DSMBs will share open 
summaries from their respective meetings.  In addition, up to two members from the ERC 
DSMB may be invited to participate as an ad hoc voting member in the NIAID DSMB.  If the 
local DSMB members are not able to participate, the NIAID DSMB will continue without a 
local in-country representative and will review the ERC DSMB open summary reports. In the 
event of differing recommendations between the two DSMBs, the two Boards and/or two 
authorized individuals will collectively determine an agreeable resolution.   
 
11.2 Sponsor Medical Monitor 
Roles and Responsibilities of the Sponsor Medical Monitor 
A Medical Monitor (MM) representing the IND Sponsor (OCRPRO), has been appointed for 
oversight of safety in this clinical study. The Sponsor MM will be responsible for performing 
periodic reviews and analyses of aggregate safety data to assure the safety of the participants 
and compliance with all safety requirements. In this capacity, the MM will: 1) review all AEs 
and UPs on a regular basis throughout the study, 2) review all SAEs, 3) be available to advise 
the investigators on study-related medical questions or problems, and 4) evaluate cumulative 
subject safety data and make recommendations regarding the safe continuation of the study. 
A Safety Monitoring Plan that has been written for this protocol provides more specific 
information about the frequency of the data reviews, content to be reviewed, and the format 
of the meetings between the PI and the Sponsor MM to discuss the results of the analysis of 
the safety data. 
11.3 Safety Review and Communications Plan  
A Safety Review and Communication Plan (SRCP) has been developed for the protocol. The 
SRCP is an internal communications document between the PI and the CSO, which 
delineates the safety oversight responsibilities of the principal investigator, the CSO, and 
other stakeholders. The SRCP also includes the overall plan for conducting periodic safety 
surveillance assessments.  
 
 
12 Protection of Human Subjects 
 
12.1 Institutional Review Boards 
The PI will be responsible for obtaining IRB approvals.  Before the start of the study, the 
appropriate documents (including, but not limited to, the Protocol, Investigator’s Brochure, 
Informed Consent Form, information sheets, and advertisements) will be submitted to the 
IRBs/ECs, DSMBs, and the Sponsor for approval.  A copy of the study approvals (including 
approval of the informed consent form) is to be maintained in the Investigator study 
document binder, and a copy will be supplied to the Sponsor.  During the study, the 
Investigator is responsible for providing the IRBs/ECs, and the Sponsor with all documents 
subject to review (e.g., Protocol Amendments, informed consent form updates, 

 
84 
PR-15097  
 
V2.10: 16Jun17 
 
advertisements, and any written information that may be provided to the participant).  Annual 
reports on the progress of the study will be made to the IRBs/ECs by the Investigator in 
accordance with IRB guidelines and government regulations. 
 
Administrative changes generally do not require IRB approval but the University of Vermont 
IRB and local ERC should be notified. The Sponsor must be immediately notified of all 
administrative changes, including minor and administrative modifications, prior to any action 
being taken. Any non-administrative changes which may impact study participants’ safety, 
the trial design, or achievement of study objectives will require approval from the Sponsor 
and the IRBs/ECs. The only exception is a protocol modification that is required immediately 
to eliminate apparent immediate hazards to a participant. Such a modification may be 
implemented by the PI and reported to the UVM IRB and local ERC within 48 hours. 
 
The ICF and/or assent forms must be revised to reflect any significant amendment which 
directly affects volunteers and must also be reviewed and IRB approved with the amendment. 
Study participants will be consented using the most recently-approved ICF and assent. 
Participants already enroled in the study will be informed about revisions to the protocol and, 
depending on the impact of the amendment, may be asked to re-consent. Re-consenting may 
be accomplished by repeating the informed consent process using the revised ICF, with 
attention given to the changes, or it may be done using an addendum to the ICF, clearly 
stating any revisions or new information and summarizing the potential effect upon 
participation in the study. The new ICF document must be signed and placed into the study 
record. A signed copy is given to the volunteer. 
 
The PI will be responsible for submitting the Continuing Review Report (CRR) to the icddr,b 
EC with the assistance of the UVM Study Coordinator. A close-out report should be 
submitted to the UVM IRB and the icddr,b EC at the completion of the study. 
 
12.2 Informed Consent 
In obtaining and documenting informed consent, the Investigator and study staff must comply 
with the applicable regulatory requirements, GCP guidelines, and ethical principles.  The 
written informed consent form and the assent form (for participants 11-17 years of age) must 
be approved by the IRBs/ECs prior to its use.  The ICF or AF describe the study objectives, 
risks and potential benefits to participation, and study participant and parent requirements for 
participation. The ICF will be signed by adult study participants. The AF will be signed by at 
least 1 parent or acceptable legal representative. Children between the ages of 11 and 17 must 
mark the AF. Both an ICF signed by the parent/legal guardian and the AF signed by the 
minor must be signed for a study participant less than 18 years of age to participate in the 
study. If the parent or legal guardian gives consent for a minor, but the minor does not 
provide assent, the participant will not be enroled in the study.   
 
Adult participants or the parent or legal guardian of a minor participant may withdraw 
consent at any time during the course of the trial. A minor study participant may withdraw 
assent at any time.  A copy of the informed consent and assent documents will be offered to 
the study participant or to the participant’s parents or legal guardian. The rights and welfare 
of the study participants will be protected by emphasizing that the quality of the medical care 
provided will not be adversely affected if an individual declines participation in the study.  
 
If a child reaches the age of 18 while on study, informed consent will be obtained at that time.  
Similarly, if a child reaches the age of 11 while on study, assent will be obtained at that time. 

 
85 
PR-15097  
 
V2.10: 16Jun17 
 
Illiterate persons will be asked to mark their thumbprint in lieu of a signature and an 
impartial witness will also sign.  
 
If a study participant is injured in the process of a study-specific procedure or activity, 
reasonable and necessary medical care to treat the injury will be provided to the participant at 
no charge. 
 
12.3 Risks 
Risks to the study participants are mainly associated with venipuncture and immunization and 
are outlined below.  Female study participants will be cautioned of the unknown risk of study 
vaccines to the fetus and will be required to use effective birth control methods through study 
day 28.  Study participants are counselled on pregnancy prevention methods and risks to 
pregnancy through day 28 of the study. 
 
12.3.1 Venipuncture 
The total amount of blood to be drawn throughout the 1080-day duration of the study is 
approximately 257 mL for cohort 01 (18 – 50), 121 mL for cohort 02 (11 - < 18 years of age), 
and 33 mL for cohorts 03 and 04 (5 - < 11, and 1 - < 5 years of age respectively). These 
volumes are well within the Office for Human Research Protections (OHRP) and NIH 
guidelines (Clinical Center Medical Administrative Policy M 95-9) for blood volumes in 
clinical research and should not compromise the health of study participants. The total 
volume of blood to be drawn from paediatric study participants  (age <11 years) will be 
3mL/kg or less at any one time, and no more than 46 mL of blood will be drawn over any 
single 8-week period in the paediatric cohorts. 
 
Risks occasionally associated with venipuncture include bleeding, pain, bruising, or 
hematoma at the site of venipuncture; light headedness; syncope (rarely); and infection 
(rarely). 
 
12.3.2 Immunization 
Possible local vaccine reactions include pain, swelling, or erythema usually lasting no more 
than 2 to 3 days, lymphadenopathy, or pruritus at the injection site.  Systemic reactions such 
as macular papular rash and transient benign neutropenia have been observed in some study 
participants infected with other recombinant dengue vaccine candidates.  Other potential 
systemic reactions that may occur include symptoms of dengue such as fever, headache, eye 
pain, photophobia, generalized myalgias, arthralgias, elevated ALT, neutropenia, elevated 
PTT, or decreased platelet count.  Immediate hypersensitivity reactions including urticaria, 
anaphylaxis, or other IgE-mediated responses are possible, as with any vaccine.  Study 
participants who receive the TV005 vaccine may theoretically be at increased risk for more 
severe disease (DHF/DSS) if they respond poorly to 1 or more of the components of the 
vaccine and become infected with a wild type DENV in the future. 
 
Any viral vaccine carries a small risk of Guillian-Barré syndrome (GBS).  GBS is a reactive, 
self-limited, autoimmune disease triggered by a preceding bacterial or viral infection.  The 
clinical manifestations begin gradually, are symmetric, and consist of mild sensory deficits, 
cranial nerve involvement, and motor weakness that may lead to paralysis.  The paralysis will 
typically spontaneously resolve.  This has not been reported following naturally acquired 
dengue infection or after any investigational dengue vaccination, but remains a small but 
potential risk. 
 

 
86 
PR-15097  
 
V2.10: 16Jun17 
 
There is also a theoretical risk associated with breach of confidentiality by participating in 
this study, although every measure will be taken to assure the confidentiality of participants 
in this study. 
 
As with any investigational vaccine, there may be risks that are currently unknown.  Study 
participants will be informed of any such risks should further information become available. 
 
Receiving information concerning positive hepatitis status may cause significant 
emotional distress and relationship conflicts.  Any such study participant shall be 
referred to an appropriate health care provider for definitive diagnosis and subsequent 
counseling and treatment. These volunteers will not eligible for participation in the 
study. 
12.4 Benefits 
 
Benefits from participation in this study will include enhanced access to medical care during 
the screening visit and all subsequent visits. Study participants will receive a full physical 
exam which may detect medical problems which were previously unknown to the 
participants, and this may allow earlier diagnosis and treatment of any significant previously 
unidentified illnesses or health care problems.   
 
Adult and adolescent study participants will receive laboratory screening for hepatitis C 
infection, and all participants will receive laboratory screening for hepatitis B infection, 
which could benefit the participant if a previously undiagnosed disease is identified.  These 
individuals will be referred to a medical provider for counselling and treatment.  If a study 
participant has a febrile illness during the course of the study, participation may lead to 
earlier diagnosis and treatment of dengue fever.  Enroled study participants will also receive 
primary care up to Study Day 180. 
 
 
 
12.5 Compensation 
Enroled study participants will be compensated with food at all scheduled clinic visits and 
with 500 Tk (at current exchange rates, approximately $7.00 US dollars) per study visit 
including the screening visit.  The compensation is intended to help offset the cost of travel to 
the clinic site for study visits and to compensate for loss of income during visits that occur 
during work hours.  Enroled study participants will also receive primary care up to Study Day 
180. 
 
12.6 Confidentiality 
All study-related information will be stored securely at the study site.  All participant 
information will be stored in locked file cabinets in areas with access limited to study staff.  
All laboratory specimens, reports, study data collection, and process and administrative forms 
will be identified by coded number only to maintain participant confidentiality.  Forms, lists, 
logbooks, appointments books, and any other listings that link subject study ID numbers to 
other identifying information will be stored in a separate locked file in an area with limited 
access.  A participant’s study information will not be released without the written permission 
of the participant, except as necessary for monitoring by the Sponsor and/or its contractors 
and the FDA. 
 

 
87 
PR-15097  
 
V2.10: 16Jun17 
 
Subject identification codes will consist of the study code, TV005, followed by a number 
designating the age cohort (01 for the adult study participants, 02 for the cohort age 11-<18, 
03 for the cohort age 5-<11, and 04 for the cohort age 1-<5), and then by a unique 3 digit 
identifier, XXX. Three digit subject identifier codes will be assigned in order of enrolment; 
for example, subject TV005-01-001 will represent the first adult participant enroled into the 
study to receive an injection. 
 
Data and study files may be accessed by authorized Investigators, UVM and icddr,b study 
staff, the Sponsor’s Clinical Research Associate (CRA) and/ IRB members, the Sponsor 
(OCRPRO/DCR/NIAID/NIH), the icddr,b ERC personnel, and other regulatory personnel 
and authorized study personnel as documented on the Study Task Delegation and Signature 
List, a regulatory document which will be kept on file in the icddr,b Clinical Research 
Coordinator Section and updated as needed.   
 
12.7 Biohazard Containment 
As the transmission of HIV and other 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 during the drawing of blood and during the shipping and 
handling of all specimens for this study, as currently recommended by regional Occupational 
Health guidelines and the U.S. Centers for Disease Control and Prevention.  
 
All infectious specimens will be transported using packaging mandated in the Code of 
Federal Regulations, 42 CFR Part 72.  Please also refer to individual carrier guidelines (e.g., 
Federal Express, World Courier) for specific instructions. 
 
Use of Animals 
 
No animals will be used in the execution of this protocol.  
 
 
13 Study Specific Policies and Procedures 
 
13.1 Collaborations 
This study represents collaboration between the OCRPRO/DCR/NIAID/NIH, Johns Hopkins 
University School of Public Health (JHSPH), University of Vermont Vaccine Testing Center, 
and International Center for Diarrhoeal Disease Research, Bangladesh (iccdr,b) with roles 
and responsibilities of each party detailed in the contract agreement.  Each organization has 
specific roles and responsibilities which overlap in certain areas.  In summary, icddr,b field 
clinic, research and clinical laboratories, clinical trial personnel are responsible for preparing 
for and executing clinical operations;  the OCRPRO/DCR/NIAID/NIH through the 
Regulatory Compliance and Human Subject Protection Program (RCHSPP), Leidos 
Biomedical Research, Inc., will provide Sponsor support, to include financial, logistic, 
regulatory, monitoring, and quality assurance sustainment in accordance with regulatory 
authority guidelines. Financial support and funding distribution and management for the 
study has been executed in conjunction with JHU.   
 
13.2 Study Sponsor and Funding 
The study is sponsored and funded by OCRPRO/DCR/NIAID/NIH through a contract 
agreement. 
 

 
88 
PR-15097  
 
V2.10: 16Jun17 
 
Office of Clinical Research Policy and Regulatory Operations (OCRPRO) 
Division of Clinical Research (DCR) 
National Institute of Allergy and Infectious Diseases (NIAID) 
National Institutes of Health (NIH) 
OCRPRO/DCR/NIAID/NIH 
5601 Fishers Lane 
Room 4B11, MSC 9820 
Bethesda, MD 20892 
Phone: 301-451-5136 
Fax: 301-480-1765 
 
Fund transfer between the Sponsor and the icddr,b clinical site will be managed by a contract 
with Johns Hopkins University School of Public Health. 
 
 
 

 
89 
PR-15097  
 
V2.10: 16Jun17 
 
 
14 Facilities 
The icddr,b has a large multi-disciplinary international and national scientific research staff. 
Existing field, hospital, laboratory, and office facilities will be used for this study. Scientists 
at icddr,b have conducted a variety of observational field studies and clinical trials in the 
Mirpur community where the field site is located.  
 
Existing laboratory facilities of the Immunology and Parasitology Laboratories, Centre for 
Vaccine Sciences, icddr,b will be used for all clinical aspects of this study, including 
recruitment, screening visits, administration of the vaccine, follow-up clinic visits, and febrile 
illness surveillance. 
 
Vaccine storage, accountability, and preparation will be conducted at icddr,b research 
laboratories with training and assistance from the University of Vermont Vaccine Testing 
Center.   
 
PRNT assays and viremia assays will be conducted at the icddr,b research laboratories with 
assistance and training from the University of Vermont Vaccine Testing Center.   
Exploratory immunology assays will be performed at the University of Vermont Vaccine 
Testing Center.  
 
Literature Cited 
 
1. Beatty ME, Stone A, Fitzsimons DW, et al. Best practices in dengue surveillance: a report 
from the Asia-Pacific and Americas Dengue Prevention Boards. PLoS neglected tropical 
diseases 2010; 4:e890. 
 
2. Rahman M, Rahman K, Siddique AK, Shoma S, Kamal AHM, Ali KS, Nisaluk A, & 
Breiman RF. First outbreak of dengue haemorrhagic fever, Bangladesh. Emerging Infectious 
Diseases 2002; 8:738-740. 
 
3.  Monath T, Heinz F (1996) Flaviviruses. In: Fields BN, Knipe DM, Howley PM, Chanock 
RM, Melnick JL et al., editors. Fields Virology. third ed. Philadelphia: Lipincott-Raven. pp. 
961-1034. 
4.  WHO (1997) Dengue haemorrhagic fever:  diagnosis, treatment, prevention and control. 
Geneva: WHO. 
5.  Podder G, Breiman RF, Azim T, Myatthu H, Velathanthiri N, Mai LQ, Lowry K, & 
Aaskov JG. Short report: Origin of dengue type 3 viruses associated with the dengue 
outbreak in Dhaka Bangladesh, in 2000 and 2001. Am J Trop Med Hyg 2006; 74(2): 263.-
265. 
 
6.  Sabin A (1955) Recent advances in our knowledge of Dengue and Sandfly Fever. Amer J 
Trop Med Hyg 4: 198-207. 
7.  Burke DS, Nisalak A, Johnson DE, Scott RM (1988) A prospective study of dengue 
infections in Bangkok. Am J Trop Med Hyg 38: 172-180. 

 
90 
PR-15097  
 
V2.10: 16Jun17 
 
8.  Sohn YM, Tandan JB, Yoksan S, Ji M, Ohrr H (2008) A 5-year follow-up of antibody 
response in children vaccinated with single dose of live attenuated SA14-14-2 Japanese 
encephalitis vaccine: immunogenicity and anamnestic responses. Vaccine 26: 1638-1643. 
9.  Wu SJ, Grouard-Vogel G, Sun W, Mascola JR, Brachtel E, et al. (2000) Human skin 
Langerhans cells are targets of dengue virus infection [see comments]. Nat Med 6: 816-820. 
10.  Couvelard A, Marianneau P, Bedel C, Drouet MT, Vachon F, et al. (1999) Report of a 
fatal case of dengue infection with hepatitis: demonstration of dengue antigens in hepatocytes 
and liver apoptosis. Hum Pathol 30: 1106-1110. 
11.  Kuo CH, Tai DI, Chang Chien CS, Lan CK, Chiou SS, et al. (1992) Liver biochemical 
tests and dengue fever. Am J Trop Med Hyg 47: 265-270. 
12.  Marianneau P, Steffan AM, Royer C, Drouet MT, Jaeck D, et al. (1999) Infection of 
primary cultures of human Kupffer cells by Dengue virus: no viral progeny synthesis, but 
cytokine production is evident. J Virol 73: 5201-5206. 
13.  Whitehead SS, Hanley KA, Blaney JE, Jr., Gilmore LE, Elkins WR, et al. (2003) 
Substitution of the structural genes of dengue virus type 4 with those of type 2 results in 
chimeric vaccine candidates which are attenuated for mosquitoes, mice, and rhesus monkeys. 
Vaccine 21: 4307-4316. 
14.  Sabin AB, Schlesinger RW (1945) Production of immunity to dengue with virus 
modified by propagation in mice. Science 101: 640-642. 
15.  Schlesinger RW, Gordon I, Frankel JW, Winter JW, Patterson PR, et al. (1956) Clinical 
and serologic response of man to immunization with attnuated dengue and yellow fever 
viruses. J Immunol 77: 352-364. 
16.  Wisseman CL, Jr., Sweet BH, Rosenzweig EC, Eylar OR (1963) Attenuated Living Type 
1 Dengue Vaccines. Am J Trop Med Hyg 12: 620-623. 
17.  Bellanti JA, Bourke RTA, Rosenzweig EC, Eylar OR (1966) Report of dengue vaccine 
trial in the Caribbean, 1963:  a collaborative study. Bull World Health Organ 35: 93. 
18.  Edelman R, Tacket CO, Wasserman SS, Vaughn DW, Eckels KH, et al. (1994) A live 
attenuated dengue-1 vaccine candidate (45AZ5) passaged in primary dog kidney cell culture 
is attenuated and immunogenic for humans. J Infect Dis 170: 1448-1455. 
19.  Edelman R, Wasserman SS, Bodison SA, Putnak RJ, Eckels KH, et al. (2003) Phase I 
trial of 16 formulations of a tetravalent live-attenuated dengue vaccine. Am J Trop Med Hyg 
69: 48-60. 
20.  Kanesa-thasan N, Sun W, Kim-Ahn G, Van Albert S, Putnak JR, et al. (2001) Safety and 
immunogenicity of attenuated dengue virus vaccines (Aventis Pasteur) in human volunteers. 
Vaccine 19: 3179-3188. 
21.  Sun W, Edelman R, Kanesa-Thasan N, Eckels KH, Putnak JR, et al. (2003) Vaccination 
of human volunteers with monovalent and tetravalent live-attenuated dengue vaccine 
candidates. Am J Trop Med Hyg 69: 24-31. 

 
91 
PR-15097  
 
V2.10: 16Jun17 
 
22.  Bhamarapravati N, Sutee Y (2000) Live attenuated tetravalent dengue vaccine. Vaccine 
18: 44-47. 
23.  Sabchareon A, Lang J, Chanthavanich P, Yoksan S, Forrat R, et al. (2002) Safety and 
immunogenicity of tetravalent live-attenuated dengue vaccines in Thai adult volunteers: role 
of serotype concentration, ratio, and multiple doses. Am J Trop Med Hyg 66: 264-272. 
24.  Durbin AP, Kirkpatrick BD, Pierce KK, Elwood D, Larsson CJ, et al. (2013) A Single 
Dose of Any of Four Different Live Attenuated Tetravalent Dengue Vaccines Is Safe and 
Immunogenic in Flavivirus-naive Adults: A Randomized, Double-blind Clinical Trial. J 
Infect Dis 207: 957-965. 
25.  Sabchareon A, Wallace D, Sirivichayakul C, Limkittikul K, Chanthavanich P, et al. 
(2012) Protective efficacy of the recombinant, live-attenuated, CYD tetravalent dengue 
vaccine in Thai schoolchildren: a randomised, controlled phase 2b trial. Lancet 380: 1559-
1567. 
26.  Blaney JE, Jr., Sathe NS, Goddard L, Hanson CT, Romero TA, et al. (2008) Dengue 
virus type 3 vaccine candidates generated by introduction of deletions in the 3' untranslated 
region (3'-UTR) or by exchange of the DENV-3 3'-UTR with that of DENV-4. Vaccine 26: 
817-828. 
27.  Durbin AP, Karron RA, Sun W, Vaughn DW, Reynolds MJ, et al. (2001) Attenuation 
and immunogenicity in humans of a live dengue virus type-4 vaccine candidate with a 30 
nucleotide deletion in its 3'-untranslated region. Am J Trop Med Hyg 65: 405-413. 
28.  Whitehead SS, Falgout B, Hanley KA, Blaney Jr JE, Jr., Markoff L, et al. (2003) A Live, 
Attenuated Dengue Virus Type 1 Vaccine Candidate with a 30-Nucleotide Deletion in the 3' 
Untranslated Region Is Highly Attenuated and Immunogenic in Monkeys. J Virol 77: 1653-
1657. 
29.  Hanley KA, Manlucu LR, Manipon GG, Hanson CT, Whitehead SS, et al. (2004) 
Introduction of mutations into the non-structural genes or 3' untranslated region of an 
attenuated dengue virus type 4 vaccine candidate further decreases replication in rhesus 
monkeys while retaining protective immunity. Vaccine 22: 3440-3448. 
30.  Blaney JE, Jr., Matro JM, Murphy BR, Whitehead SS (2005) Recombinant, Live-
Attenuated Tetravalent Dengue Virus Vaccine Formulations Induce a Balanced, Broad, and 
Protective Neutralizing Antibody Response against Each of the Four Serotypes in Rhesus 
Monkeys. J Virol 79: 5516-5528. 
31.  Vaughn DW. Antibody responses, viral serotypes and the viral burden in dengue 
fever/dengue haemorrhagic fever; 2000 20-24 November; Chiang Mai, Thailand. 
32.  Murgue B, Roche C, Chungue E, Deparis X (2000) Prospective study of the duration and 
magnitude of viraemia in children hospitalised during the 1996-1997 dengue-2 outbreak in 
French Polynesia. J Med Virol 60: 432-438. 
33.  Kalayanarooj S, Vaughn DW, Nimmannitya S, Green S, Suntayakorn S, et al. (1997) Early 
clinical and laboratory indicators of acute dengue illness. J Infect Dis 176: 313-321. 

 
92 
PR-15097  
 
V2.10: 16Jun17 
 
34.  Blaney JE, Jr., Durbin AP, Murphy BR, Whitehead SS (2006) Development of a live 
attenuated dengue virus vaccine using reverse genetics. Viral Immunol 19: 10-32. 
35.  Durbin AP, Whitehead SS, Shaffer D, Elwood D, Wanionek K, et al. (2011) A single 
dose of the DENV-1 candidate vaccine rDEN1Δ30 is strongly immunogenic and induces 
resistance to a second dose in a randomized trial. PLoS neglected tropical diseases 5: e1267. 
36.  Durbin AP, McArthur JH, Marron JA, Blaney JE, Thumar B, et al. (2006) 
rDEN2/4Delta30(ME), A Live Attenuated Chimeric Dengue Serotype 2 Vaccine Is Safe and 
Highly Immunogenic in Healthy Dengue-Naive Adults. Hum Vaccin 2: 255-260. 
37.  Durbin AP, Kirkpatrick BD, Pierce KK, Schmidt AC, Whitehead SS (2011) 
Development and clinical evaluation of multiple investigational monovalent DENV vaccines 
to identify components for inclusion in a live attenuated tetravalent DENV vaccine. Vaccine 
29: 7242-7250. 
38.  Durbin AP, Whitehead SS, McArthur J, Perreault JR, Blaney JE, Jr., et al. (2005) rDEN4 
Delta 30, a Live Attenuated Dengue Virus Type 4 Vaccine Candidate, Is Safe, Immunogenic, 
and Highly Infectious in Healthy Adult Volunteers. J Infect Dis 191: 710-718. 
39.  Kirkpatrick BD, Durbin AP, Pierce KK, Carmolli MP, Whitehead SS et al. (2015) 
Robust and balanced responses to all 4 dengue virus serotypes following administration of a 
single dose of a live attenuated tetravalent dengue vaccine to healthy, flavivirus-naïve adults. 
Journal of Infectious Diseases 212: 702-710. 
 
40.  Durbin AP, McArthur J, Marron JA, Blaney JE, Jr., Thumar B, et al. (2006) The live 
attenuated dengue serotype 1 vaccine rDEN1Delta30 is safe and highly immunogenic in 
healthy adult volunteers. Hum Vaccin 2: 167-173. 
41.  Troyer JM, Hanley KA, Whitehead SS, Strickman D, Karron RA, et al. (2001) A live 
attenuated recombinant dengue-4 virus vaccine candidate with restricted capacity for 
dissemination in mosquitoes and lack of transmission from vaccinees to mosquitoes. Am J 
Trop Med Hyg 65: 414-419. 
42.  Blaney JE, Jr., Hanson CT, Firestone CY, Hanley KA, Murphy BR, et al. (2004) 
Genetically modified, live attenuated dengue virus type 3 vaccine candidates. Am J Trop 
Med Hyg 71: 811-821 
 
43.  ICDDR,B Clinical Laboratory Services, (March 16, 2016) Unit and Reference Values.