A Phase 1, Randomized, Open-label, Comparison of Heterologous Prime-Boost Vaccination Schedules of Tetravalent Dengue Virus Purified Inactivated Vaccine (PIV) and Tetravalent Dengue Virus Live Attenuated Vaccine (LAV) in Healthy Adults
Phase: PHASE1
Conditions: Dengue Virus
Sponsor: U.S. Army Medical Research and Development Command

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
1 
US Government Proprietary 
 
 
 
 
 
 
A PHASE 1, RANDOMIZED, OPEN-LABEL, SINGLE- 
CENTER, COMPARISON OF HETEROLOGOUS PRIME- 
BOOST VACCINATION SCHEDULES OF 
TETRAVALENT DENGUE VIRUS PURIFIED 
INACTIVATED VACCINE (PIV) AND TETRAVALENT 
DENGUE VIRUS LIVE ATTENUATED VACCINE (LAV) 
IN HEALTHY ADULTS IN A NONENDEMIC REGION 
 
 
 
 
 
 
NCT03141138 
 
 
IND 16122; S-16-23 Study 
 
 
Protocol version 4.0 dated 06NOV2019 
 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
2 
US Government Proprietary 
 
 
 
 
 
 
A PHASE 1, RANDOMIZED, OPEN-LABEL, SINGLE- 
CENTER, COMPARISON OF HETEROLOGOUS PRIME- 
BOOST VACCINATION SCHEDULES OF 
TETRAVALENT DENGUE VIRUS PURIFIED 
INACTIVATED VACCINE (PIV) AND TETRAVALENT 
DENGUE VIRUS LIVE ATTENUATED VACCINE (LAV) 
IN HEALTHY ADULTS IN A NONENDEMIC REGION 
 
 
 
 
 
Sponsor 
The Surgeon General, Department of the Army 
Sponsor’s Representative 
Mark S. Paxton JD, MS 
US Army Medical Research and Development 
Command (USAMRDC) 
1430 Veterans Drive, Ft. Detrick, MD 21702-5009 
Telephone: 301-619-0317; DSN: 343-0317 
Fax: 301-619-0197 
Email: usarmy.detrick.medcom- 
usamrmc.mbx.regulatory-affairs@mail.mil 
Research Monitor 
Primary: LCDR Tida Lee MD 
Naval Medical Research Center(NMRC) 
503 Robert Grant Avenue 
Silver Spring, MD 20910-7500 
Telephone: 301-319-9260 
Email: tida.k.lee.mil@mail.mil 
Alternate: CDR Ramiro Gutierrez MD 
Naval Medical Research Center(NMRC) 
503 Robert Grant Avenue 
Silver Spring, MD 20910-7500 
Telephone: 301-319-3193 
Email: Ramiro.l.gutierrez.mil@mail.mil 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
3 
US Government Proprietary 
 
 
 
Principal Investigator 
Simon Pollett, MBBS 
WRAIR 
503 Robert Grant Avenue 
Silver Spring, MD 20910-7500 
Telephone: 301-319-2059 
Email: simon.d.pollett.ctr@mail.mil 
Subinvestigators 
LTC James E. Moon, MD 
WRAIR 
503 Robert Grant Avenue 
Silver Spring, MD 20910-7500 
Telephone: 301-319-9176 
Email: james.e.moon.mil@mail.mil 
Christine Lee, M.D. 
Clinical Trials Center 
Walter Reed Army Institute of Research 
503 Robert Grant Ave. 
Silver Spring, MD 20910 
Phone: 301-319-9660 
Email: christine.e.lee12.ctr@mail.mil 
Melinda Hamer, M.D. 
Chief, Army Clinical Trials Center 
Department of Translational Medicine 
Walter Reed Army Institute of Research, 
503 Robert Grant Ave. 
Silver Spring, MD 20910 
Phone: 301-319-3136 
Email: Melinda.j.hamer.mil@mail.mil 
Jack Hutter, M.D. 
Infectious Disease Department 
Walter Reed National Military Medical Center 
8901 Rockville Pike 
Bethesda, MD 20889 
Phone: 504-234-0735 
Email: jack.n.hutter.mil@mail.mil 
Justin Curley, M.D. 
Research Physician 
Walter Reed Army Institute of Research 
503 Robert Grant Ave. 
Silver Spring, MD 20910 
Phone: 301-319-9687 
Email: Justin.m.curley.mil@mail.mil 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
4 
US Government Proprietary 
 
 
 
Clinical Trial Site 
Walter Reed Army Institute of Research (WRAIR) 
Clinical Trials Center (CTC) 
503 Robert Grant Avenue 
Silver Spring, MD 20910-7500 
Telephone: 301-319-9335 
Fax: 301-319-9585 
Email: usarmy.detrick.medcom-wrair.mbx.clinical- 
trials@mail.mil 
Qualified Physician Responsible for All 
Trial-Site-Related Medical Decisions 
Simon Pollett, MBBS 
Clinical Investigator 
WRAIR Clinical Trials Center 
Telephone: 301-319-2059 
Email: simon.d.pollett.ctr@mail.mil 
Clinical Laboratories and Other Departments/Institutions Involved in the Trial 
Site Investigational Product 
Accountability 
Sandra Gibson 
Quality Assurance Manager 
WRAIR Pilot Bioproduction Facility 
Telephone: 301-319-7417 
Fax: 301-319-9360 
Email: sandra.j.gibson11.ctr@mail.mil 
Clinical Laboratory 
Quest Diagnostics Incorporated 
1901 Sulphur Spring Road 
Halethorpe, MD 21227 
Telephone: 410-247-9100 
Research Laboratories 
WRAIR Viral Diseases Branch 
503 Robert Grant Avenue 
Silver Spring, MD 20190 
POC: LTC Richard Jarman 
Telephone: 301-319-9223 
Email: richard.g.jarman.mil@mail.mil 
Statistician 
Devin Hunt 
Walter Reed Army Institute of Research (WRAIR) 
Clinical Trials Center (CTC) 
503 Robert Grant Avenue 
Silver Spring, MD 20910-7500 
Telephone: 301-319-9571 
Email: devin.j.hunt.ctr@mail.mil 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
5 
US Government Proprietary 
 
 
 
Data Management 
USAMRDC Data Management 
United States Army Medical Research and Development 
Command (USAMRDC) 
1430 Veterans Drive 
Fort Detrick, MD 21702-5009 
Telephone: 301-619-1106 
Fax: 301-619-7790 
Institutional Review Board 
WRAIR IRB 
503 Robert Grant Avenue 
Silver Spring, MD 20910-7500 
Phone: 301-319-9940 
Fax: 301-319-9961 
Email: usarmy.detrick.medcom- 
wrair.mbx.hspb@mail.mil 
USAMRDC Office of Research 
Protections 
Human Research Protection Office 
US Army Medical Research and Development 
Command, 
ATTN: MCMR-RPH 
504 Scott Street 
Fort Detrick, MD 21702-5012 
Fax: 301-619-7803 
Telephone: 301-619-2165 
Email: usarmy.detrick.medcom- 
usamrmc.other.hrpo@mail.mil 
Funding 
Military Infectious Diseases Research Program 
(MIDRP) 
FOR OFFICIAL USE ONLY 
Information and data included in this document contain privileged and/or proprietary information, 
which is the property of the United States Army. No person is authorized to make it public without 
express written permission of the United States Army. These restrictions on disclosure will apply 
equally to all future information, which is indicated as privileged or proprietary. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
6 
US Government Proprietary 
 
 
Investigator’s Agreement 
A PHASE 1, RANDOMIZED, OPEN-LABEL, SINGLE- 
CENTER, COMPARISON OF HETEROLOGOUS PRIME- 
BOOST VACCINATION SCHEDULES OF 
TETRAVALENT DENGUE VIRUS PURIFIED 
INACTIVATED VACCINE (PIV) AND TETRAVALENT 
DENGUE VIRUS LIVE ATTENUATED VACCINE (LAV) 
IN HEALTHY ADULTS IN A NONENDEMIC REGION 
 
 
 
 
“I have read this protocol and agree to conduct the study as outlined herein in accordance with 
International Conference on Harmonisation Good Clinical Practice Guideline and FDA, DoD, 
and United States Army Regulations.” 
 
 
 
Simon Pollett, MBBS 
Date 
Principal Investigator 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
7 
US Government Proprietary 
 
 
EMERGENCY CONTACT INFORMATION 
 
Role in Study 
Name 
Address and Telephone Number 
Principal Investigator 
Dr. Simon Pollett 
Simon Pollett, MBBS 
WRAIR 
503 Robert Grant Avenue 
Silver Spring, MD 20910-7500 
Telephone: 301-319-2059 
Email: 
simon.d.pollett.ctr@mail.mil 
Subinvestigators 
Dr. Christine Lee 
301-319-9660 
Dr. James Moon 
Cell phone: 240-406-6497 
Study Coordinator/Protocol 
Nurse 
Donna Tosh 
Office: 301-319-9332 
Research Monitor 
Dr. Tida Lee 
Office: 301-319-9260 
USAMRDC Clinical Services 
Support Division 
Deputy of Regulatory 
Clinical Operations Branch 
Clinical Services Support 
Division 
United States Army Medical 
Research and Development 
Command (USAMRDC) 
1430 Veterans Drive 
Fort Detrick, MD 21702-5009 
Telephone: 301-619-1106Fax: 
301-619-7790 
Sponsor’s Representative 
Kenneth A. Bertram, MD, 
PhD 
Principal Assistant for 
Acquisition 
1430 Veterans Drive 
Ft. Detrick, MD 21702-5009 
301-619-0317 
Email: usarmy.detrick.medcom- 
usamrmc.mbx.regulatory- 
affairs@mail.mil 
WRAIR Institutional Review 
Board 
Walter Reed Army Institute of 
Research (WRAIR) 
Institutional Review Board 
(IRB) 
503 Robert Grant Avenue 
Silver Spring, MD 20910-7500 
Phone: 301-319-9940 
Fax: 301-319-9961 
Email: usarmy.detrick.medcom- 
wrair.mbx.hspb@mail.mil 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
8 
US Government Proprietary 
 
 
1. 
SYNOPSIS 
 
Name of Sponsor: 
The Surgeon General, Department of the Army 
Name of Investigational Product: 
1. Tetravalent Dengue Virus Purified Inactivated Vaccine (PIV) with Alum adjuvant 
2. Tetravalent Dengue Virus Live Attenuated Vaccine (LAV) Formulation 17 post transfection 
Name of Active Ingredient: 
1. Inactivated dengue virus types 1-4 
2. Live attenuated dengue virus types 1-4 
Title of Study: 
A PHASE 1, RANDOMIZED, OPEN-LABEL, SINGLE-CENTER, COMPARISON OF HETEROLOGOUS 
PRIME-BOOST VACCINATION SCHEDULES OF TETRAVALENT DENGUE VIRUS PURIFIED 
INACTIVATED VACCINE (PIV) AND TETRAVALENT DENGUE VIRUS LIVE ATTENUATED VACCINE 
(LAV) IN HEALTHY ADULTS IN A NONENDEMIC REGION 
Study Center(s): Clinical Trials Center, Walter Reed Army Institute of Research (CTC, WRAIR) 
Principal Investigator: 
Simon Pollett, MBBS 
Subinvestigators: 
LCDR Marvin Sklar; COL Paul Keiser, MD; Simon Pollett, MBBS; LTC James Moon, MD;, Kristin Mills, DO; 
MAJ Nathaniel Copeland, MD; MAJ Melinda Hamer, MD; Christine Lee, MD; CPT Jack Hutter, MD; MAJ Justin 
Curley, MD; 
Study Period (years): 
Estimated date first subject enrolled: July 2017 
Estimated date last subject will completed: January 2019 
Estimated time all study related testing and data analysis will be 
complete: January 2021 
Phase of development: 1 
Objectives: 
Primary: 
• 
To further evaluate the safety and reactogenicity of 2 tetravalent dengue vaccine (TDENV) candidates 
administered in dengue naïve subjects in a heterologous prime boost fashion with a PIV followed by LAV 
180 days later. 
• 
To evaluate the safety and reactogenicity of a previously untested vaccination schedule in dengue naïve 
subjects consisting of PIV followed by LAV 90 days later 
Secondary: 
• 
To further evaluate the humoral and cell-mediated immunogenicity of the PIV/LAV Day 0,180 
administration schedule and compare it to the humoral and cell-mediated immunogenicity of the PIV/LAV 
Day 0,90 schedule 
Exploratory: 
• 
To evaluate the rate of decay and durability of humoral immunity in the Day 0,90 PIV/LAV prime-boost 
vaccination schedule and the Day 0,180 PIV/LAV prime-boost vaccination schedule 
• 
To characterize the cell-mediated immune response of the Day 0,90 PIV/LAV prime-boost vaccination 
schedule compared to the 0, 180 day PIV/LAV prime-boost vaccination schedule 
Methodology: 
This study is a Phase 1, randomized, open-label, study with 2 treatment groups (N=40): 
Group 1 (n=20): TDENV-PIV 4 µg + Alum adjuvant (Day 0), TDENV-LAV F17 (Day 180) 
Group 2 (n=20): TDENV-PIV 4 µg + Alum adjuvant (Day 0), TDENV-LAV F17 (Day 90) 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
9 
US Government Proprietary 
 
 
 
Estimated Number of Subjects to Screen: 80-120 
Maximum Number of Subjects to Enroll: 40 
Main Criteria for Inclusion/Exclusion: 
Healthy male or female dengue naïve subjects between 18 and 42 years of age (inclusive) at the time of consent. 
Investigational Product Dosage, Schedule, and Mode of Administration: 
TDENV-PIV 
Dosage: 0.5 mL of DENV serotypes 1-4 (4 µg / serotype) in alum adjuvant 
Mode of administration: intramuscular (IM) into the subject’s upper arm, deltoid area of the subject’s arm; 
vaccination will be given in the non-dominant arm whenever possible 
TDENV-LAV F17 
Dosage: 0.5 mL of the post-transfection LAV F17 vaccine 
Mode of administration: subcutaneously into the upper-outer triceps/deltoid area of the subject’s arm; vaccination 
will be given in the non-dominant arm whenever possible 
Schedule 
Varied, 2 vaccinations per volunteer in a heterologous prime-boost strategy. First dose on Day 0 (PIV) AND a 
second dose (LAV) on Day 180 for Group 1. First dose on Day 0 (PIV) AND a second dose (LAV) on Day 90 for 
group 2. 
Duration of Subject Participation: 
Approximately 18 Months 
Criteria for Evaluation: 
Safety: 
• 
Solicited adverse events (AEs) during the 28 days follow up after prime vaccination and 28 days following 
boost vaccination. 
• 
Unsolicited adverse events during the 28 days following each vaccination. 
• 
Hematological and biochemical abnormalities on vaccination day, Day 7, and Day 28 after each 
vaccination. 
• 
Occurrence of serious adverse events (SAEs) during the entire study. 
• 
Occurrence of potential immune-mediated diseases (pIMDs) during the entire study. 
• 
Occurrence of medically attended adverse events (AEs) during the entire study. 
• 
Vaccine induced viral replication measured by DENV serotype specific reverse transcription-polymerase 
chain reaction (RT-PCR) or equivalent assay at Days 6 thru 14 after LAV vaccination (5 consecutive days 
of testing per subject within this window excluding weekends) 
Immunogenicity: 
• 
Microneutralizing (MN) and flow cytometry based neutralizing dengue antibody titers before first (prime) 
vaccination, 28 days after, immediately prior to the second (boost) vaccination (90 or 180 days), then 28 
days, 90 days, 180 days, 270 days, and 360 days following the second(boost) vaccination. 
• 
Geometric mean titers (GMTs) of neutralizing antibody against each DENV serotype. 
• 
Seropositivity rates for each DENV type 1-4. 
• 
Trivalent and tetravalent rates of seroconversion. 
• 
CMI assays 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
10 
US Government Proprietary 
 
 
Statistical Methods: 
Demography: 
Demographic characteristics (age at first visit in years, sex, race (geographic ancestry), and ethnicity of each study 
cohort will be tabulated. The distribution of subjects enrolled will be tabulated as a whole and per study arm. For 
continuous variables (ie, age), mean or medians will be presented with range and standard deviations or interquartile 
range depending on the normality of the data. For categorical variables, proportions will be presented. Proportions, 
means and/or medians will be compared between the 2 arms using chi-squared, t-test, or Mann-Whitney U 
respectively. 
Safety and reactogenicity: 
The following results will be tabulated by group on the total vaccinated cohort: 
• 
The number and proportion of subjects with at least one local solicited adverse event (AE), at least one 
general solicited AE and any solicited AE during the 7 day and 28-day follow-up periods for prime and 
boost vaccination will be tabulated with 95% confidence interval (CI) after each vaccine dose and overall. 
• 
The number and proportion of subjects reporting each individual solicited local (any, grade 3) and general 
AE (any, grade 3, any related and grade 3 related) during the 7-day and 28-day follow-up periods after 
each vaccination will be tabulated with 95% CIs. Occurrence of fever will be reported in 0.5°C cumulative 
increments. Duration of fever will be recorded as the number of consecutive days of fever. 
• 
The number and proportion of subjects reporting at least one of each type of unsolicited AE during the 28- 
day (Day 0 to Day 27) follow-up period after each vaccination will be tabulated with 95% CIs. The 
verbatim reports of unsolicited AEs will be reviewed by a physician and the signs and symptoms will be 
coded according to the Medical Dictionary for Regulatory Activities (MedDRA). Every verbatim term will 
be matched with the appropriate Preferred Term and categorized by system organ class. Similar tabulations 
will be done for grade 3 unsolicited AEs and, or unsolicited AEs related to vaccination. 
• 
The proportion of subjects with at least one report of any SAE classified by the MedDRA and reported up 
to 28 days (Day 0-Day 27) after each vaccination will be tabulated with 95% CIs. 
• 
SAEs, withdrawals due to AEs, pIMDs, pregnancies, and all related AEs will be described in detail. 
• 
The number and proportion of subjects who receive at least one concomitant medication/vaccination 
during the 28 days follow-up period (Day 0 to Day 27) after each vaccination will be tabulated with 95% 
CIs. Additionally, the number and percentage of subjects receiving non-steroidal anti-inflammatory drugs 
(NSAIDs) or any prophylactic antipyretics will be calculated by group for the first 7 days after each dose. 
• 
The proportion of subjects with abnormal hematological or biochemical laboratory values will be presented 
with 95% CIs at baseline and at each specified time point. These results, for laboratory tests listed on the 
toxicity grading scale in Appendix D, will be reported by severity grade according to the FDA’s toxicity 
grading scale adjusted for harmony with Quest normal lab values. 
• 
The number and proportion along with mean/median of subjects with detectable dengue viremia days after 
each vaccination including day(s) of detectable viremia and peak viremia will be described. 
Humoral Immunogenicity: 
The GMTs of anti-dengue neutralization antibodies at Day 0 and Day 28 after each immunization and immediately 
prior to the boost vaccination (Day 90 or 180) for the prime vaccination and at Days 90, 180, 270 and 360 following 
boost vaccination will be calculated by group with 95% CIs. The proportion of volunteers with anti-dengue 
neutralizing antibody titers above the assay cut-off at these time points will be calculated with 95% CIs. The 
proportion of volunteers with neutralizing antibody titers above the assay cut-off to 3 or 4 DENV types (trivalent or 
tetravalent response rates) at these time points will be calculated by group with 95% CIs. 
CMI: 
CMI responses will be described using ELISPOT, Cytokine flow cytometry, flow cytometry base 
immunophenotyping, Fluorescence-activated cell sorting, In vitro proliferation assays, antibody-dependent 
cellular cytotoxicity by flow cytometry, multiplexed ELISA, immune genotyping and systems biology 
analyses. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
11 
US Government Proprietary 
 
 
2. 
TABLE OF CONTENTS, LIST OF TABLES, AND LIST OF 
FIGURES 
TABLE OF CONTENTS 
TITLE PAGE ...................................................................................................................................1 
EMERGENCY CONTACT INFORMATION ................................................................................6 
1. 
SYNOPSIS .................................................................................................................7 
2. 
TABLE OF CONTENTS, LIST OF TABLES, AND LIST OF FIGURES .............10 
TABLE OF CONTENTS ...............................................................................................................10 
LIST OF TABLES .........................................................................................................................13 
LIST OF FIGURES .......................................................................................................................14 
3. 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS ..........................15 
4. 
INTRODUCTION ....................................................................................................18 
5. 
INVESTIGATIONAL PRODUCT ..........................................................................20 
5.1. 
Investigational Product Packaging and Labeling .........................................20 
5.2. 
Investigational Product Storage and Packaging ...........................................21 
5.3. 
Investigational Product Preparation .............................................................21 
5.4. 
Summary of Clinical Trials with Investigational Products ..........................21 
5.4.1. TDENV-LAV ...................................................................................21 
5.4.2. TDENV-PIV .....................................................................................23 
5.4.3. PIV/LAV Prime-boost ......................................................................24 
6. 
TRIAL DESIGN .......................................................................................................25 
6.1. 
Overall Study Design and Rationale ............................................................25 
6.2. 
Trial Objectives and Purpose .......................................................................26 
6.2.1. Primary Objective .............................................................................26 
6.2.2. Secondary Objective .........................................................................26 
6.2.3. Exploratory Objectives .....................................................................26 
6.3. 
Study Endpoints ...........................................................................................26 
6.3.1. Primary Endpoints ............................................................................26 
6.3.2. Secondary Endpoints ........................................................................26 
6.3.3. Exploratory Endpoints ......................................................................27 
6.4. 
Study Site .....................................................................................................27 
6.5. 
Study Population ..........................................................................................27 
6.6. 
Duration of Subject Participation .................................................................27 
6.7. 
Measures Taken to Minimize/Avoid Bias ....................................................27 
6.7.1. Assignment .......................................................................................27 
6.7.2. Open-label Study ..............................................................................28 
6.8. 
Route of Administration, Dosage Regimen, Treatment Period, and 
Justification ..................................................................................................28 
6.9. 
Investigational Product Accountability ........................................................28 
6.9.1. Risks/Discomfort  to  Subjects  and   Precautions   to   Minimize 
Risk ...................................................................................................29 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
12 
US Government Proprietary 
 
 
6.9.1.1. 
Local Reactions ..............................................................29 
6.9.1.2. 
Systemic Reactions.........................................................29 
6.9.1.3. 
LAV and DPIV SAE and Grade 3 AEs ..........................29 
6.9.1.4. 
Pregnancy .......................................................................30 
6.9.1.5. 
Lactation .........................................................................30 
6.9.1.6. 
Venipuncture ..................................................................31 
6.9.1.7. 
Allergic Reactions and Immune Mediated 
Reactions ........................................................................31 
6.9.1.8. 
Bovine Spongiform Encephalopathy..............................31 
6.9.1.9. 
Natural Dengue Virus Infection .....................................31 
6.9.1.10. 
Unknown Risks ..............................................................32 
6.9.2. Alternatives to this IND Product or Study .......................................32 
6.9.3. Intended Benefit for Subjects ...........................................................32 
6.9.4. Risks to the Study Personnel and the Environment..........................32 
7. 
SELECTION OF SUBJECTS ..................................................................................32 
7.1. 
Subject Exclusion Criteria ............................................................................33 
8. 
STUDY PROCEDURES ..........................................................................................35 
8.1. 
Recruitment of Subjects ...............................................................................35 
8.2. 
Informed Consent .........................................................................................44 
8.3. 
Study Visits ..................................................................................................45 
8.4. 
Subject ID Cards ..........................................................................................45 
8.5. 
Memory Aids ................................................................................................45 
8.6. 
Biological Samples .......................................................................................46 
8.7. 
Concomitant Medications or Vaccinations ..................................................46 
8.8. 
Procedures for Monitoring Subject Compliance ..........................................46 
9. 
DOSE ADJUSTMENT AND STUDY TERMINATION PROCEDURES .............47 
9.1. 
Contraindications to Subsequent Vaccination ..............................................47 
9.2. 
Safety Criteria for Stopping Doses ...............................................................47 
9.3. 
Study Termination Criteria ...........................................................................48 
9.3.1. Subject Withdrawal Criteria .............................................................49 
9.3.2. How to Withdraw Subjects...............................................................49 
9.3.3. Data Collected for Withdrawn Subjects ...........................................49 
9.3.4. Replacement of Subjects ..................................................................49 
9.3.5. Follow-up for Withdrawn Subjects ..................................................50 
10. 
IMMUNOGENICITY ASSESSMENTS .................................................................50 
10.1. 
Geometric Mean Titers and Seroconversion ................................................50 
10.2. 
Cell-Mediated Immunity (CMI) Assessment ...............................................51 
10.2.1. CMI Assays and Assay Systems ......................................................52 
10.2.1.1. 
ELISPOT ........................................................................52 
10.2.1.2. 
Cytokine Flow Cytometry (CFC) ...................................52 
10.2.1.3. 
Flow Cytometrycytometry-based 
Immunophenotypingimmunophenotyping and 
Fluorescencefluorescence-activated Cell 
Sortingcell sorting (FACS) .............................................52 
10.2.1.4. 
In vitro proliferation assays ............................................52 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
13 
US Government Proprietary 
 
 
10.2.1.5. 
Antibody-dependent Cellular Cytotoxicity 
(ADCC) ..........................................................................52 
10.2.1.6. 
Multiplexed Enzyme Linked Immunosorbent 
Assay (ELISA) ...............................................................53 
10.2.1.7. 
Immune Genotyping .......................................................53 
10.2.1.8. 
Systems Biology Analyses .............................................53 
11. 
SAFETY ASSESSMENT ........................................................................................53 
11.1. 
Specification of Safety Assessments ............................................................54 
11.1.1. Demographic Data ............................................................................54 
11.1.2. Physical Examination, Vital Signs, and Medical History ................54 
11.1.3. Clinical Laboratory Assessments .....................................................55 
11.1.4. Post-vaccination Viremia .................................................................55 
11.1.5. Definition of Adverse Events ...........................................................56 
11.1.6. Unanticipated Problems Involving Risks To Subjects Or 
Others ...............................................................................................58 
11.1.7. Potential Immune-mediated Diseases ..............................................58 
11.2. 
Relationship to Investigational Product .......................................................58 
11.3. 
Severity Assessment .....................................................................................60 
11.4. 
Assessment of Outcome ...............................................................................63 
11.5. 
Duration of Follow-Up of Subjects after Adverse Events............................63 
11.6. 
Recording Adverse Events ...........................................................................63 
11.7. 
Reporting Adverse Events ............................................................................64 
11.7.1. Reporting Serious and Unexpected Adverse Events ........................64 
11.7.1.1. 
Reporting to the Sponsor ................................................64 
11.7.1.2. 
Reporting to the IRB ......................................................66 
11.7.2. Reporting Additional Immediately Reportable Events to the 
Sponsor’s Safety Office and WRAIR IRB .......................................66 
11.7.2.1. 
Pregnancy .......................................................................66 
11.7.2.2. 
AE-related Withdrawal of Consent ................................66 
11.7.2.3. 
Additional Immediately Reportable Event .....................67 
11.7.2.4. 
Pending Inspections/Issuance of Reports .......................67 
11.7.3. IND Annual Report and Final Clinical Study Report ......................67 
11.7.3.1. 
IND Annual Report ........................................................67 
11.7.3.2. 
Final Report ....................................................................67 
12. 
STATISTICS ............................................................................................................67 
12.1. 
Description of Statistical Methods ...............................................................67 
12.1.1. Analysis Addressing the Primary Study Objective ..........................68 
12.1.2. Analysis Addressing the Secondary Study Objective ......................68 
12.1.3. Analysis Addressing Exploratory Objective ....................................68 
12.1.4. Clinical Laboratory Data Analyses ..................................................69 
12.1.5. Schedule of Analyses .......................................................................70 
12.2. 
Planned Enrollment and Reason for Sample Size ........................................70 
12.3. 
Level of Significance to be Used .................................................................71 
12.4. 
Statistical Criteria for the Termination of the Trial ......................................71 
12.4.1. Stopping Rules .................................................................................71 
12.5. 
Accounting for Missing, Unused, and Spurious Data ..................................71 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
14 
US Government Proprietary 
 
 
12.6. 
Procedures for Reporting Deviations from the Original Statistical 
Plan ...............................................................................................................71 
12.7. 
Selection of Subjects to be Included in Analyses .........................................71 
12.7.1. Total Vaccinated Population ............................................................71 
12.7.2. According to Protocol Population ....................................................71 
13. 
ETHICS ....................................................................................................................72 
13.1. 
Compliance Statement ..................................................................................72 
13.2. 
Ethics Review ...............................................................................................72 
13.2.1. Review/Approval of Study Protocol ................................................73 
13.2.2. Confidentiality ..................................................................................73 
13.2.3. Compensation for Participation ........................................................74 
13.3. 
Biological Sample Handling and Analysis ...................................................75 
13.4. 
Specimen Storage and Labeling ...................................................................75 
14. 
DATA HANDLING AND RECORDKEEPING .....................................................76 
14.1. 
Inspection of Records ...................................................................................76 
14.2. 
Retention of Records ....................................................................................76 
15. 
QUALITY CONTROL, QUALITY ASSURANCE, AND DIRECT 
ACCESS TO SOURCE DATA/DOCUMENTS ......................................................77 
15.1. 
Study Monitoring .........................................................................................77 
15.2. 
Audits and Inspections .................................................................................78 
15.3. 
Institutional Review Board ...........................................................................78 
15.4. 
Protocol Suspensions or Discontinuation .....................................................78 
16. 
PROTOCOL MODIFICATIONS AND DEVIATIONS ..........................................79 
16.1. 
Protocol Modifications .................................................................................79 
16.2. 
Protocol Deviation Procedures .....................................................................79 
17. 
PUBLICATION POLICY ........................................................................................80 
17.1. 
Initial Analysis .............................................................................................80 
18. 
LIST OF REFERENCES .........................................................................................81 
19. 
APPENDICES ..........................................................................................................83 
APPENDIX A. 
ROLES AND RESPONSIBILITIES ............................................................84 
APPENDIX B. 
LABELS .......................................................................................................89 
APPENDIX C. 
RECRUITMENT SCRIPT ...........................................................................90 
APPENDIX D. 
MODIFIED FDA LABORATORY TOXICITY SCALES..........................93 
 
LIST OF TABLES 
Table 1: 
Investigational Product ...............................................................................................20 
Table 2: 
Clinical Studies of LAV ................................................................................................. 22 
Table 3: 
Clinical Studies of TDENV-PIV ................................................................................24 
Table 4: 
Vaccination Schedule .................................................................................................28 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
15 
US Government Proprietary 
 
 
Table 5: 
Study Events Schedule for Group 1: Day 0 – PIV, Day 180 – LAV ..........................37 
Table 6: 
Study Events Schedule for Group 2: Day 0 – PIV, Day 90 – LAV ............................40 
Table 7: 
Adverse Event Study Hold Rules ...............................................................................48 
Table 8: 
Laboratory Assay Study Hold Rules ..........................................................................48 
Table 9: 
Humoral Immunity Assessments (Antibody Determination) .....................................51 
Table 10: List of Potential Immune-mediated Diseases .............................................................59 
Table 11: Solicited Adverse Event Intensity Scales ...................................................................61 
Table 12: Intensity Scale for All Other Adverse Events .............................................................63 
Table 13: Study Contacts for Reporting Serious Adverse Events and Unanticipated 
Problems Involving Risk to Patients or Others ...........................................................65 
Table 14: SAE Information to be Reported to the Sponsor's Safety Office ................................65 
Table 15: Precision  for  the  95%  Confidence  Interval  for  Varying  Frequency  Rates 
(N=20).........................................................................................................................71 
 
LIST OF FIGURES 
Figure 1: 
Representative Label for TDENV-LAV F17 ..............................................................20 
Figure 2: 
Representative Label for TDENV-PIV .......................................................................21 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
16 
US Government Proprietary 
 
 
3. 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
 
Abbreviation 
Explanation 
ADCC 
antibody dependent cellular cytotoxicity 
ADL 
Activities of daily living 
AE 
adverse event 
ALT 
alanine aminotransferase 
ANC 
absolute neutrophil count 
AST 
aspartate aminotransferase 
ATP 
according to protocol 
BD 
Becton Dickinson 
BSE 
bovine spongiform encephalopathy 
C 
Celsius 
CFC 
cytokine flow cytometry 
CFR 
Code of Federal Regulation 
CI 
confidence interval 
CMI 
cell-mediated immunity 
CTC 
Clinical Trials Center, WRAIR 
DENV 
dengue virus 
DHF 
dengue hemorrhagic fever 
DoD 
Department of Defense 
DPIV 
dengue purified inactivated vaccine 
eCRF 
electronic case report form 
ELISPOT 
enzyme-linked immunospot 
F 
Fahrenheit 
FACS 
fluorescence-activated cell sorting 
FDA 
US Food and Drug Administration 
Fox 
Forkhead box 
GATA 
guanine-adenine-guanine-adenine-binding transcription factor 
GCP 
Good Clinical Practice 
G-CSF 
granulocyte colony-stimulating factor 
GM-CSF 
granulocyte macrophage colony-stimulating factor 
GMT 
geometric mean titer 
GSK 
Glaxo Smith Kline 
HCV 
hepatitis C virus 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
17 
US Government Proprietary 
 
 
 
Abbreviation 
Explanation 
HIPAA 
Health Insurance Portability Accountability Act 
HIV 
human immunodeficiency virus 
HLA 
human lymphocyte antigen 
ICH 
International Conference on Harmonisation 
IM 
intramuscular(ly) 
IND 
investigational new drug 
IRB 
Institutional Review Board 
LAV F17/19 
tetravalent dengue live-attenuated virus formulation (either 17 or 19 is referenced) 
LLN 
lower limit of normal 
mAb 
monoclonal antibody 
MCP 
membrane cofactor protein 
MedDRA 
Medical Dictionary for Regulatory Activities 
mg 
milligram(s) 
MIG 
monokine induced by gamma interferon 
MIP 
macrophage inflammatory protein 
mL 
Milliliter(s) 
mm 
millimeter(s) 
MN 
Microneutralizing 
mRNA 
messenger ribonucleic acid 
NCT 
ClinicalTrials.gov study identifier 
ORA 
ORP HRPO 
Office of Regulated Activities 
Office of Research Protections, Human Research Protection Office 
PBF 
Pilot Bioproduction Facility, WRAIR 
PBMC 
peripheral blood mononuclear cells 
PI 
principal investigator 
PIV 
tetravalent dengue virus purified inactivated vaccine 
PRN 
pro re nata (as needed, whenever necessary) 
PSSO 
Product Safety Surveillance Office, USAMRDC 
RANTES 
regulated on activation, normal T cell expressed and secreted 
RORC 
retinoic acid receptor-related orphan receptor C 
RPMI 
Roswell Park Memorial Institute medium 
RT-PCR 
reverse transcription-polymerase chain reaction 
SAE 
serious adverse event 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
18 
US Government Proprietary 
 
 
 
Abbreviation 
Explanation 
SAP 
statistical analysis plan 
SFC 
spot forming cells 
SOP 
standard operating procedure 
SQ 
subcutaneous 
SUN 
serum urea nitrogen 
TCR 
T cell receptor 
TDENV 
tetravalent dengue virus 
TGF 
transforming growth factor 
TNF 
tumor necrosis factor 
ULN 
upper limit of normal 
UPIRTSO 
unanticipated problems involving risks to subjects or others 
URI 
University of Rhode Island 
USAMRDC 
United States Army Medical Research and Development Command 
USAMRMC 
United States Army Medical Research and Materiel Command 
WBC 
white blood cell count 
WHO 
World Health Organization 
WRAIR 
Walter Reed Army Institute of Research 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
19 
US Government Proprietary 
 
 
4. 
INTRODUCTION 
Dengue is a mosquito-borne disease caused by serologically related but antigenically distinct 
dengue viruses (DENV) grouped into 4 types (DENV-1 to DENV-4) and is endemic in 
100 countries in the tropical and subtropical regions of Asia-Pacific, the Americas, the Middle 
East, and Africa. The World Health Organization (WHO) estimates over 40% of the world’s 
population to be at risk for dengue infection. An estimated 100 million cases of infection occur 
each year, resulting in 500,000 hospitalizations with estimated 2.5% mortality (WHO Dengue 
2016). Other models have yielded much higher estimates of infection rates and unreported 
illnesses (Bhatt-2013). In recent years, dengue outbreaks have been increasing and expanding 
geographically. Local transmission has been reported in Europe and the continental United States 
(Florida, Texas, and New Mexico). 
While dengue is a threat to endemic populations, travelers and military personnel deploying to 
endemic areas are also at significant risk. A recent GeoSentinel analysis of 82,825 ill Western 
travelers identified 1,910 with acute dengue infection of which 0.9% developed hemorrhagic 
fever, resulting in one death (Jansenius-2013). The annual proportionate morbidity due to dengue 
in Southeast Asia during an epidemic is estimated to be 159 cases per 1,000 travelers. The risk is 
likely higher for deployed military personnel due to longer exposure periods. US military 
operations over the past century have experienced numerous disruptions due to dengue (Gibbons 
et al-2012). With continued military focus in the Pacific region and the Americas, the number of 
military and civilian personnel traveling to dengue endemic regions is also expected to rise 
significantly. 
No specific treatment is available for dengue, but symptomatic and supportive measures (eg, 
intravenous [IV] fluid management) significantly reduce the morbidity and mortality rates. It is 
estimated that intensive and careful fluid management and inpatient monitoring can lower 
mortality rates in severe Dengue from greater than 20% to less than 1% (WHO Dengue 2016). 
Currently, vector control is the only method with a demonstrated, though limited efficacy in 
prevention. Mosquito control does not offer satisfactory control of dengue; additional prevention 
methods are required. No antiviral drugs are approved for clinical use, but several candidates are 
under evaluation at the preclinical stage. However, available data suggest that treatment at the 
onset of symptoms is unlikely to have significant impact on viral dynamics (Clapham et al- 
2014). Several vaccine candidates are at various stages of development including a Sanofi 
Pasteur vaccine that is approved for use in several countries (Punee et al-2016). However, this 
vaccine has significantly decreased efficacy in non-immune populations and concerns remain 
about potential disease enhancement in some groups including those previously unexposed to 
any dengue infection (Punee et al-2016). Therefore, it is not a suitable vaccine for the US 
Military population. The ideal dengue vaccine protects against all 4 serotypes of DENV and is 
effective in endemic and non-endemic populations. Current Challenges to dengue vaccine 
development include the lack of a good animal model, lack of an established correlate of 
protection, and an incomplete understanding of the human immune response profile after dengue 
infection or vaccination. 
Many approved vaccines are administered in multiple doses. The first dose primes the immune 
system, and the second dose serves to boost the immune response. A homologous prime-boost 
strategy uses the same vaccine for both doses. Live-attenuated vaccines and purified inactivated 
vaccines can activate different parts of the human immune system (Lu-2009; Woodland-2004). 
Using both types of vaccines in a heterologous prime-boost strategy has proven effective at 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
20 
US Government Proprietary 
 
 
boosting cellular responses against other pathogens (Woodland-2004). A heterologous prime- 
boost dengue vaccine strategy was evaluated previously using 4 different vaccination schedules 
(WRAIR #2136). This study involved 4 groups of 20 subjects each who received either 
PIV/LAV or LAV/PIV on a 0, 28 day or a 0, 180 day schedule. Overall the prime-boost 
schedules were safe and well tolerated in all groups (unpublished data, Lin et al). Results from 
this study indicated the greatest immunogenicity was achieved using PIV followed by LAV. 
Both the 0,28 day and 0, 180 day schedule achieved greater than 95% quadrivalent 
seroconversion rates(unpublished data Lin et al) assessed by micronuetralizing antibody titers. 
However, there were differences between the antibody titers and durability of antibody responses 
6 months post completion of all vaccinations. The 0, 180 day immunization regimen 
demonstrated significantly higher mean antibody titers at 6 months post-vaccination compared to 
the 0, 28 day regimen (unpublished data Lin et al). The cell-mediated immune responses did not 
demonstrate significant differences. Overall it appears that the 0, 180 day regimen is superior at 
least in durability of antibody response so this regimen was selected for further evaluation. 
However, a shorter interval between doses would be significantly more practical for travelers and 
soldiers for whom this vaccine is intended. Therefore, the previously untested 0, 90 day regimen 
is under consideration as a means to combine the durability of the 0, 180 day regimen with a 
more practical dosing schedule. Potential protective efficacy of the prime-boost strategy has also 
been suggested by challenge in non-human primates previously as published in 2010. This study 
showed sterilizing immunity from challenge with dengue serotypes 1-4 in non-human primates 
following vaccination with a heterologous PIV/LAV prime-boost vaccination schedule 
(Simmon-2010). This further suggests that the immune response generated and measured in 
humans with this vaccination strategy will provide protective efficacy. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
21 
US Government Proprietary 
 
 
5. 
INVESTIGATIONAL PRODUCT 
PIV consists of 4 purified, inactivated dengue-virus vaccines (DENV serotypes 1-4) adjuvanted 
with alum. LAV is a live, attenuated DENV vaccine that is composed of 4 monovalent 
attenuated strains (DENV serotypes 1-4 formulation F17). 
Table 1 presents a summary description of the investigational products. 
Table 1: 
Investigational Product 
 
Product Name 
Adjuvanted Tetravalent Dengue 
Virus Purified Inactivated 
Vaccine (TDENV-PIV) 
Tetravalent Dengue Virus Live- 
attenuated Vaccine (TDENV-LAV, 
formulation F17) 
Dosage Form 
Vaccine 
Vaccine 
Unit Dose 
0.5 mL 
(4 μg/serotype/dose) 
0.5 mL 
Route of 
Administration 
Intramuscular injection, 1-inch, 22- 
gauge needle 
Subcutaneous injection, 5/8-inch 25- 
gauge needle 
Physical Description 
Liquid suspension 
Freeze-dried cake 
Manufacturer 
Pilot Bioproduction Facility (PBF), 
Walter Reed Army Institute of 
Research (WRAIR) 
PBF, WRAIR 
Lot Number 
Lot 1725 
Lot 1856 
Product Indication 
Prevention of dengue infection 
Prevention of dengue infection 
 
5.1. 
Investigational Product Packaging and Labeling 
TDENV-LAV and TDENV-PIV are packaged in sterile vials sealed with plug stoppers and 
aluminum crimps. 
The investigational products are covered under separate Investigational New Drug (IND) 
applications. Each vial will be labeled for human administration and will include the following 
statement: “Caution: New Drug − Limited by Federal Law to Investigational Use.” Example 
labels for each of the products are provided in Figure 1 and Figure 2. 
Figure 1: 
Representative Label for TDENV-LAV F17 
 
Tetravalent Dengue Virus Vaccine, 
Live-attenuated, Formulation17 
BPR No.: BPR-844-01 
Lot No.: 1856 
Contents: 0.7 mL (Freeze-Dried) 
Caution: New drug limited by Federal (or 
United States) law to investigational use 
Date of Mfg.: 31 Aug 07 
Storage: -15 to -30°C 
Mfg. By: WRAIR, Silver Spring, MD 20910, USA 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
22 
US Government Proprietary 
 
 
Figure 2: 
Representative Label for TDENV-PIV 
 
Tetravalent Dengue Virus Purified Inactivated 
Vaccine, 4 mcg/serotype/0.5 mL dose 
BPR No.: BPR-1040-00 
Lot No.: 1725 
Contents: 0.7 mL (Alum Adsorbed) 
Caution: New drug - Limited by Federal (or 
United States) law to investigational use 
Date of Mfg.: 30Sep 11 
Storage: 2 - 8°C 
Mfg. By: WRAIR, Silver Spring, MD 20910, USA 
 
5.2. 
Investigational Product Storage and Packaging 
LAV is stored at -15°C to -30°C and PIV is stored at 2°C-8°C at the Pilot Bioproduction Facility 
(PBF), Walter Reed Army Institute of Research (WRAIR) and will be transported to the clinical 
site according to standard operating procedures (SOP G-060). 
At the study site the investigator or his or her designee will be responsible for product 
management. The lyophilized LAV F17 to be administered to the subjects must be stored at -15 
to -30°C and PIV at 2-8°C in a safe and locked place with restricted, controlled access. The 
temperature of the storage unit will be monitored daily with a validated temperature monitoring 
device(s) and documented. The diluent (WFI) used for reconstitution of LAV F17 will be stored 
at 20- 25°C. Rehydration is performed at 20- 25°C and once vial is rehydrated it is kept at 2-8°C 
until administration or up to 24 hours. Any temperature excursion (ie, temperature outside the 
defined range of storage) must be reported to the sponsor’s representative within 24 hours of 
knowledge of the excursion. After exposure to a temperature excursion, the product will not be 
used until written approval has been given by the sponsor’s representative. 
 
5.3. 
Investigational Product Preparation 
LAV F17 is provided in single dose vials and is freeze dried. A syringe must be filled to 0.7 mL 
with sterile water-for-injection and used for reconstitution. The vaccine vial must be swirled until 
the freeze-dried cake is completely dissolved. 
To prepare PIV, the vial must be inverted 3 times to ensure that the alum is resuspended, before 
the appropriate injection volume is withdrawn with a syringe for injection. 
 
5.4. 
Summary of Clinical Trials with Investigational Products 
5.4.1. 
TDENV-LAV 
Three phase 2 studies have used LAV. The first study (TDEN-001) vaccinated 86 healthy 
volunteers in Silver Spring, MD and was conducted between April 2006 and March 2008. This 
placebo-controlled, randomized, observer-blind study evaluated pre-transfection LAV and 2 
post-transfection formulations including the current LAV (F17) and another designated LAV 
F19 given in a 0-, 6-month vaccination schedule. LAV F19 contained 6-fold lower concentration 
of DENV-4 than the other serotypes. LAV F17 demonstrated an acceptable safety profile based 
on physical examination, clinical laboratory determinations, and the lack of symptomatic viremia 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
23 
US Government Proprietary 
 
 
during follow-up visits. There were 7 total grade 3 AEs reported over 79 received doses of 
vaccine. These included one of each of the following: injection site swelling, arthralgia, fatigue, 
headache, muscle ache and 2 reports of redness at injection site. Neither dengue-related illness 
nor vaccine-related serious adverse events (SAEs) were reported (Thomas et al-2013). The rate 
of tetravalent seropositivity among unprimed subjects, as determined by neutralizing antibody 
testing (MN50) after 2 doses of LAV F17, was 60%. 
Two additional studies that compared LAV F17 (pre-transfection and post-transfection) and 
LAV F19 (post-transfection) were conducted; one in Thailand and one in Puerto Rico (Bauer et 
al-2015 and Watanaveeradej et al-2014). Because these are dengue-endemic areas, the majority 
of subjects were flavivirus-primed. In the study conducted in Thailand there were no grade 3 
local solicited AEs. Out of 40 subjects receiving F17 LAV, there were 7 systemic grade 3 AEs 
reported. These included one each of fatigue, fever, headache, nausea, pruritus, and 2 reported 
rashes. There were no cases of vaccine-related SAEs. Low levels of monotypic viremia were 
observed, but these were not associated with illness. The vaccine improved tetravalent response 
rates among the primed subjects, and tetravalent antibody responses were observed after 2 doses 
in unprimed subjects. The Puerto Rico study included multiple sites and included young children 
for a total 211 subjects receiving F17 LAV, 212 receiving F19 LAV and 213 receiving placebo. 
No related SAEs were reported, and solicited AEs did not increase from Dose 1 to Dose 2. 
Among the unprimed subjects, tetravalent MN50 antibody response rates and geometric mean 
titers (GMTs) were higher in the formulation F17 cohorts versus the formulation F19 cohorts. 
These studies demonstrated that the re-derived, post-transfection LAV F17 is safe and 
immunogenic in flavivirus-naïve and flavivirus-primed volunteers. 
Table 2: 
Clinical Studies of LAV 
 
Type 
of 
Study 
Study No. 
Study Design and 
Type of Control 
Test Product(s); Dosage 
Regimen; Route of Admin. 
No. of 
Subject 
s 
Study 
Population 
Phase 
2 
TDEN- 
001 
Observer blind, 
randomized, 
placebo-controlled 
Pre-transfection LAV F17, 
post-transfection LAV F17, 
post-transfection LAV F19, 
placebo. 
-0,6 months 
-Subcutaneous (SQ) 
86 
Healthy adults 
(US) 
Phase 
2 
TDEN- 
002 
Observer blind, 
randomized, 
placebo-controlled 
Pre-transfection LAV F17, 
post-transfection LAV F17, 
post-transfection LAV F19, 
placebo. 
-0,6 months 
-SQ 
120 
Healthy 
flavivirus- 
primed adults 
(Thailand) 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
24 
US Government Proprietary 
 
 
 
Type 
of 
Study 
Study No. 
Study Design and 
Type of Control 
Test Product(s); Dosage 
Regimen; Route of Admin. 
No. of 
Subject 
s 
Study 
Population 
Phase 
2 
TDEN- 
003 
Observer blind, 
randomized, 
placebo- 
controlled, age-de- 
escalation 
Pre-transfection LAV F17, 
post-transfection LAV F17, 
post-transfection LAV F19, 
placebo. 
-0,6 months 
-SQ 
636 
Flavivirus- 
primed, 12 
mos to 50 
years (Puerto 
Rico) 
 
5.4.2. 
TDENV-PIV 
The WRAIR PIV combined with GSK adjuvant or alum has been used in 2 Phase 1 clinical 
trials. Both studies evaluated PIV (1 or 4 µg ) combined with alum adjuvant or one of GSK’s 
proprietary adjuvant systems (AS01E and AS03B). Both studies are currently in the active 
follow-up phase. The first study population (S-11-23; NCT01666652; DPIV-001) was comprised 
of healthy US adults who were mostly flavivirus naïve. The second study (S-12-12; 
NCT01702857; DPIV-002) is being conducted in Puerto Rico, a dengue endemic area, where 
most subjects are dengue-primed. 
All 4 PIV vaccine formulations were generally well-tolerated. The Puerto Rico study had a 
higher incidence of solicited AEs (Day 0-7 post-vaccination) and more Grade 3 solicited AEs 
(none for the continental US study) compared with the US mainland study. No Grade 3 vaccine- 
related unsolicited AEs have been reported in either study. No vaccine-related SAEs in either 
study have been reported. No subjects withdrew from either study due to vaccine-related events. 
No unexpected adverse events or new pIMD were observed in either study. 
For the US mainland study, balanced neutralizing antibody responses (MN assay and microPRN 
assay) were seen at Day 56. GMTs at Day 56 were robust for the AS01E, AS03B (1 μg), and alum 
adjuvanted 4-µg dose groups (Schmidt et al). Data analysis is ongoing for both studies. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
25 
US Government Proprietary 
 
 
Table 3: 
Clinical Studies of TDENV-PIV 
 
Type 
of 
Study 
IND / 
Study 
No. 
Study Design and Type 
of Control 
Test Product(s); 
Dosage Regimen; 
Route of Admin. 
No. of 
Subjects 
Study Population 
Phase 
1 
IND 
15112 
DPIV- 
001; 
S-11-23 
Randomized, placebo- 
controlled, observer- 
blind with 5 parallel 
groups. 11 visits over 13 
months 
Test Products: 
1 µg TDENV-PIV 
with Alum adjuvant 
4 µg TDENV-PIV 
with Alum adjuvant 
1 µg TDENV-PIV 
with AS01E adjuvant 
1 µg TDENV-PIV 
with AS03B adjuvant 
Placebo (saline) 
Regimen: Day 0 and 
Day 28 
Route of 
Administration: 
Intramuscular 
100 total; 
20/group 
Healthy adults in 
Dengue non- 
endemic region 
(mainland US) 
Phase 
1 
IND 
15112 
DPIV- 
002; 
S-12-12 
Randomized, placebo- 
controlled, observer- 
blind with 5 parallel 
groups. 
11 visits over 13 months 
with additional follow 
up through 2 dengue 
seasons 
Test Products: 
1 µg TDENV-PIV 
with Alum adjuvant 
4 µg TDENV-PIV 
with Alum adjuvant 
1 µg TDENV-PIV 
with AS01E adjuvant 
1 µg TDENV-PIV 
with AS03B adjuvant 
Placebo (saline) 
Regimen: Day 0 and 
Day 28 
Route of 
Administration: 
Intramuscular 
100 total; 
20/group 
Healthy Adults in 
Dengue endemic 
area (Puerto Rico) 
 
5.4.3. 
PIV/LAV Prime-boost 
From 2014 to 2015, 80 subjects were enrolled in an initial evaluation of the prime-boost 
vaccination strategy (S-13-10 IND16122). They were separated into 4 vaccine schedule groups 
as below: 
Group 1 (n=20): LAV (Day 0), PIV 4 μg + alum (Day 28) 
Group 2 (n=20): PIV 4 µg + alum (Day 0), LAV (Day 28) 
Group 3 (n=20): LAV (Day 0), PIV 4 μg + alum (Day 180) 
Group 4 (n=20): PIV 4 µg + alum (Day 0), LAV (Day 180) 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
26 
US Government Proprietary 
 
 
The prime-boost regimen was well tolerated in all 4 groups. There were no grade 3 local AEs. 
Grade 3 related, systemic AEs occurred in small numbers. One volunteer in Group 2 reported 
grade 3 headache and one reported grade 3 GI upset. One volunteer in Group 3 reported grade 3 
GI upset. One volunteer in Group 4 reported grade 3 fever and another volunteer in Group 4 
reported grade 3 fatigue, GI upset, headache, myalgia arthralgia. There were no grade 3 systemic 
related AEs in Group 1. The PIV followed by LAV schedule resulted in the highest rate of 
seroconversion in Group 2 with 100% tetravalent seroconversion and 94% tetravalent 
seroconversion in Group 4 at 28 days post-vaccination. Group 4 volunteers maintained very high 
geometric mean antibody titers at the final 6 month post-vaccination time point. Group 2 
volunteers began to show some significant degradation of antibody titers by 6 month post boost. 
 
6. 
TRIAL DESIGN 
6.1. 
Overall Study Design and Rationale 
This study is a Phase 1, randomized, open-label study of the prime-boost vaccine candidates 
given in the prime-boost regimen previously demonstrated to have a high level of 
immunogenicity and immune durability: Day 0 prime (PIV) and Day 180 boost (LAV), and 
compare it with a previously untested schedule: Day 0 prime (PIV) and Day 90 boost (LAV) in 
order to define the potential tradeoff between potential immunogenicity, including cell-mediated 
immunity, and a more practical dosing schedule. 
Heterologous Prime-boost: Compared with inactivated vaccines, live-attenuated vaccines have 
the advantage of inducing high-avidity antibodies and T lymphocyte responses (Rothman-2004). 
Disadvantages include risks to severely immunocompromised recipients and increased 
reactogenicity. Most approved vaccines and previous experiments with dengue vaccine 
candidates have used a homologous boosting strategy (repeated administration of the same 
antigen and carrier). While this is effective for boosting humoral responses, it is relatively 
inefficient for boosting cellular immunity because prior immunity tends to impair robust antigen 
presentation and activation of the immune system. Giving the same antigen via different antigen- 
delivery systems (heterologous prime-boost) can engage multiple parts of the immune system to 
establish a balanced immune response. Although serotype-specific neutralizing antibodies 
remain the most accepted method to assess post-vaccination and post-infection immunity, as the 
dengue community learns about the human response to natural infection and vaccination, the role 
of cell-mediated immunity in dengue infection is emerging (Woodland-2004). 
The rationale behind this study of heterologous prime-boost vaccination is to determine if 
humoral and cell-mediated immunity can be optimized while reducing reactogenicity by 
adjusting the vaccination schedule. By conducting a thorough immunological assessment, a 
heterologous prime-boost dengue vaccination study may also contribute to a better understanding 
of the immunological profile induced by dengue infection and vaccination. The necessity of a 
heterologous prime-boost study is further supported by the absence of a validated correlate of 
protection and recent demonstration of the inability of a live attenuated tetravalent dengue 
vaccine (CYD, Sanofi Pasteur) to protect against a specific serotype of circulating dengue virus, 
despite robust neutralizing antibody response from a specific dengue serotype (Sabchareon- 
2012). The current study will use the re-derived LAV F17 and 4-µg PIV with alum adjuvant 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
27 
US Government Proprietary 
 
 
similar toS-13-10. The safety risks are not expected to exceed those demonstrated previously in 
S-13-10. 
 
6.2. 
Trial Objectives and Purpose 
6.2.1. 
Primary Objective 
• To further evaluate the safety and reactogenicity of 2 tetravalent dengue vaccine 
(TDENV) candidates administered in a heterologous prime boost fashion with PIV 
followed by LAV 180 days later 
• To evaluate the safety and reactogenicity of a previously untested vaccination schedule 
consisting of PIV followed by LAV 90 days later 
 
6.2.2. 
Secondary Objective 
• Evaluate the humoral and cell-mediated immunogenicity of the PIV/LAV Day 0, 180 
administration schedule and compare it to the humoral and cell-mediated immunogenicity 
of the PIV/LAV Day 0, 90 schedule 
 
6.2.3. 
Exploratory Objectives 
• To evaluate the rate of decay and durability of humoral immunity in the Day 0, 90 
PIV/LAV prime-boost vaccination schedule and the Day 0,180 PIV/LAV prime-boost 
vaccination schedule 
• To characterize the cell-mediated immune response of the Day 0, 90 PIV/LAV prime- 
boost vaccination schedule compared to the 0,180 Day PIV/LAV prime-boost 
vaccination schedule 
 
6.3. 
Study Endpoints 
6.3.1. 
Primary Endpoints 
The primary safety and reactogenicity endpoints are as follows: 
• Solicited adverse events during the 7 days follow up period after each vaccination 
• Solicited adverse events during the 28 day follow up period after each vaccination 
• Unsolicited adverse events during the 28 days following each vaccination 
• Hematological and biochemistry abnormalities on day of vaccination and at 7 days and 
28 days post each vaccination 
• Occurrence of serious adverse events (SAEs) during the entire study 
• Occurrence of pIMDs during the entire study 
• Occurrence of medically attended AEs during the study 
6.3.2. 
Secondary Endpoints 
• Microneutralizing (MN) and PRNT dengue antibody titers before vaccination and 
28 days after each vaccination for each volunteer. Additional antibody levels will be 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
28 
US Government Proprietary 
 
 
assessed at 90, 180, 270, and 360 days following booster vaccination vaccine 
administration. 
− GMTs of neutralizing antibodies to each DENV serotype 
− Seropositivity rates for each DENV type 
− Trivalent and tetravalent seroconversion rates(Defined by Ab titers >1:10 to 3 
dengue virus types or 4 dengue virus types respectively) 
 
6.3.3. 
Exploratory Endpoints 
• CMI markers assessment by, but not limited to, the following methods: 
− Enzyme-linked immunospot (ELISPOT), flow cytometry, carboxyfluorescein 
succinimidyl ester, dye-dilution proliferation, Luminex, transcriptomics, 
proteomics, glycomics 
• Decline in GMT neutralizing antibody titers from Day 28 to Day 360 following 
booster vaccinations 
• Vaccine induced viral replication measured by DENV serotype specific RT-PCR 
Day 6-14(5 sequential days will be tested when possible. Only 5 tests per patient will 
be obtained) after LAV vaccination 
 
6.4. 
Study Site 
This study will be conducted at the WRAIR Clinical Trials Center (CTC) (Silver Spring, MD, 
USA). 
 
6.5. 
Study Population 
This study will enroll healthy male and healthy non-pregnant, non-breastfeeding female dengue 
naïve subjects between the ages of 18 and 42 (inclusive) at the time of screening. 
The subjects will be residents of the Washington, DC, metropolitan area. Dengue antibody 
screening will be conducted. It is expected that the majority of volunteers will be flavivirus- 
naïve. 
A maximum of 40 subjects will be enrolled. Refer to Section 12.2 for a statistical justification of 
the sample size. 
 
6.6. 
Duration of Subject Participation 
Each subject will participate for approximately 18 months in Group 1 or 15 months in Group 2 
(360 days following final vaccine receipt). 
 
6.7. 
Measures Taken to Minimize/Avoid Bias 
6.7.1. 
Assignment 
Subjects will be assigned as listed in Table 4. Subjects will be randomized to the 2 groups in a 
1:1 distribution. Block randomization will be used to allocate subjects to each study cohort. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
29 
US Government Proprietary 
 
 
Eligible subjects will be randomized before vaccination on visit 1. Each volunteer will be 
assigned a subject number at initial screening starting with 0001 based on time of enrollment. 
The subject number may be different from volunteer screening number. 
 
6.7.2. 
Open-label Study 
This will be an open-label study. 
 
6.8. 
Route of Administration, Dosage Regimen, Treatment Period, and 
Justification 
PIV vaccinations will be administered intramuscularly (IM) in the deltoid region of the non- 
dominant arm whenever possible and LAV F17 vaccinations will be administered 
subcutaneously (SQ) in the upper-outer triceps/deltoid area of the non-dominant arm when 
possible. 
Previous trials with the LAV vaccine used a Day 0 and Day 180 vaccination strategy. The Day 
180 homologous boost is widely used in vaccination of live-attenuated vaccines for dengue 
(Sanofi’s CYD vaccine) and other pathogens. The gap between priming and boosting for 
inactivated vaccines is typically shorter. Previous clinical trials of the PIV candidates have used 
the Day 0 and Day 28 vaccination schedule. 
Vaccination will be given according to the regimen shown in Table 4. 
Table 4: 
Vaccination Schedule 
 
Group 
No. of 
Subjects 
Day 0 
Day 90 
Day 180 
1 
20 
PIV 
- 
LAV F17 
2 
20 
PIV 
LAV F17 
- 
 
6.9. 
Investigational Product Accountability 
The sponsor’s representative is responsible for distributing the investigational product to the 
study site and has delegated this responsibility to the WRAIR CTC; however, the sponsor’s 
representative has ultimate responsibility for product accountability. After the investigational 
product is distributed, the Principal Investigator is responsible for and will maintain logs of 
investigational product receipt, storage, reconstitution, accountability by subject, and 
investigational product remaining before final disposition. Product accountability logs will be 
maintained by the CTC. The Principal Investigator (PI) may delegate, in writing, this 
responsibility to another individual, but the PI is ultimately responsible for the investigational 
product and its proper storage upon receipt at the study site until it is transferred back to the 
sponsor’s representative or designee or is destroyed, as directed by the sponsor’s representative. 
Residual vaccine will be retained separately in the CTC under controlled conditions until the end 
of the study or until the study monitor can perform inspections of residual vaccine during 
periodic monitoring visits to confirm correct dosing. Once either of the above have been met, the 
sponsor will be contacted to determine if the vials may be destroyed. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
30 
US Government Proprietary 
 
 
Once product accountability is completed by the study monitor, and with approval by the 
sponsor, the residual vials may be sent back to the PBF for destruction or disposed of in a rigid 
biohazard container at the CTC. Damaged vaccine will be stored and held until the study monitor 
completes product accountability. After reconciliation is complete and a transfer memorandum 
has been received from the sponsor, damaged vials will be returned to the PBF Known and 
Potential Risks and Benefits to Human Subjects 
 
6.9.1. 
Risks/Discomfort to Subjects and Precautions to Minimize Risk 
Outlined below are anticipated and unexpected adverse reactions, and a brief description of 
procedures to ameliorate risks and symptoms. All known risks and precautions described here 
are explained in detail in the informed consent. 
 
6.9.1.1. 
Local Reactions 
Local reactions may include pain, redness, and swelling at the injection site. Study staff 
including physicians will be available at follow up visits and via phone in between visits to 
provide advice on any appropriate treatment of any reactions that may occur. 
 
6.9.1.2. 
Systemic Reactions 
General reactions may include fatigue, fever, rash, pruritus, headache with potential for retr- 
orbital pain, photophobia, conjunctival injection, gastrointestinal symptoms (nausea, vomiting, 
loss of appetite diarrhea, and abdominal pain), muscle ache, joint pain, cough, transient mild to 
moderate leukopenia and transient mild to moderate elevation in serum alanine aminotransferase 
and aspartate aminotransferase. Study staff including physicians will be available at follow up 
visits and via phone in between visits to provide advice on any appropriate treatment of any 
reactions that may occur. 
 
6.9.1.3. 
LAV and DPIV SAE and Grade 3 AEs 
There have been no SAEs related to these vaccines in prior clinical studies. The most common 
AEs following vaccination have included headaches, myalgia, pruritus, fever, nausea, 
lymphadenopathy, rash, and arthralgia. Grade 3 symptoms in US adults following subcutaneous 
(SC) administration included Redness at injection site (4.5%), swelling(2.3%), arthralgia(2.3%), 
fatigue(2.3%), headache(2.3%), muscle aches(2.3%) (Thomas et al-2013). 
Grade 3 local AEs in Thai children included redness following dose 1 and swelling following 
dose 2 each in 1 of 7 children vaccinated. There were no systemic grade 3 symptoms reported in 
this group (Simasathien et al-2008). 
Among 40 Thai adults who received F17 LAV there were no grade 3 local AEs. Reported 
systemic grade 3 AEs included fatigue (2.5%), fever (2.5%), headache (2.5%), nausea (2.5%), 
pruritus (2.5%) and rash (5%) (Watanaveeradej et al -2014). 
Among 636 Puerto Rican subjects including children, the most frequently reported grade 3 local 
symptom was redness, reported after 2.4% of F17 LAV doses and 1.9% of placebo doses 
respectively. Grade 3 swelling was reported after 1.4% of F17 doses and after 0.5% of placebo 
doses. Grade 3 pain was reported by one subject in the F17 group after dose 2 (0.2% overall per 
dose) and was not reported in the placebo group. Overall, the most frequently reported grade 3 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
31 
US Government Proprietary 
 
 
general symptom was fever (4.8% F17 LAV vs. 3.8% placebo group) and Grade 3 headache 
(1.0% F17 LAV vs. 2.9% placebo group). Other solicited grade 3 general symptoms were 
reported with a very low incidence (<0.2% of doses) (Bauer et al-2015). 
Grade 3 related adverse events with dengue inactivated vaccine have been only rarely reported. 
In DPIV-001 there was one grade 3 related fever among all volunteers receiving 2 doses and one 
report of grade 3 myalgias among one volunteer receiving 3 doses. Among Puerto Rican 
recipients in the DPIV-002 trail, grade 3 solicited general AEs ranged between 0% and 2.5% for 
most AEs. Grade 3 headache was reported following 5% of AS03-adjuvanted vaccine doses, and 
grade 3 myalgia was reported following 10% of AS01-adjuvanted vaccine doses. No vaccine- 
related SAEs have been reported in either study. 
Among 80 volunteers previously vaccinated using the prime-boost strategy there were 11 grade 3 
adverse events. There were only 5 individuals who experienced grade 3 related adverse events. 
Two of these were in the 0, 28 Day PIV/LAV groups and included one grade 3 headache and one 
grade 3 GI upset. In the 0, 180 Day PIV/LAV there was one individual who experienced grade 3 
fever and another individual who experienced grade 3 fatigue, headache, myalgia, arthralgia and 
GI upset. One individual in the 0, 180 Day LAV/PIV group experienced grade 3 GI upset. 
Overall, fatigue, fever, headache, muscle aches were commonly reported among the PIV/LAV 
group occurring in 20-50% of volunteers vaccinated with this schedule. There were no SAEs and 
no pIMDs in this prime-boost (WRAIR 2136) trial (Lin et al. unpublished data). 
Therefore the vaccine reactions that have been observed are sufficiently mild to warrant 
continued investigation of vaccines given they have potential to protect against the severe 
morbidity and mortality caused by dengue infections. Study staff including physicians will be 
available at follow up visits and via phone in between visits to provide advice on any appropriate 
treatment of any reactions that may occur in order to minimize the reactions for participating 
subjects. 
 
6.9.1.4. 
Pregnancy 
Risks to fetal development are unknown at this time. Pregnant females will be excluded from this 
study. Study subjects should not become pregnant for at least 3 months after the last study 
vaccination. The following methods of contraception are considered adequate for female subjects 
of childbearing potential: abstinence; oral contraceptive, either estrogen progesterone combined, 
or progesterone alone; injectable progesterone; implants of levonorgestrel; estrogenic vaginal 
ring; contraceptive patches; intrauterine device or intrauterine system; double-barrier method 
[condom and occlusive cap (diaphragm or cervical vault caps) with or without spermicidal agent 
(foam, gel, film, cream, suppository)]; male partner sterilization prior to the female subject’s 
entry into the study, and this male is the sole partner for that subject. 
 
6.9.1.5. 
Lactation 
Risks to nursing infants are unknown at this time. Females who are currently breastfeeding their 
infant/child and plan to continue to do so during the study period will be excluded from this 
study. Lactating females must agree not to breast feed while in the study and for 3 months after 
the last study vaccination in order to be included. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
32 
US Government Proprietary 
 
 
6.9.1.6. 
Venipuncture 
Blood sampling carries a minimal risk of minor discomfort and the possibility of minor bruising 
at the site of the needle puncture and, rarely, the possibility of infection and/or nerve damage at 
the needle puncture site. All phlebotomists drawing blood in conjunction with this trial will be 
certified and experienced in proper phlebotomy techniques in order to minimize these risks. 
 
6.9.1.7. 
Allergic Reactions and Immune Mediated Reactions 
As with any Investigational New Drug (IND) product administration and no matter what 
precautions are taken, there is always the risk of a serious, or even life-threatening, allergic 
reaction. Medical emergency equipment is located at the study site. This is available to handle 
emergencies, such as anaphylaxis, angioedema, bronchospasm, and laryngospasm. There also 
exists a theoretical chance of immune mediated events including Guillain-Barre syndrome. 
However, neither the PIV nor LAV used in this study has ever been associated with Guillain- 
Barre syndrome or any other immune mediated adverse event. 
 
6.9.1.8. 
Bovine Spongiform Encephalopathy 
Bovine (cow)-derived materials were used in the manufacture of the PIV and LAV vaccines in 
this research study. Certified, fetal bovine serum used in the manufacture of the vaccines was 
purchased from a company in New Zealand where herds are free from bovine spongiform 
encephalopathy (BSE) disease. As a result, the risk of contamination of the vaccines with BSE 
has been minimized. 
 
6.9.1.9. 
Natural Dengue Virus Infection 
Observations from natural dengue infections have shown that the greatest risk for severe dengue 
infection comes when a person is infected by one dengue type and then infected again, at a later 
time, by a different dengue type. One proposed pathophysiologic mechanism underlying severe 
dengue disease onset is antibody-dependent enhancement (ADE) whereby pre-existing 
antibodies from a previous heterotypic dengue serotype fail to protect against infection with a 
different serotype but rather cause enhancement of infection. It is possible, if a vaccine candidate 
does not protect against at least 3 dengue types after vaccination, a more severe case of dengue 
may develop if the subject becomes infected in the future, compared with if the subject had never 
been vaccinated at all. This risk is theoretical based on what we know about natural infections. In 
the Sanofi-Pasteur vaccine trials there was some signal towards increased rates of hospitalization 
in the <5 year old vaccinated subjects at Asian sites (Halstead 2016). However, other than this 
signal in a small segment of the vaccinated cohort, there is no clear evidence that dengue 
vaccines (monovalent or tetravalent) place people at increased risk for severe dengue. It is 
unclear if this potential increased risk is specific to Dengvaxia or whether other dengue vaccines 
will have similar effects. In either case, volunteers in this trial are not expected to be exposed to 
natural dengue virus infection since vaccination will be occurring in a non-endemic setting 
(USA). Cases of vaccine-induced viremia after vaccination with LAV appeared to be infrequent 
and were not associated with clinical symptoms or laboratory abnormalities (Thomas-2013, 
TDEN-002, 003 study reports). Among subjects residing in dengue endemic areas who have 
been vaccinated with LAV, no increased risk of severe dengue or dengue hemorrhagic fever 
(DHF) has been identified. This study will be conducted in a dengue non-endemic region, further 
reducing any theoretical risk. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
33 
US Government Proprietary 
 
 
6.9.1.10. Unknown Risks 
As with all research there is the remote possibility of risks that are unknown or that cannot be 
foreseen at this time. Each vaccine candidate in this study has been safely administered to human 
subjects. Any unknown risks identified in this study will be reported to the sponsor and research 
monitor. 
 
6.9.2. 
Alternatives to this IND Product or Study 
An alternative is to not participate in this study. 
 
6.9.3. 
Intended Benefit for Subjects 
There is no intended direct medical benefit for subjects participating in this study. Subjects may 
be protected against future dengue infections; however, this cannot be assured. 
 
6.9.4. 
Risks to the Study Personnel and the Environment 
The principal risks in the clinical setting include handling of needles that may be contaminated 
and the attendant risks including infection with hepatitis, human immunodeficiency virus (HIV), 
and other human pathogens. Adherence to universal precautions will reduce significantly the risk 
of exposure. 
All biohazardous waste will be disposed of as stipulated by local, state, and federal regulations. 
 
7. 
SELECTION OF SUBJECTS 
It is estimated that 80 to 120 volunteers will need to be screened in order to enroll the target of 
40 volunteers meeting all inclusion and exclusion criteria. Subject Inclusion Criteria 
Subjects must meet all of the following criteria to be included in the study: 
1. Male or female between 18 and 42 years of age (inclusive) at the time of consent 
2. Able to provide written informed consent 
3. Healthy as established by medical history and clinical examination and basic hematologic 
laboratory analysis before entering into the study 
4. Able and willing to comply with the requirements of the protocol (eg, document events in 
memory aid, return for follow-up visits, etc.) 
5. Dengue exposure naïve as established by pre-enrollment dengue antibody testing and 
questioning of volunteer 
6. Female subject of non-childbearing potential (non-childbearing potential is defined as 
having either a current bilateral tubal ligation at least 3 months prior to enrollment or a 
history of an hysterectomy, bilateral oophorectomy, or is post-menopause(12 months or 
more since last menstrual period)) or Female subject of childbearing potential may be 
enrolled in the study, if the subject has: 
− Practiced adequate contraception for 30 days prior to vaccinations, and 
− A negative urine pregnancy test on each day of vaccination, and 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
34 
US Government Proprietary 
 
 
− Agreed to continue adequate contraception through at least 3 months following 
last vaccination. 
 
7.1. 
Subject Exclusion Criteria 
Subjects meeting any of the following criteria will be excluded from the study: 
1. Use of any investigational or non-registered product (drug or vaccine or device) other 
than the study vaccines during the period starting 30 days preceding the first dose of 
study vaccine and/or planned use during the study period 
2. Chronic administration (defined as more than 14 days in total) of prescription 
immunosuppressants or other prescription immune-modifying drugs during the period 
starting 180 days prior to the first vaccine dose 
− For corticosteroids, this will mean prednisone ≥ 20 mg/d or equivalent 
− Inhaled and topical steroids are allowed 
3. History of or active use of cancer chemotherapy or radiation therapy for the treatment of 
cancer 
4. Receipt or planned receipt of a vaccine/product outside the study protocol within 30 days 
of each scheduled dose of an investigational product 
5. Planned administration of any flavivirus vaccine, to include licensed vaccines for Yellow 
Fever or Japanese Encephalitis Virus as well as other investigational vaccines for dengue, 
Zika, West Nile, other flavivirus, for the entire study duration 
6. Previous receipt of a foreign or investigational dengue vaccine 
7. Concurrently participating in another clinical study, at any time during the study period, 
in which the subject has been or will be exposed to an investigational or a non- 
investigational product (pharmaceutical product or device) 
8. Any confirmed or suspected immunosuppressive or immunodeficient condition, based on 
medical history and physical examination 
9. History of, or current, auto-immune disease 
10. History of any reaction or hypersensitivity likely to be triggered by any component of the 
vaccines or related to a study procedure (This includes hypersensitivity reactions to alum, 
streptomycin, neomycin, or any other flavivirus vaccine, such as Yellow Fever virus and 
Japanese Encephalitis virus vaccines) 
11. Major congenital defects or serious chronic illness 
12. History of any chronic neurological disorders or chronic and/or uncontrolled seizures 
13. Acute infectious disease and/or fever (oral body temperature ≥ 100.4°F/38.0°C) at the 
time of enrollment (a subject with a minor illness, ie, mild diarrhea, mild upper 
respiratory infection, etc, without fever, may be enrolled at the discretion of the 
investigator) 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
35 
US Government Proprietary 
 
 
14. Acute or chronic, clinically significant pulmonary, cardiovascular, hepatic, or renal 
functional abnormality, as determined by history, physical examination or laboratory 
screening tests 
15. Receipt of immunoglobulins or any blood products during the period starting 90 days 
preceding the first dose of study vaccine or planned receipt during the study period 
16. Donated blood within 8 weeks before first scheduled investigational vaccine receipt or 
planned donation of blood products throughout the study period 
17. History of chronic alcohol abuse and/or drug abuse that, in the opinion of the 
investigator, could result in poor compliance with study requirements. 
18. Pregnant or breastfeeding female or female planning to become pregnant or planning to 
discontinue contraceptive precautions 
19. A planned move to a location that will prohibit compliance with the requirements of the 
trial 
20. Subject seropositive for hepatitis B surface antigen (HBsAg), hepatitis C virus antibodies 
(anti-HCV), or human immunodeficiency virus antibodies (anti-HIV) 
21. Safety laboratory test results that are outside the acceptable values at screening: 
− > 110% upper limit of normal (ULN) for alanine aminotransferase (ALT), 
aspartate aminotransferase (AST), alkaline phosphatase, creatinine, serum urea 
nitrogen (SUN) and bilirubin (total and direct) 
− < 90% lower limit of normal (LLN) or > 120% ULN for hemoglobin, hematocrit 
and platelet count 
− < 75% LLN or >110% ULN for total white blood cell count (WBC) 
Note: Per guidance in Section 8.1, abnormal lab(s) may be repeated x1 in the 
screening window provided the total amount of blood drawn for all screening labs 
does not exceed 50 mL. 
22. Active Diabetes or active peptic ulcer disease (PUD) 
23. Diagnosis with Bipolar Disorder or Schizophrenia, hospitalization in the past year for a 
mental health disorder, or any other psychiatric condition, which in the opinion of the 
investigator prevents the subject from meeting all requirements of the study 
24. Chronic migraine headaches, defined as more than 15 headache days per month over a 
3-month period of which more than 8 are migrainous, in the absence of medication over 
use 
25. Chronic medical condition that, in the opinion of the investigator, impacts subject safety 
26. Any other condition which, in the opinion of the investigator, prevents the subject from 
meeting all requirements of the study. 
27. Do not wish to have their blood stored and used for future research 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
36 
US Government Proprietary 
 
 
8. 
STUDY PROCEDURES 
Detailed study procedures are provided in Table 5 and Table 6. 
 
8.1. 
Recruitment of Subjects 
Subjects will be recruited by the WRAIR CTC through the use of newspaper ads, flyers, and 
posters along with ads on craigslist, facebook or other social media websites. Web-based 
advertising (clinicaltrials.org), word of mouth, and the CTC database will be used to contact 
clinical trial volunteers. Potential volunteers of various ethnic backgrounds, education, and 
income will be residents of the Washington, DC, metropolitan area. An institutional review 
board (IRB)-approved recruitment script (Appendix C) will be read during all phone and face-to- 
face contacts with potential study subjects. All recruitment materials will be submitted to the 
WRAIR IRB for review and approval prior to use. CTC study subjects may also recruit new 
subjects. Recruitment will be by non-coercive means. 
Informed consent will be obtained prior to any study procedures. Prior to agreeing to participate 
in the study, volunteers will participate in an information session about the study. The 
investigator or designee will explain the study, outline participation requirements, review the 
consent form in detail with volunteers, and then answer any questions. Volunteers will then be 
afforded ample time to read the informed consent form and ask questions. If a volunteer decides 
to participate he/she will sign and date the informed consent document. 
The recruitment process will in general be similar for both military and civilian personnel. 
However, special effort will be made to ensure that military personnel are not unduly influenced 
or coerced into participating by individuals of superior rank or position. All contact recruiting 
methods (eg, oral presentations and correspondence) for military personnel will be performed 
either by non-military personnel or military personnel who are being observed by an independent 
ombudsman during the contact to ensure that no undue influence occurs. During oral 
presentations to groups of military personnel, this ombudsman will also act to ensure that the 
briefed population is not influenced or coerced by NCOs or Officers not associated with the 
study. Acceptable ombudsmen for this protocol include Michael Hrycyk and Joel Gonzales. 
A signed copy of the informed consent will be provided to the volunteer before any study 
procedure is performed. 
Investigators will also provide the opportunity to participate in the study to military personnel at 
the WRAIR and NMRC using the same IRB-approved recruitment script. The research monitor 
of the study will be present at the time of the study introduction and will serve as an ombudsman, 
except when addressing soldiers from the monitor’s own branch. Additionally, no senior non- 
commissioned officers (NCOs) or officers from any junior enlisted soldier’s chain of command 
will be present during the recruitment brief of junior enlisted soldiers to ensure that no undue 
pressure is exerted upon them. 
The following procedures will be conducted no more than 90 days and no fewer than 4 days 
prior to study start for an individual subject. The results of these assessments will determine 
subject eligibility. 
• Informed consent 
• Medical history 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
37 
US Government Proprietary 
 
 
• Inclusion and exclusion criteria 
• Height and weight 
• Demographic information 
• Urine pregnancy test 
• Physical exam and vital signs 
• Concomitant or recent medications/ vaccination 
• Blood sampling for safety laboratory analyses (30 mL) 
If the investigator feels that there may be a reason that the screening tests are abnormal due to 
laboratory error or normal physiological variance, or a temporary self-limited condition any or 
all of the screening laboratory tests may be repeated once as necessary to allow an accurate 
determination of a potential subject’s eligibility. Individuals who have minor abnormalities that 
are deemed not clinically significant by the physician investigator and do not meet exclusion 
criteria otherwise defined, may be enrolled in the study. 
Subjects found to be seropositive for HIV-1 or Hepatitis B (HBV) or C (HCV) will be counseled 
and referred to the regular health care provider or public health clinic for further evaluation, and 
according to the Code of Maryland Regulations (COMAR) 10.06.01.03 C. Positive HIV, HBV 
and HCV results will be reported by the study team to the Maryland AIDS Administration, 
Department of Health and Mental Hygiene within 24 hours of being confirmed as per Health 
General Article 18-205. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
37 
US Government Proprietary 
 
 
Table 5: 
Study Events Schedule for Group 1: Day 0 – PIV, Day 180 – LAV 
 
Visit 
Screen 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
Optimal Visit Day 
Day 
-90 to 
-4 
Day 0 
PIV 
Primee 
Day 
7 
Day 
28 
Day 
180 
LAV 
Booste 
Day 
186 a 
Day 
187 a 
Day 
188 a 
Day 
189 a 
Day 
190 a 
Day 
208 
Day 
270 
Day 
360 
Day 
450 
Day 
540 
Allowed Interval 
for Visits (day) 
N/A 
±2 
±5 
±7 
6-10 
days 
from 
visit 4 
7-11 
days 
from 
visit 4 
8-12 
days 
from 
visit 4 
9-13 
days 
from 
visit 4 
10-14 
days 
from 
visit 4 
26-42 
days 
from 
visit 4 
70- 
110 
days 
from 
visit 4 
160- 
200 
days 
from 
visit 4 
250- 
290 
days 
from 
visit 
4 
340- 
380 
days 
from 
visit 
4 
General Procedures 
Informed consent 
• 
Randomization 
• 
Medical history 
• 
• b 
Confirm inclusion/ 
exclusion criteria 
• 
• 
• 
Check 
contraindications to 
subsequent 
vaccination 
• 
Measure/record 
height and weight; 
collect demographics 
• 
Urine pregnancy test, 
as applicable 
• 
• 
• 
Allocate 
experimental 
vaccination number 
• 
• 
Receive verbal 
consent to vaccinate 
• 
• 
Vaccination 
• 
• 
Distribution of 
subject card 
• 
Distribution of 
memory aid 
• 
• 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
38 
US Government Proprietary 
 
 
 
Visit 
Screen 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
Optimal Visit Day 
Day 
-90 to 
-4 
Day 0 
PIV 
Primee 
Day 
7 
Day 
28 
Day 
180 
LAV 
Booste 
Day 
186 a 
Day 
187 a 
Day 
188 a 
Day 
189 a 
Day 
190 a 
Day 
208 
Day 
270 
Day 
360 
Day 
450 
Day 
540 
Allowed Interval 
for Visits (day) 
N/A 
±2 
±5 
±7 
6-10 
days 
from 
visit 4 
7-11 
days 
from 
visit 4 
8-12 
days 
from 
visit 4 
9-13 
days 
from 
visit 4 
10-14 
days 
from 
visit 4 
26-42 
days 
from 
visit 4 
70- 
110 
days 
from 
visit 4 
160- 
200 
days 
from 
visit 4 
250- 
290 
days 
from 
visit 
4 
340- 
380 
days 
from 
visit 
4 
Return of memory 
aid 
• 
• 
Review memory aid 
• 
• 
• 
• 
• 
• 
• 
• 
Record concomitant 
medications/ 
vaccinations 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
Record intercurrent 
medical conditions 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
Safety Assessments 
Physical examination 
and vital signsf 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
Prevaccination body 
temperature 
• 
• 
Blood sample for 
safety laboratory 
analyses (mL)c 
30 
10 
10 
10 
10 
10 
10 
Blood sample for 
viral replication 
assay (mL) 
4 
4 
4 
4 
4 
Document local and 
general solicited 
symptoms (Days 0- 
28 postvaccination) 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
Document 
unsolicited AEs 
(Days 0-28 
postvaccination) 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
39 
US Government Proprietary 
 
 
 
Visit 
Screen 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
Optimal Visit Day 
Day 
-90 to 
-4 
Day 0 
PIV 
Primee 
Day 
7 
Day 
28 
Day 
180 
LAV 
Booste 
Day 
186 a 
Day 
187 a 
Day 
188 a 
Day 
189 a 
Day 
190 a 
Day 
208 
Day 
270 
Day 
360 
Day 
450 
Day 
540 
Allowed Interval 
for Visits (day) 
N/A 
±2 
±5 
±7 
6-10 
days 
from 
visit 4 
7-11 
days 
from 
visit 4 
8-12 
days 
from 
visit 4 
9-13 
days 
from 
visit 4 
10-14 
days 
from 
visit 4 
26-42 
days 
from 
visit 4 
70- 
110 
days 
from 
visit 4 
160- 
200 
days 
from 
visit 4 
250- 
290 
days 
from 
visit 
4 
340- 
380 
days 
from 
visit 
4 
Record SAEs 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
Record pregnancy 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
Record pIMDs 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
Immunogenicity Assessments 
Blood sample for 
neutralizing 
antibodies d 
10 
10 
10 
10 
10 
10 
10 
10 
10 
Blood sample for 
CMI assays (mL) 
(section 10.2) 
56 
16 
40 
40 
16 
40 
40 
40 
40 
40 
Daily Blood Volume 
(mL) 
40 
76 
26 
60 
70 
4 
30 
4 
4 
4 
60 
50 
50 
50 
50 
Cumulative Blood 
Volume (mL) 
40 
116 
142 
202 
272 
276 
306 
310 
314 
318 
378 
428 
478 
528 
578 
a 5 consecutive days preferred but weekends will not be included. Goal is as close as possible to get 5 consecutive draws between 6-14 days after vaccination. 
Visits 5-9 are intended to occur 6-14 days after the administration of LAV boost. The day numbers listed for visit day in the table above for visits after second 
vaccination only apply if vaccination occurs exactly on day 180. If the LAV boost is shifted back or forward as allowed per the protocol, visits 5-14 may be 
shifted along with the corresponding windows by the same number of days and still remain in line with the planned activities of this protocol. 
b Record changes between screening and Visit 1 in the eCRF. 
c Safety assessments include complete blood count [white blood cell count (WBC), hemoglobin (Hgb), platelet count, differential, and a calculated absolute 
neutrophil count (ANC)], aspartate aminotransferase (AST) and alanine aminotransferase (ALT), serum urea nitrogen (SUN), creatinine, alkaline phosphatase, 
total and direct bilirubin. The maximum amount of blood collected for safety assessments will be 30 mL at screening and 15 mL at other study visits. Day 0 
safety laboratory tests will not be used to determine eligibility. 
d Initial blood neutralizing antibody testing will include flaviviruses: dengue, zika, yellow fever, west nile virus, Japanese encephalitis virus. Only positive results 
for dengue will be used as exclusionary criteria. The others will only help to define previous exposures and potential differences in immune response to 
vaccination 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
40 
US Government Proprietary 
 
 
e Vital signs on vaccination days will be taken prior to dosing and at least 30 minutes post dose, to include heart rate, blood pressure, temperature, and respiratory 
rate. 
f At time of screening a complete physical exam will be performed. At subsequent visits a focused physical exam will be performed based upon any subject 
reported symptoms. If no symptoms are reported at follow up visits no additional physical exam may be performed if not indicated based on investigator 
discretion. 
Table 6: 
Study Events Schedule for Group 2: Day 0 – PIV, Day 90 – LAV 
 
Type of Contact 
Screen 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
Optimal Visit Day 
Day 
-90 to 
-4 
Day 0 
PIV 
Primee 
Day 
7 
Day 
28 
Day 
90 
LAV 
Booste 
Day 
96 a 
Day 
97 a 
Day 
98 a 
Day 
99 a 
Day 
100 a 
Day 
118 
Day 
180 
Day 
270 
Day 
360 
Day 
450 
Allowed Interval for 
Visits (day) 
N/A 
±2 
±5 
±7 
6-10 
days 
from 
visit 4 
7-11 
days 
from 
visit 4 
8-12 
days 
from 
visit 4 
9-13 
days 
from 
visit 4 
10-14 
days 
from 
visit 4 
26-42 
days 
from 
visit 4 
70- 
110 
days 
from 
visit 4 
160- 
200 
days 
from 
visit 4 
250- 
290 
days 
from 
visit 4 
340- 
380 
days 
from 
visit 4 
General Procedures 
Informed consent 
• 
Randomization 
• 
Medical history 
• 
•b 
Confirm 
inclusion/exclusion 
criteria 
• 
• 
• 
Check 
contraindications to 
subsequent 
vaccination 
• 
Measure/record height 
and weight; collect 
demographics 
• 
Urine pregnancy test, 
as applicable 
• 
• 
• 
Allocate experimental 
vaccination number 
• 
• 
Vaccination 
• 
• 
Distribution of subject 
card 
• 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
41 
US Government Proprietary 
 
 
 
Type of Contact 
Screen 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
Optimal Visit Day 
Day 
-90 to 
-4 
Day 0 
PIV 
Primee 
Day 
7 
Day 
28 
Day 
90 
LAV 
Booste 
Day 
96 a 
Day 
97 a 
Day 
98 a 
Day 
99 a 
Day 
100 a 
Day 
118 
Day 
180 
Day 
270 
Day 
360 
Day 
450 
Allowed Interval for 
Visits (day) 
N/A 
±2 
±5 
±7 
6-10 
days 
from 
visit 4 
7-11 
days 
from 
visit 4 
8-12 
days 
from 
visit 4 
9-13 
days 
from 
visit 4 
10-14 
days 
from 
visit 4 
26-42 
days 
from 
visit 4 
70- 
110 
days 
from 
visit 4 
160- 
200 
days 
from 
visit 4 
250- 
290 
days 
from 
visit 4 
340- 
380 
days 
from 
visit 4 
Distribution of 
memory aid 
• 
• 
Return of memory aid 
• 
• 
Review memory aid 
• 
• 
• 
• 
• 
• 
• 
• 
Record concomitant 
medications/ 
vaccinations 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
Record intercurrent 
medical conditions 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
Safety Assessments 
Physical examination 
and vital signsf 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
Prevaccination body 
temperature 
• 
• 
Blood sample for 
safety laboratory 
analyses (mL)d 
30 
10 
10 
10 
10 
10 
10 
Blood sample for viral 
replication assay (mL) 
4 
4 
4 
4 
4 
Document local and 
general solicited 
symptoms (Days 0-28 
postvaccination) 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
Document unsolicited 
AEs (Days 0-28 
postvaccination) 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
Record SAEs 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
Record pregnancy 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
42 
US Government Proprietary 
 
 
 
Type of Contact 
Screen 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
Optimal Visit Day 
Day 
-90 to 
-4 
Day 0 
PIV 
Primee 
Day 
7 
Day 
28 
Day 
90 
LAV 
Booste 
Day 
96 a 
Day 
97 a 
Day 
98 a 
Day 
99 a 
Day 
100 a 
Day 
118 
Day 
180 
Day 
270 
Day 
360 
Day 
450 
Allowed Interval for 
Visits (day) 
N/A 
±2 
±5 
±7 
6-10 
days 
from 
visit 4 
7-11 
days 
from 
visit 4 
8-12 
days 
from 
visit 4 
9-13 
days 
from 
visit 4 
10-14 
days 
from 
visit 4 
26-42 
days 
from 
visit 4 
70- 
110 
days 
from 
visit 4 
160- 
200 
days 
from 
visit 4 
250- 
290 
days 
from 
visit 4 
340- 
380 
days 
from 
visit 4 
Record pIMDs 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
Immunogenicity Assessments 
Blood sample for 
neutralizing antibodies 
d 
10 
10 
10 
10 
10 
10 
10 
10 
10 
Blood sample for CMI 
assays (mL) (section 
10.2) 
56 
16 
40 
40 
16 
40 
40 
40 
40 
40 
Daily Blood Volume 
(mL) 
40 
76 
26 
60 
70 
4 
30 
4 
4 
4 
60 
50 
50 
50 
50 
Cumulative Blood 
Volume (mL) 
40 
116 
142 
202 
272 
276 
306 
310 
314 
318 
378 
428 
478 
528 
578 
a 5 consecutive days preferred but weekends will not be included. Goal is as close as possible to get 5 consecutive draws between 6-14 days after vaccination. 
Visits 5-9 are intended to occur 6-14 days after the administration of LAV boost. The day numbers listed for visit day in the table above for visits after second 
vaccination only apply if vaccination occurs exactly on day 90. If the LAV boost is shifted back or forward as allowed per the protocol, visits 5-14 may be 
shifted along with the corresponding windows by the same number of days and still remain in line with the planned activities of this protocol. 
b Record changes between screening and Visit 1 in the eCRF. 
c Safety assessments include complete blood count [white blood cell count (WBC), hemoglobin (Hgb), platelet count, differential, and a calculated absolute 
neutrophil count (ANC)], aspartate aminotransferase (AST) and alanine aminotransferase (ALT), serum urea nitrogen (SUN), creatinine, alkaline phosphatase, 
total and direct bilirubin. The maximum amount of blood collected for safety assessments will be 30 mL at screening and 15 mL at other study visits. Day 0 
safety laboratory tests will not be used to determine eligibility. 
d Initial blood neutralizing antibody testing will include flaviviruses: dengue, zika, yellow fever, west nile virus, Japanese encephalitis virus. Only positive results 
for dengue will be used as exclusionary criteria. The others will only help to define previous exposures and potential differences in immune response to 
vaccination. 
e Vital signs on vaccination days will be taken prior to dosing and at least 30 minutes post dose, to include heart rate, blood pressure, temperature, and respiratory 
rate. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
43 
US Government Proprietary 
 
 
f At time of screening a complete physical exam will be performed. At subsequent visits a focused physical exam will be performed based upon any subject 
reported symptoms. If no symptoms are reported at follow up visits no additional physical exam may be performed if not indicated based on investigator 
discretion. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
44 
US Government Proprietary 
 
 
 
8.2. 
Informed Consent 
The informed consent process and document will be reviewed and approved by the WRAIR IRB 
and sponsor’s representative prior to initiation of the study. The consent document contains a full 
explanation of the possible risks, advantages, and alternate treatment options, and availability of 
treatment in the case of injury, in accordance with 21 CFR 50. The consent document indicates 
that by signature, the subject permits witnessing of applicable study procedures by the sponsor’s 
representative, as well as access to relevant medical records by the sponsor’s representative and 
by representatives of the US Food and Drug Administration (FDA). The sponsor’s representative 
will submit a copy of the initial IRB- and sponsor’s representative-approved consent form to the 
FDA and will maintain copies of revised consent documents that have been reviewed and 
approved by the WRAIR IRB and the ORP. 
A written informed consent document, in compliance with 21 CFR Part 50, 32 CFR Part 219, 
and the Belmont Principles will be signed by the subject before any study-related procedures are 
initiated for that subject. The consent process will follow the WRAIR CTC SOP (UWD-C 206). 
All potential subjects will receive a formal briefing in the CTC by an investigator. The 
investigators will present the protocol in lay terms to individual subjects. The briefing will 
describe the rationale for and the key features of the study, the nature of subject participation, 
inclusion and exclusion criteria, and potential risks and benefits to the subject. Questions on the 
purpose of the protocol, protocol procedures, and risks to the subjects will then be solicited. Any 
question that cannot be answered will be referred to the principal investigator. This briefing will 
involve a combination of standardized presentation materials and spontaneous interaction 
between investigator and potential subjects. All standardized materials will have been approved 
by the WRAIR IRB prior to their use. No subject should grant consent until questions have been 
answered to his/her satisfaction. The subject will be informed that the study products are 
investigational vaccines and not licensed by the FDA for commercial use, but permitted to be 
used in this clinical research. Informed consent includes the principle that it is critical the subject 
be informed about the potential risks and benefits. This information will allow the subject to 
make a personal risk versus benefit decision and understand the following: 
• Participation is entirely voluntary, 
• Subjects may withdraw from participation at any time, 
• Refusal to participate involves no penalty, and 
• The individual is free to ask any questions that will allow him/her to understand the 
nature of the protocol. 
Should the protocol be modified, the subject consent document must be revised to reflect the 
changes to the protocol. If a previously enrolled subject is directly affected by the change, the 
subject will receive a copy of the revised informed consent document. The approved revision 
will be read, signed, and dated by the subject. The original consent document must be retained 
by the investigator as part of the study records. Each subject will receive a copy of the signed 
informed consent document. At each vaccination visit, inclusion and exclusion criteria will be 
reviewed and verbal consent to receive the vaccine will be obtained. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
45 
US Government Proprietary 
 
 
All subjects will be required to sign an Information Sheet and indicate whether or not they are a 
federal employee (Active Duty or Civilian) when they participate in research as human subjects 
to demonstrate that they have received the compensation policies for federal employees for 
research subjects. Military subjects will also need to submit a signed statement of supervisor’s 
approval. 
The subject will be informed that a description of this clinical trial will be available on 
http://www.ClinicalTrials.gov. 
 
8.3. 
Study Visits 
Refer to Table 5 through Table 6 for study procedures for each of vaccination Groups 1 and 2. 
 
8.4. 
Subject ID Cards 
A subject card will be provided to each subject once they are enrolled in the study. This card will 
include the address and telephone number of the main contact for information about the clinical 
study. In an emergency situation, this card serves to inform the responsible attending physician 
that the subject is in a clinical study and that relevant information may be obtained by contacting 
the investigator. Subjects will be instructed to keep the subject card in their possession at all 
times. 
 
8.5. 
Memory Aids 
Memory aids will be provided to each subject after each vaccination to record body 
temperature through 7 days post vaccination and any solicited AEs occurring within 28 days. 
After 7 days post vaccination subjects will only be asked to record a temperature if they feel 
feverish. The subject will be instructed to bring the completed memory aid to the investigator at 
the 7 day for review and at Day 28 day for final review. Memory aids will be reviewed by an 
investigator at these visits and any symptoms or signs recorded by the subject on their memory 
aid will be discussed for clarification. 
Collection and verification of completed memory aid will be done during discussion with the 
subject. The investigator will review the memory aid with the subject, to ensure that all 
information is accurate. The investigator will have latitude in determination of severity through 
questioning of the subject. Any unreturned memory aid will be sought from the subject through 
telephone call(s) or any other convenient methods. 
If a subject does not return the memory aid or the memory aid is returned incomplete, the study 
staff will verbally solicit the information on pain, general symptoms, inter-current illnesses, and 
concomitant medications during the study visit and record them in the study record. If the subject 
fails to fill out part of the memory aid or fails to return it he or she will not be asked to fill out the 
data retrospectively. Instead, after discussion any recalled adverse events will be recorded in the 
subject’s chart per their recollection at the time of the visit. Temperature, redness, and induration 
will be considered lost data and documented as a protocol deviation since they require real time 
measurement by the study subject. If a subject’s memory aid is lost and not returned a protocol 
deviation will be noted. Upon completion of 28 days post vaccination, the memory aids will be 
returned and placed into the subject’s chart for inclusion as part of the subject’s source 
documents. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
46 
US Government Proprietary 
 
 
8.6. 
Biological Samples 
All sample testing will be done in accordance with the consent of the individual subject. 
Samples collected under this protocol will be used to conduct protocol-related safety and 
immunogenicity evaluations. 
Collected samples may also be used in other assays, for test improvement or test development of 
analytical methods related to the study vaccine and its constituents or the disease under study to 
allow a more reliable measurement of the vaccine response. Under these circumstances, 
additional testing on the samples may be performed by the sponsor outside the scope of this 
protocol. 
Any study requiring future use of these biological samples will require IRB approval. In 
addition, a subject may decide at any point during the duration of the study to withdraw consent 
for the future use of his/her samples. Should a subject withdraw consent for the future use of his 
or her samples, the samples will be destroyed after the conclusion of this study. 
Collected samples will be stored until exhausted through use in laboratory testing, unless local 
rules, regulations or guidelines require different timeframes or different procedures, which will 
then be in line with the subject consent. 
 
8.7. 
Concomitant Medications or Vaccinations 
This protocol places no restrictions on rescue medications; the investigator will recommend 
medication for symptomatic relief, if necessary. 
When contacted by the investigator, the subject will be questioned about any medications taken 
and vaccinations received. All concomitant medication and (non-study) vaccinations, with the 
exception of vitamins and dietary supplements, are to be recorded in the eCRF. 
Concomitant medication administered prophylactically in anticipation of reaction to the 
vaccination and any medication intended to treat an AE will also be recorded. A prophylactic 
medication is a medication administered in the absence of any symptom and in anticipation of a 
reaction to the vaccination (eg, an anti-pyretic is considered to be prophylactic when it is given 
in the absence of fever and any other symptom, to prevent fever from occurring). The use of 
prophylactic medications is discouraged. 
Prescription immunosuppressants or other prescription immune-modifying drugs or cancer 
chemotherapy drugs will not be permitted during the study. Any volunteer who initiates 
treatment with medications of this nature will be excluded from any further vaccinations. Inhaled 
or topical corticosteroids will be allowed and do not require exclusion from vaccination or 
enrollment. 
FDA approved vaccinations may proceed during the study but subjects will be asked about these 
at each visit and they will be recorded in the subject’s chart. Subjects will be asked not to receive 
any vaccinations within 30 days before or after any study vaccinations if possible. 
 
8.8. 
Procedures for Monitoring Subject Compliance 
If a subject meets withdrawal conditions for a concomitant medication violation or 
noncompliance, this should be clearly stated in the source document and the study termination 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
47 
US Government Proprietary 
 
 
electronic CRF. The study termination eCRF will be completed, with the reason for withdrawal 
specified. Study termination procedures include recording: date of last contact, major reason for 
termination, concomitant vaccination and medication, AEs, SAEs, pregnancy, study conclusion 
and signature of the investigator. 
 
9. 
DOSE ADJUSTMENT AND STUDY TERMINATION 
PROCEDURES 
9.1. 
Contraindications to Subsequent Vaccination 
The following events constitute absolute contraindications to the second planned vaccination. If 
any of these events occur during the study, the subject must not receive additional doses of 
vaccine but may continue other study procedures at the discretion of the investigator. 
• Hypersensitivity reaction, including anaphylaxis, following the administration of the first 
dose of study vaccine. 
• Pregnancy 
• Autoimmune disease or pIMD 
• Discovery of any health condition which, in the investigator’s opinion, places the subject 
at increased risk from receipt of further investigational product; or discovery of a change 
in the subject’s status which renders him/her unable to comply with protocol-mandated 
safety follow-up 
• Initiation of new immunosuppressive or immune modulating drug or therapy 
• Noncompliance that requires withdrawal from the study or precludes subsequent 
vaccination 
The investigational product(s) can be administered to subjects with a minor illness (ie, mild 
diarrhea, mild upper respiratory infection, etc) without fever (fever is considered to be oral body 
temperature ≥ 37.5°C/99.5°F). If the subject has fever, with or without a minor illness, at the 
time scheduled for vaccination, the subject may be vaccinated at a later date, within the specified 
time window or withdrawn at the discretion of the investigator. 
 
Subjects may be eliminated from the according to protocol (ATP) cohort for immunogenicity if, 
during the study, they incur a condition that has the capability of altering their immune response 
or are confirmed to have an immunodeficiency condition. 
 
9.2. 
Safety Criteria for Stopping Doses 
If any of the events listed in Table 7 or Table 8 occurs, administration of investigational product 
will be discontinued for all subjects in all groups until a thorough review of the event is 
undertaken by the study team and the sponsor. Specifically, the primary investigator, the DOD 
research monitor and the sponsor’s safety physician will discuss the event to determine the likely 
cause. If after review, it is determined that the event(s) is unlikely related to vaccination and/or it 
is determined after discussion that this event does not represent a significant or unexpected risk 
to research subjects then vaccinations may be resumed. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
48 
US Government Proprietary 
 
 
Table 7: 
Adverse Event Study Hold Rules 
 
Event 
Number or Percentage 
of Subjects a 
If one subject dies or experiences any SAE related to the investigational 
products 
≥ 1 
If one subject is withdrawn from the study (by investigator request) 
following a Grade 3 AE that cannot reasonably be attributed to another 
cause 
≥ 1 
Any local or general solicited AE leading to hospitalization, or fever 
>40°C (104°F) that cannot reasonably be attributed to another cause or 
necrosis at the injection site within the 28-days (Days 0-28) post- 
vaccination period 
≥ 1 
Subjects reporting any Grade 3 solicited local AE in an investigational 
group, within the 28-day post-vaccination period 
≥ 30% of vaccines so far 
and ≥ 3 subjects 
Subjects reporting any Grade 3 solicited general AE in an investigational 
group, within the same system organ class, that cannot reasonably be 
attributed to another cause, within the 28-day post-vaccination period 
≥ 2 
Subjects reporting any Grade 3 unsolicited AE in an investigational 
group, within the same system organ class, that cannot reasonably be 
attributed to another cause, within the 28-day (Days 0-27) post- 
vaccination period 
≥ 2 
Subjects reporting any ≥ Grade 3 abnormality within the same system 
organ class in pre-specified hematological, biochemical laboratory 
parameters in an investigational group, that cannot reasonably be 
attributed to another cause, within the 28- day post-vaccination period 
≥ 2 
a The number or percentage (as marked) of subjects who must experience the event for the study to be put on hold 
until review can be performed by the PI, research monitor and sponsor to determine appropriate follow-up actions. 
 
 
Table 8: 
Laboratory Assay Study Hold Rules 
 
Test 
Analyte 
Grade ≥ 3 
Serum chemistry 
ALT 
> 5 x ULN 
AST 
> 5 x ULN 
Alkaline phosphatase > 3 x ULN 
Creatinine 
> 2 x ULN 
Hematology/Complete blood count (CBC) White Blood Cells 
> 20,000 or <1,500 cell/mm3 
Neutrophils 
< 1,000 cell/mm3 
Lymphocytes 
< 500 cell/mm3 
Eosinophils 
> 5,000 cell/mm3 
Hemoglobin female 
< 9.5 g/dL or 
Hemoglobin male 
< 10.5 g/dL or 
Platelet count 
< 100,000 cell/mm3 
 
9.3. 
Study Termination Criteria 
The PI, research monitor, sponsor’s representative, the WRAIR IRB, or the FDA may stop or 
suspend the use of these products or this study at any time. This would result in a halt to any 
further vaccinations unless or until a later determination is made to restart the study. Any 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
49 
US Government Proprietary 
 
 
previously vaccinated subjects would still be followed for safety for at least 180 days following 
receipt of any vaccine product. 
 
9.3.1. 
Subject Withdrawal Criteria 
Each subject may withdraw consent at any time during the study without penalty. Counseling 
about the subject's health will be provided if he/she decides to discontinue participation in the 
study. Medical advice regarding what is in the best interest of the subject will be provided. 
Subjects who are withdrawn because of SAEs/AEs must be clearly distinguished from subjects 
who are withdrawn for other reasons. Investigators will follow subjects who are withdrawn due 
to an SAE/AE until resolution of the event. 
The PI may discontinue the subject’s activity without the subject’s consent if any of these criteria 
is met: 
• A subject fails to comply with study procedures 
• A subject’s safety or health may be compromised by further participation (eg, an AE or 
SAE resulting in a safety concern) 
 
9.3.2. 
How to Withdraw Subjects 
If a subject withdraws, the investigator will make a reasonable effort to determine the reason for 
the withdrawal from the study and to complete termination procedures. Telephone calls, 
registered letters, and email correspondence are considered reasonable effort. For subjects 
leaving the study, a targeted examination may be performed, if medically indicated and if 
permitted by the subject. 
When a subject withdraws due to an AE or is withdrawn by the PI due to an AE, the sponsor’s 
safety office (the United States Army Medical and Research Development Command 
(USAMRDC), Office of Regulated Activities (ORA), Product Safety Surveillance Office 
(PSSO)) must be notified within 24 hours via email (usarmy.detrick.medcom-usamrmc.mbx.sae- 
reporting@mail.mil ) and investigators must follow specific policy regarding the timely 
reporting of AEs and SAEs to the WRAIR IRB (Section 11.7.1.2). In all cases, the PI will make 
a reasonable effort to complete study termination procedures. 
If a subject meets withdrawal conditions for a concomitant medication violation or 
noncompliance, the reason should clearly be stated in the source document and the study 
termination eCRF. 
 
9.3.3. 
Data Collected for Withdrawn Subjects 
All data collected up to the time of withdrawal will be reported. The study termination eCRF will 
be completed, with the reason for withdrawal specified. 
 
9.3.4. 
Replacement of Subjects 
Subjects who withdraw or are withdrawn after the first vaccination will not be replaced. 
However, subjects who withdraw or are withdrawn prior to the first vaccination will be replaced. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
50 
US Government Proprietary 
 
 
9.3.5. 
Follow-up for Withdrawn Subjects 
The investigator will make every effort to follow all subjects who are withdrawn/withdraw, after 
receiving vaccine, regardless of the reason, until the end of the study period. 
If administration of the investigational products is discontinued for a subject it will be annotated 
in the record but the subject may still be followed for safety assessments if they are amenable. 
 
10. 
IMMUNOGENICITY ASSESSMENTS 
10.1. 
Geometric Mean Titers and Seroconversion 
Assessment of neutralizing antibodies (NAb) against DENV type 1-4 will be performed by a 
qualified microneutralizing antibody assay as previously described (Putnak et al.-2008) or flow 
cytometry-based neutralization assay as previously described (Kraus et al.-2007) at WRAIR 
(Table 9). The 50% neutralization (NT50) titers will be reported by both assays. Seropositivity 
and seroconversion rates for each DENV serotype and GMTs will be evaluated. Samples will be 
used to generate data within the scope of this protocol. 
Characterization of homotypic and heterotypic NAb will be evaluated using an established assay 
for depletion of DENV-specific antibodies from human sera. The antibody depletion method will 
be carried out as described previously (de Alwis et al.-2012). Purified DENV or BSA control 
protein will be adsorbed onto 4 µm Polybead polystyrene microspheres following the 
manufacturer’s instructions (Polysciences, Inc.). The polystyrene microspheres will be washed 
3 times with 0.1M borate buffer (pH 8.5) and will be incubated with purified DENV or BSA 
control protein in borate buffer overnight at room temperature. The control and DENV-adsorbed 
beads will be blocked 3 times with BSA (10 mg/ml) in borate buffer for 30 min at room 
temperature and washed 6 times with PBS. Human immune sera will be depleted of DENV- 
specific antibody by incubation with virus-adsorbed beads for 2 hrs at 37°C with gentle mixing. 
Sequential rounds of antibody depletion will be performed and the depleted and undepleted sera 
will be tested by ELISA for determining successful removal of the desired DENV-specific 
antibodies. ELISA binding of human sera to purified DENV 1-4 will be measured as described 
previously (de Alwis et al.-2012). 
The potential of sera to mediate antibody-dependent enhancement (ADE) will be assessed using 
an established in vitro ADE assay. Two-fold serial dilutions of heat-inactivated sera will be 
mixed with an equal volume of virus (sufficient to achieve approximately 15% infection of 5x104 
K562-DC-SIGN cells) and incubated for 1 hr at 37°C. This mixture will be added to a 96-well 
plate containing medium (RPMI-1640 supplemented with 10% FBS, 1% penicillin/streptomycin, 
1% L-glutamine (200mM), and 1% non-essential amino acids (10 mM)) with 5x104 K562 cells 
per well in duplicate and incubated 18-20 hrs overnight in a 37°C, 5% CO2, humidified 
incubator. Following overnight incubation, the cells will be fixed, permeabilized and 
immunostained with flavivirus group-reactive mouse monoclonal antibody 4G2, and secondary 
polyclonal goat anti-mouse IgG PE-conjugated antibody (catalog no. 550589, BD Biosciences, 
San Jose, CA). The percent infected cells will be quantified on a BD Accuri C6 Plus flow 
cytometer (BD Biosciences, San Jose, CA). 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
51 
US Government Proprietary 
 
 
Table 9: 
Humoral Immunity Assessments (Antibody Determination) 
 
System 
Component 
Method 
Unit 
Cut-off 
Laboratory 
Serum 
Neutralizing 
antibody 
against 
DENV types 
1-4 
DENV 
neutralization 
assay in-house 
(Vero cells) 
The titer giving 50% 
reduction in viral 
infection 
MN50 ≥ 10 
WRAIRa 
Serum 
Neutralizing 
antibody 
against 
DENV types 
1-4 
DENV 
neutralization 
assay in-house 
(human U937-DC- 
SIGN) 
Reciprocal serum 
dilution giving 50% 
reduction in viral 
infection 
NT50 ≥ 40 
WRAIR VDB 
a WRAIR laboratory refers to Viral Diseases Branch or (503 Robert Grant Avenue, Silver Spring, MD 20910) 
 
10.2. 
Cell-Mediated Immunity (CMI) Assessment 
The Viral Disease Branch (VDB) Immunology Laboratory will assess vaccine-induced cell- 
mediated immunity (CMI) using a variety of assay platforms, as outlined below. Samples for 
CMI testing include peripheral blood mononuclear cells (PBMC) and serum/plasma collected 
contemporaneously and will be utilized fresh or after cryopreservation. Samples will be released 
for testing at the discretion of the PI and according to VDB Compliance Management Unit 
(CMU) procedures. All assays for CMI will be performed as exploratory studies using 
appropriate SOPs developed in the laboratory or other methods consistent with those in the 
published literature. PBMC will be assessed for markers of cellular activation and maturation as 
well as antigen-specific cytokine production. Cell subsets of interest (for example, activated T 
cells) may be sorted by fluorescence-activated cell sorting (FACS) and subjected to systems 
analysis for nucleic acid and protein expression profiling. Serum/plasma will be analyzed for 
potential biomarkers of acute phase reactant (protein) content as well as antigen-specific 
antibodies involved in NK cell function. CMI assays will be performed on pre-vaccination 
samples and at various time points post-vaccination. Pre-vaccination samples will serve as a 
control for all assays performed and hence a larger blood draw is scheduled at Day 0 (day of first 
vaccination). 
Assays for assessment of vaccine-induced CMI will include: 
• ELISPOT – for T cell function and B cell antibody secretion 
• Cytokine Flow Cytometry (CFC) – for T cell function and phenotype 
• Flow cytometry-based Immunophenotyping – for profiling of cell subset activation 
and phenotype 
• Fluorescence-activated cell sorting (FACS) – to identify specific cell subsets of 
interest for sorting and downstream detailed analysis 
• In vitro proliferation assays – for T cell function 
• Antibody-dependent Cellular Cytoxicity (ADCC) by flow cytometry – for antibody- 
dependent NK cell function 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
52 
US Government Proprietary 
 
 
• Multiplexed enzyme linked immunosorbent assay (ELISA) – for cytokine expression 
• Immune Genotyping 
• Systems Biology Analyses 
10.2.1. 
CMI Assays and Assay Systems 
 
10.2.1.1. 
ELISPOT 
Vaccine-induced T cell responses will be assessed using a standard ELISPOT assay for 
expression of IFN-gamma and/or other cytokines of interest, following SOP #UWF-D-085. 
Antigens for the T cell ELISPOTs will include overlapping peptide pools matching select 
DENV-derived proteins and when possible epitope mapping will be performed using individual 
peptides. 
B cell ELISPOTs will be performed to identify DENV-specific antibody-secreting cells, 
following SOP #UWF-D-099. The antigens used for coating plates will be either a crude 
preparation of virus from cell culture, the vaccine itself, recombinant proteins or other related 
antigens. 
 
10.2.1.2. 
Cytokine Flow Cytometry (CFC) 
For T cell function and in vitro activation studies, cryopreserved PBMC will be thawed and 
immediately stimulated with select DENV-derived peptide pools in the presence of protein 
transport inhibitors prior to staining for a variety of functional and/or cytokine markers, 
following SOP #UWF-D-079. 
 
10.2.1.3. 
Flow Cytometrycytometry-based Immunophenotypingimmunophenotyping and 
Fluorescencefluorescence-activated Cell Sortingcell sorting (FACS) 
Flow cytometry will be used to comprehensively assess cellular activation and maturation in all 
lymphoid and myeloid cell lineages in PBMC, following SOP #UWF-D-084. In select cases for 
which the minimal epitope has been identified, HLA/peptide tetramer reagents may be used to 
assess in-depth the phenotype of antigen-specific cells. After staining, sub-populations of interest 
may be sorted using FACS and subjected to subsequent systems biology analyses. 
10.2.1.4. 
In vitro proliferation assays 
Cryopreserved PBMC will be thawed and immediately stained with a cell-tracking dye (eg, 
CSFE) followed by stimulation with select DENV-derived peptide pools and staining by flow 
cytometry, following SOP #UWF-D-083. 
 
10.2.1.5. 
Antibody-dependent Cellular Cytotoxicity (ADCC) 
Serum samples will be used to determine 50% and end-point titers of vaccine-induced antibodies 
that mediate ADCC. All assays will be conducted using cell lines and methodology developed in 
the VDB Immunology Laboratory. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
53 
US Government Proprietary 
 
 
10.2.1.6. 
Multiplexed Enzyme Linked Immunosorbent Assay (ELISA) 
Commercially available multiplexed bead-based ELISAs, such as Luminex-based assays, will be 
used to measure numerous acute phase proteins, chemokines and cytokines circulating in the 
serum/plasma of vaccinees. Culture supernatants from lymphocyte proliferation assays may also 
be tested. 
 
10.2.1.7. 
Immune Genotyping 
Samples may be subject to limited genetic testing (genotyping) for certain genes and alleles (of 
these genes) that are of immunological relevance. This testing could include genes of any of the 
following loci: human leukocyte antigen (HLA) gene family, killer-cell inhibitory receptor (KIR) 
gene family, and genes that code for immunoglobulins (antibodies) and T cell receptors (TCRs). 
The results of these tests will be exclusively used for research (NOT diagnostic) purposes and 
will allow coordinated analysis of particular genotypes with immune phenotypes and responses 
characterized herein. 
 
10.2.1.8. 
Systems Biology Analyses 
The transcriptomic profile of purified subpopulations of PBMC from vaccines will be 
characterized to identify molecular signatures of vaccine-induced immunity. Such analysis can 
be performed using a variety of approaches, including microarrays and total RNA sequencing 
(RNA-Seq). 
Microarray data analysis: The Human Transcriptome Array 2.0 (Affymetrix) will be used to 
determine differentially expressed genes and differences in the transcript isoforms from each of 
the cell populations. As biological pathways are multifaceted and multiple genes/proteins are 
involved, vaccine induced differential expression of genes would by identified and evaluated by 
gene array analysis. 
Total RNA Sequencing (RNA-Seq): cDNA libraries will be prepared from purified, FACS- 
sorted subpopulations of cells for high-throughput sequencing using Illumina HiSeq and/or 
MiSeq instruments. 
 
11. 
SAFETY ASSESSMENT 
Safety monitoring will be conducted throughout the study; therefore safety concerns will be 
identified by continuous review of the data by the PI, clinic staff, clinical monitor, research 
monitor, and sponsor safety office (ORA PSSO). 
A Data and Safety Monitoring Board (DSMB) is not required for this study. 
Research Monitor 
In accordance with the DoD Instruction (DoDI) 3216.02, all DoD-conducted research studies 
determined to be greater than minimal risk [as defined by 32 CFR 219.102(i)] require the 
appointment of an independent research monitor by the IRB. 
Research monitors shall: 
1. Discuss research progress with the PI, interview subjects, consult on individual cases, or 
evaluate suspected adverse reaction reports on behalf of the IRB. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
54 
US Government Proprietary 
 
 
2. Perform, at the direction of the IRB, oversight functions (eg, observe recruitment, 
enrollment procedures, and the consent process for individuals, groups or units; oversee 
study interventions and interactions; review monitoring plans and unanticipated problems 
involving risks to subjects or others (UPIRTSO) reports; and oversee data matching, data 
collection, and analysis). 
3. Promptly report discrepancies or problems to the IRB. 
4. Have the authority to stop a research study in progress, remove individual subjects from a 
study, and take whatever steps are necessary to protect the safety and well-being of 
research subjects until the IRB can assess the research monitor's report. 
5. Review all UPIRTSOs, SAEs/SUSARs, unanticipated problems, and all subject deaths, 
and provide an unbiased written report of the events promptly to the sponsor’s safety 
office (USAMRDC ORA PSSO). The Research Monitor will provide an unbiased report 
for all UPIRTSOs, related SAEs, and all subject deaths promptly (within 48 hours) to the 
WRAIR IRB by email (usarmy.detrick.medcom-wrair.mbx.hspb@mail.mil) or facsimile 
(301) 319-9961 and they will provide a written report of these events within 10 working 
days to the WRAIR IRB. 
USAMRDC ORA PSSO: The sponsor’s safety office (ORA PSSO) is responsible for 
coordinating and integrating the review of safety data regarding The Surgeon General, 
Department of the Army (TSG-DA)-sponsored products. This office reviews each SAE reports 
for medical consistency, accuracy, and completeness and follows each event until it is 
satisfactorily resolved. The USAMRDC safety pharmacovigilance physician, as delegated by the 
sponsor, evaluates all safety cases and provides the final determination on expectedness and 
relatedness to the product, and whether expedited reporting is warranted, per current FDA 
regulation and guidance. 
 
11.1. 
Specification of Safety Assessments 
11.1.1. 
Demographic Data 
Demographic data, to include age, sex, race, and ethnicity will be collected, by study personnel 
as part of the screening process. 
 
11.1.2. 
Physical Examination, Vital Signs, and Medical History 
Physical examinations will be conducted by study investigator(s) or other qualified study 
personnel. The physical examination of the subject will include assessment of body temperature 
and resting vital signs: systolic/diastolic blood pressure and heart rate after at least 10 minutes of 
rest. Any pre-existing conditions or signs and/or symptoms will be noted, and medical history, 
height, and weight will be documented. Vital signs recorded after vaccination that are 
respectively above or below FDA scale vital sign ranges will be entered as adverse events if 
appropriate within the clinical context within which they occur and within the context of 
previously established normal range of each individual subject. Grading of abnormal vital signs 
will be guided by the ranges established by the FDA toxicity scale for abnormal vital signs. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
55 
US Government Proprietary 
 
 
11.1.3. 
Clinical Laboratory Assessments 
Clinical laboratory assessments will be performed by Quest laboratories according to standard 
operating procedure. Hematology assessments include a complete blood count: WBC, Hgb, 
platelet count, differential, and ANC. Blood chemistry assessments include AST, ALT, BUN, 
creatinine, alkaline phosphatase, total and direct bilirubin. 
Abnormal laboratory findings or other abnormal assessments that are judged by the investigator 
to be clinically significant will be recorded as AEs or SAEs if they meet the definition of an AE 
or SAE (Section 11.1.5). Only abnormal lab results determined to be clinical significant will be 
recorded as AEs or SAEs respectively. Abnormal laboratory results that are both clinically 
significant and not clinically significant will be graded per the modified FDA toxicity criteria in 
Appendix D (if the laboratory test is listed on that scale) for the Sponsor’s PSSB safety 
surveillance. Abnormal lab results will be judged by investigators to be clinically significant if 
they meet one or both of the following criteria: 
6. The abnormality suggests a disease and/or organ toxicity that is new or has worsened 
from baseline. 
7. The abnormality is of a degree that requires additional active management, eg, change of 
dose, discontinuation of the drug, close observation, more frequent follow-up 
assessments, or further diagnostic investigation. 
Clinically significant abnormal laboratory findings or other abnormal assessments that are 
detected before the first vaccination will be documented on medical history as pre-existing 
conditions and will be reported as AEs or SAEs only if the pre-existing conditions are worse 
following the vaccination. 
Clinically significant abnormal laboratory findings or other abnormal assessments that are 
detected after the first vaccination during the study will be reported as AEs or SAEs. 
The investigator will exercise his or her medical and scientific judgment in deciding whether an 
abnormal laboratory finding or other abnormal assessment is clinically significant. 
Clinically significant laboratory abnormalities will be followed up until they have returned to 
normal or to baseline, stabilized with the probability that it will become chronic, or a satisfactory 
explanation has been provided. Additional information (including but not limited to laboratory 
results) relevant to the subsequent course of such abnormalities noted for any subject must be 
made available to the study monitor. 
The sponsor may request that the investigator perform or arrange for the conduct of additional 
clinical examinations/tests and/or evaluations to elucidate as fully as possible the nature and/or 
causality of an SAE. The investigator is obliged to assist. If a subject dies during participation in 
the study or during a recognized follow-up period, the investigator will provide a copy of any 
post-mortem findings, including histopathology, to the USAMRDC PSSO (Safety Office). 
11.1.4. 
Post-vaccination Viremia 
Assessment of viremia will be conducted at WRAIR using quantitative RT-PCR or by detecting 
viable virus through culture in vero cells. Based on results from WRAIR 2136 RNAemia appears 
to occur most frequently between d]Days 7-14. However, it is often very transient. We will 
attempt to capture the greatest number of positive samples by testing daily between 6 and 14 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
56 
US Government Proprietary 
 
 
days following vaccination with LAV. Sequential days of testing will be encouraged as much as 
possible but weekend testing will be excluded due to logistical constraints. Testing for viremia 
will occur for a total of 5 days per subject. Viremia is not expected after PIV vaccinations so 
viremia testing will occur only after LAV vaccinations. RNAemia will be reviewed to asses for 
correlation with solicited and unsolicited adverse events along with subsequent immune 
responses elicited by vaccination. 
 
11.1.5. 
Definition of Adverse Events 
An ADVERSE EVENT (AE) is any untoward medical occurrence associated with the use of the 
investigational product in humans, whether or not considered related to the investigational 
product. An AE can therefore be any unfavorable and unintended sign (including an abnormal 
laboratory finding), symptom, or disease (new or exacerbated) temporally associated with the use 
of a medicinal (investigational) product. 
SUSPECTED ADVERSE REACTION means any adverse event for which there is a reasonable 
possibility that the vaccine caused the adverse event. For the purposes of IND safety reporting, 
“reasonable possibility” means there is evidence to suggest a causal relationship between the 
vaccine and the adverse event. Suspected adverse reaction implies a lesser degree of certainty 
about causality than adverse reaction, which means any adverse event caused by a study product. 
A SOLICITED AE is a predetermined event, identified in the investigator’s brochure, which 
may reflect safety concerns related to the investigational product. The solicited AEs for this 
study include: 
• Local solicited adverse events: 
− Pain at injection site 
− Redness at injection site 
− Swelling at injection site 
• General solicited adverse events: 
− Fatigue 
− Fever 
− Headache 
− Gastrointestinal symptoms (nausea, vomiting, diarrhea, and/or abdominal pain) 
− Joint pain 
− Muscle aches 
− Rash 
Solicited symptoms will be recorded by subjects on memory aids from the day of vaccination 
(Day 0) through the end of Day 27 after each vaccination. Redness and swelling will be 
measured in millimeters, using a metric ruler provided by the CTC, and the greatest surface 
diameter of each will be recorded. Body temperature (oral route preferred) will be recorded in 
the evening, using a thermometer provided by the CTC. Actual temperatures will only be 
recorded in the eCRF if they are abnormal (per grading in Table 11). Volunteers will only be 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
57 
US Government Proprietary 
 
 
asked to record daily temperatures for the first 7 days following each vaccination. Following that 
they will be asked to record a temperature only if they feel feverish for Days 8-28. If multiple 
temperatures are recorded by a volunteer in a single day, the highest temperature measured on 
that day will be recorded in the eCRF. 
UNSOLICITED AEs are all other adverse events not listed as a solicited AE, occurring from the 
day of vaccination through 28 days after vaccination. 
A SERIOUS ADVERSE EVENT (SAE)/SERIOUS SUSPECTED ADVERSE REACTION is an 
AE or suspected adverse reaction that, in the view of either the PI or medical monitor, results in 
any of the following outcomes: 
• death 
• a life-threatening adverse event 
− An adverse event or suspected adverse reaction is considered “life-threatening” if, 
in the view of either the investigator or sponsor, its occurrence places the patient 
or subject at immediate risk of death. It does not include an adverse event or 
suspected adverse reaction that, had it occurred in a more severe form, might have 
caused death. 
• Inpatient hospitalization or an event associated with the investigational product that 
results in hospitalization or prolongation of hospitalization 
• a persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions 
• a congenital anomaly/birth defect 
• Important Medical Events when meeting the below criteria: 
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered serious when, based upon appropriate medical judgment, they 
may jeopardize the patient and may require medical or surgical intervention to prevent one of the 
outcomes listed in this definition. 
An UNEXPECTED ADVERSE EVENT/UNEXPECTED SUSPECTED ADVERSE 
REACTION is an AE or suspected adverse reaction that is not listed in the investigator’s 
brochure or is not listed at the specificity or severity that has been observed; or, if an investigator 
brochure is not required or available, is not consistent with the risk information described in the 
general investigational plan or elsewhere in the current application, as amended. For example, 
under this definition, hepatic necrosis would be unexpected (by virtue of greater severity) if the 
investigator brochure referred only to elevated hepatic enzymes or hepatitis. Similarly, cerebral 
thromboembolism and cerebral vasculitis would be unexpected (by virtue of greater specificity) 
if the investigator brochure listed only cerebral vascular accidents. “Unexpected,” as used in this 
definition, also refers to adverse events or suspected adverse reactions that are mentioned in the 
investigator brochure as occurring with a class of study products or as anticipated from the 
pharmacological properties of the study product, but are not specifically mentioned as occurring 
with the particular study product under investigation. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
58 
US Government Proprietary 
 
 
11.1.6. 
Unanticipated Problems Involving Risks To Subjects Or Others 
Federal regulations require that UPIRTSOs be promptly reported to the IRB within the IRB 
specified time frame. These events encompass a broader category of events than SAEs and may 
include issues such as problems with loss of control of subject data or the investigational 
product; adverse psychological reactions; or breach of confidentiality. Risks to others (eg, study 
personnel) must also be reported. 
The WRAIR IRB requires prompt reporting of UPIRTSOs (using the Deviation/Unanticipated 
Problem Report Form) to the HSPB (within 48 hours) upon becoming aware of the event by 
telephone, facsimile, or email, and recording it in the study unanticipated problem log 
(Deviation/Unanticipated Problem Report Form). A written report is required to be submitted by 
the PI to the HSPB within 10 working days of knowledge of the event. 
All other unanticipated problems should be included in the deviation/unanticipated problem log 
and reported to the IRB as part of the protocol continuing review, as per the SOP UWZ-C-618, 
Continuing Review and Continuation Determinations. 
Unanticipated problems involving risks to subjects or others are any incident, experience, or 
outcome that meets all of the following criteria: 
• Unexpected (in terms of nature, severity, or frequency) given (a) the procedures that are 
described in the protocol, investigators brochure or informed consent document; and (b) 
the characteristics of the subject population; 
• Related or possibly related to a subject’s participation in the study; and 
• Suggests that the study places subjects or others at a greater risk of harm than was 
previously known or recognized. 
The IRB and the ORP will evaluate the PI’s and research monitor’s reports to determine whether 
a given incident, experience or outcome constitutes an unanticipated problem involving risk to 
subjects or others and, in coordination with the sponsor’s safety office, ensure reporting of the 
unanticipated problems involving risk to subjects or others to the appropriate investigative sites 
and regulatory offices, as applicable. 
 
11.1.7. 
Potential Immune-mediated Diseases 
Potential immune-mediated diseases (pIMDs) are a subset of AEs that include autoimmune 
diseases and other inflammatory and/or neurologic disorders of interest which may or may not 
have an autoimmune etiology. AEs that need to be recorded and reported as pIMDs include those 
listed in Table 10. The investigator will exercise his/her medical and scientific judgment in 
deciding whether other immune-mediated diseases have an autoimmune origin (ie, 
pathophysiology involving systemic or organ-specific pathogenic auto antibodies) and should 
also be recorded as a pIMD. 
 
11.2. 
Relationship to Investigational Product 
The investigator must assign a relationship of each AE to the receipt of the investigational 
product. The investigator will use clinical judgment in conjunction with the assessment of a 
plausible biologic mechanism, a temporal relationship between the onset of the event in relation 
to receipt of the investigational product, and identification of possible alternate etiologies 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
59 
US Government Proprietary 
 
 
including underlying disease, concurrent illness or concomitant medications. A determination of 
‘related to investigational product’ or ‘not related to investigational product’ will be made along 
with a narrative and supporting documentation. The determination that an AE is ‘not related to 
investigational product’ will be made if the AE is clearly not related to the administration of the 
study treatment, another cause of the event is most plausible, a clinically plausible temporal 
sequence is inconsistent with the onset of the event and the study treatment, and/or a causal 
relationship is considered biologically implausible. The determination that an AE is ‘related to 
the investigational product’ will be made if the AE is temporally related to the administration of 
the study treatment, a causal relationship is considered biologically plausible, and there is no 
alternative cause that provides a more likely explanation. 
Table 10: 
List of Potential Immune-mediated Diseases 
 
Neuroinflammatory 
disorders 
Musculoskeletal disorders 
Skin disorders 
Cranial nerve disorders, 
including paralyses/paresis 
(eg, Bell’s palsy), and 
neuritis (eg, optic neuritis) 
Multiple sclerosis (including 
variants) 
Transverse myelitis 
Guillain-Barré syndrome, 
(including Miller Fisher 
syndrome and other variants) 
Other demyelinating diseases 
(including acute 
disseminated 
encephalomyelitis) 
Myasthenia gravis (including 
Lambert-Eaton myasthenic 
syndrome) 
Non-infectious encephalitis/ 
encephalomyelitis 
Neuritis (including peripheral 
neuropathies) 
Narcolepsy 
Systemic lupus erythematosus 
Scleroderma (including, 
CREST syndrome and 
morphea) 
Systemic sclerosis 
Dermatomyositis 
Polymyositis 
Antisynthetase syndrome 
Rheumatoid arthritis, 
Juvenile chronic arthritis, 
(including Still’s disease) 
Polymyalgia rheumatica 
Reactive arthritis 
Psoriatic arthropathy 
Ankylosing spondylitis 
(including undifferentiated 
spondyloarthritides) 
Relapsing polychondritis 
Mixed connective tissue 
disorder 
Psoriasis 
Vitiligo 
Raynaud’s phenomenon 
Erythema nodosum 
Autoimmune bullous skin diseases 
(including pemphigus, pemphigoid 
and dermatitis herpetiformis) 
 
Cutaneous lupus erythematosus 
Alopecia areata 
Lichen planus 
Sweet’s syndrome 
Liver disorders 
Gastrointestinal disorders 
Metabolic diseases 
Autoimmune hepatitis 
Crohn’s disease 
Autoimmune thyroiditis (including 
Hashimoto thyroiditis) 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
60 
US Government Proprietary 
 
 
 
Primary biliary cirrhosis 
Primary sclerosing 
cholangitis 
Autoimmune cholangitis 
Ulcerative colitis 
Ulcerative proctitis 
Celiac disease 
Grave's or Basedow’s disease 
Diabetes mellitus type I 
Addison’s disease 
Vasculitides 
Others 
Large vessels vasculitis including: giant cell 
arteritis such as Takayasu's arteritis and 
temporal arteritis. 
Autoimmune hemolytic anemia 
Autoimmune thrombocytopenia 
Medium sized and/or small vessels 
vasculitis including: polyarteritis nodosa, 
Kawasaki's disease, microscopic 
polyangiitis, Wegener's granulomatosis, 
Churg–Strauss syndrome (allergic 
granulomatous angiitis), Buerger’s disease 
(thromboangiitis obliterans), necrotizing 
vasculitis and anti-neutrophil cytoplasmic 
antibody (ANCA) positive vasculitis (type 
unspecified), Henoch-Schonlein purpura, 
Behcet's syndrome, leukocytoclastic 
vasculitis. 
Antiphospholipid syndrome 
Pernicious anemia 
Autoimmune glomerulonephritis (including IgA 
nephropathy, glomerulonephritis rapidly progressive, 
membranous glomerulonephritis, 
membranoproliferative glomerulo-nephritis, and 
mesangioproliferative glomerulonephritis) 
Uveitis 
Autoimmune myocarditis/cardiomyopathy 
Sarcoidosis 
Stevens-Johnson syndrome 
Sjögren’s syndrome 
Idiopathic pulmonary fibrosis 
Goodpasture syndrome 
 
11.3. 
Severity Assessment 
All AEs will be assessed for severity by the investigator. Inherent in this assessment is the 
medical and clinical consideration of all information surrounding the event including any 
medical intervention required. Solicited adverse events will be graded according to the scale 
shown in Table 11. All other events will be graded according to the scale in Table 12. The eCRF 
for AEs will reflect only the highest severity for continuous days an event occurred. 
If a subject is evaluated in an emergency room for non-life threatening illness or symptoms (ie, 
visits emergency department on weekend for mild problems because the physician’s office is 
closed), the information from that visit will be reviewed and severity of the adverse event will be 
assessed according to the subject’s clinical signs and symptoms. 
Clinically significant abnormal laboratory values will be graded by study investigators based 
upon a modified FDA toxicity scale (Appendix D). All abnormal lab values will graded (based 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
61 
US Government Proprietary 
 
 
on table in Appendix D) upon entry into the database for PSSO safety surveillance. The tables in 
Appendix D were modified to come into harmony with established laboratory normal values per 
Quest Diagnostics guidelines. That is, changes were made to the FDA toxicity scale to insure 
that no lab values are marked as “normal” per the Quest Diagnostics laboratory standards but are 
considered “abnormal” by the grading scale which could create a conflict or possibly missed 
laboratory related adverse events. 
As defined by the International Conference on Harmonisation (ICH) guideline for Good Clinical 
Practice (GCP), the term “severe” is often used to describe intensity (severity) of a specific event 
(as in mild, moderate, or severe myocardial infarction); the event itself however, may be of 
relatively minor medical significance (such as severe headache). This is not the same as 
“serious”, which is based on subject/event outcome or action criteria usually associated with 
events that pose a threat to a subject’s life or functioning. Seriousness (not severity) serves as a 
guide for defining regulatory reporting obligations. In addition to the severity scales listed below 
any adverse event that is deemed by the investigator to be potentially life threatening will be 
graded as Grade 4 and any adverse event that results in death will be graded as Grade 5. 
Table 11: 
Solicited Adverse Event Intensity Scales 
 
Adverse Event 
Intensity 
Grade 
Parameter 
Pain at injection site 
1 
Mild: Any pain neither interfering with nor preventing normal every 
day activities. 
2 
Moderate: Painful when limb is moved and interferes with every day 
activities. 
3 
Severe: Significant pain at rest. Prevents normal every day activities. 
Fevera 
1 
37.5◦C – 38.4◦C (99.5°F – 101.1◦F) 
2 
38.5◦C – 38.9◦C (101.2°F – 102.0°F) 
3 
≥39.0◦C (102.1°F) 
Redness at injection 
site 
1 
Greatest surface diameter ≥ 25 to 50 mm 
2 
Greatest surface diameter ≥ 51 to 100 mm 
3 
Greatest surface diameter ≥ 101 mm 
Swelling at injection 
site 
1 
Greatest surface diameter ≥ 25 to 50 mm 
2 
Greatest surface diameter ≥ 51 to 100 mm 
3 
Greatest surface diameter ≥ 101 mm 
Headache 
1 
Mild: Headache that is easily tolerated 
2 
Moderate: Headache that interferes with normal activity 
3 
Severe: Headache that prevents normal activity 
Fatigue 
1 
Mild: Fatigue that is easily tolerated 
2 
Moderate: Fatigue that interferes with normal activity 
3 
Severe: Fatigue that prevents normal activity 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
62 
US Government Proprietary 
 
 
 
Adverse Event 
Intensity 
Grade 
Parameter 
Gastrointestinal 
symptoms (nausea, 
vomiting, diarrhea 
and/or abdominal 
pain) 
1 
Mild: Gastrointestinal symptoms that are easily tolerated 
2 
Moderate: Gastrointestinal symptoms that interfere with normal 
activity 
3 
Severe: Gastrointestinal symptoms that prevent normal activity 
Joint pain 
1 
Mild: Joint pain that is easily tolerated 
2 
Moderate: Joint pain that interferes with normal activity 
3 
Severe: Joint pain that prevents normal activity 
Muscle aches 
1 
Mild: Muscle aches that is easily tolerated 
2 
Moderate: Muscle aches that interferes with normal activity 
3 
Severe: Muscle aches that prevents normal activity 
Rash 
1 
Eruption covering <30% of total body surface area and not limiting 
ADLs 
2 
Eruption covering <50% of total body surface area and/or limiting 
ADLs 
3 
Eruption covering >50% of total body surface area and/or limiting 
self-care 
a The preferred route for recording body temperature in this study is oral. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
63 
US Government Proprietary 
 
 
Table 12: 
Intensity Scale for All Other Adverse Events 
 
Grade 
Description 
1 (mild) 
An AE which is easily tolerated by the subject, causing minimal 
discomfort and not interfering with everyday activities. 
2 (moderate) 
An AE which is sufficiently discomforting and interferes with normal 
everyday activities. 
3 (severe) 
An AE which prevents normal, everyday activities (ie, prevent attendance 
at work/school) and would necessitate the administration of corrective 
therapy 
 
11.4. 
Assessment of Outcome 
Outcome of any non-serious AE occurring within 28 days post-vaccination (ie, unsolicited AE) 
or any SAE/pIMD reported during the entire study will be assessed as: 
• Recovered/resolved 
• Recovering/resolving 
• Not recovered/not resolved 
• Recovered /resolved with sequelae 
• Fatal (SAEs only) 
11.5. 
Duration of Follow-Up of Subjects after Adverse Events 
Investigators are required to follow SAEs to resolution, even if this extends beyond the 
prescribed reporting period. Resolution is the return to baseline status or stabilization of the 
condition with the probability that it will become chronic. The SAE outcomes will be reported to 
the sponsor’s safety office (ORA PSSO) using the Serious Adverse Event Report Form. 
Investigators are not obligated to actively seek SAEs in former subjects; however, if a SAE, 
considered to be related to the investigational product is brought to the attention of the 
investigator at any time following completion of the study, the event will be reported to the 
sponsor’s safety office as defined in Section 12.7.1. 
Adverse events will otherwise be followed either until resolution or until end of study at which 
time the current status of the adverse event will be recorded as described in Section 12.4. 
 
11.6. 
Recording Adverse Events 
AEs and SAEs will be assessed at all study visits. AEs will be recorded on the eCRF only on day 
of vaccination through to 28 days following each vaccination respectively. SAEs will be 
recorded at all study visits. These AEs and SAEs will be documented in the source records, and 
recorded on the eCRFs using accepted medical terms and/or the diagnoses that accurately 
characterize the event. It should be noted that the form for collection of SAE information is not 
the same as the AE eCRF. When the same data are collected on both forms, the forms must be 
completed in a consistent manner. For example, the same event term should be used on both 
forms. New or updated information will be recorded in the originally completed CRF using 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
64 
US Government Proprietary 
 
 
Good Documentation Practices. The investigator will submit any updated SAE data to the 
sponsor’s safety office (ORA PSSO) within 24 hours of receipt of the information. 
When a diagnosis is known, the AE term recorded on the eCRF will be the diagnosis rather than 
a constellation of symptoms. The investigator will assess all AEs for seriousness, relationship to 
investigational product, severity, and other possible etiologies. When an event has not resolved 
by study closure, it will be documented on the AE eCRF as “not recovered/not resolved.” 
 
11.7. 
Reporting Adverse Events 
The PI will report all AEs to USAMRDC, the WRAIR IRB, and the USAMRMC ORP in the 
appropriate safety, annual, and/or final reports. After appropriate data cleaning and query 
resolution between the clinical site, sponsor’s clinical monitor, and clinical data manager, SAEs 
from the clinical database will be reconciled with the sponsor’s SAE database. SAEs and AEs for 
inclusion in annual and final reports to the FDA will be provided from the clinical database by 
the clinical data manager in the Clinical Services Support Division. 
 
11.7.1. 
Reporting Serious and Unexpected Adverse Events 
Contact information for reporting SAEs is provided in Table 13. Email reporting of SAE 
should be sent to: usarmy.detrick.medcom-usamrmc.mbx.sae-reporting@mail.mil. 
 
11.7.1.1. Reporting to the Sponsor 
All SAEs must be reported promptly (within 24 hours) to the sponsor’s safety office (ORA 
PSSO) per 21 CFR 312.64, whether or not the event is considered related to study product using 
a sponsor-approved Serious Adverse Event Form. Further, the investigator should comply with 
relevant study site standard operating procedures (SOP) on reporting SAEs. 
The minimum information that the investigator will provide to the ORA PSSO is specified in 
Table 14. The sponsor’s representative may request additional information for purposes of the 
study. 
In order to comply with regulations mandating sponsor notification of specified SAEs to the 
FDA within 7 calendar days, investigators must submit additional information as soon as it is 
available. The sponsor’s representative will report unexpected SAEs associated with the use of 
the vaccine to the FDA as specified at 21 CFR 312.32 (c). 
Investigators must follow all relevant regulatory requirements as well as specific policy 
regarding the timely reporting of SAEs to the research monitor, the WRAIR IRB. 
Reporting to the sponsor’s safety office does not fulfill the investigator’s duty to report all 
unanticipated problems involving risk to human subjects or others to the IRB. The PI will notify 
the WRAIR IRB, and the research monitor. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
65 
US Government Proprietary 
 
 
Table 13: 
Study Contacts for Reporting Serious Adverse Events and Unanticipated 
Problems Involving Risk to Patients or Others 
 
Sponsor’s Safety Office 
US Army Medical Research and Development Command 
Office of Regulated Activities 
Product Safety Surveillance Office ATTN: MCMR-UMR 
1430 Veterans Drive 
Fort Detrick, MD 21702-5009 
Fax: 301-619-7790 
Telephone: 301-619-1106 
Email: usarmy.detrick.medcom-usamrmc.mbx.sae- 
reporting@mail.mil 
Institutional Review Board 
503 Robert Grant Avenue 
Silver Spring, MD 20910-7500 
Phone: 301-319-9940 
Fax: 301-319-9961 
Email: usarmy.detrick.medcom-wrair.mbx.hspb@mail.mil 
Research Monitor 
LCDR Tida Lee MD 
Naval Medical Research Center(NMRC) 
503 Robert Grant Avenue 
Silver Spring, MD 20910-7500 
Telephone: 
Fax: 
Email: tida.k.le.mil@mail.mil 
 
Table 14: 
SAE Information to be Reported to the Sponsor's Safety Office 
 
Notification Method Information to be Provided 
Email or Telephone 
(within 24 hours of 
knowledge of event) 
IND number, sponsor study number, name of the investigational product, and 
investigator name and contact number 
Subject identification number 
SAE term, description, onset date, date(s)of investigational product 
administration, severity, relationship to investigational product, and subject’s 
current status 
AND 
Email or Fax 
Cover sheet or letter 
Sponsor-approved Serious adverse event report form 
Concomitant medication case report form or a list of concomitant medications 
taken within 30 days of the event 
Discharge summary or Medical record progress notes including pertinent 
laboratory/diagnostic test results 
 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
66 
US Government Proprietary 
 
 
11.7.1.2. Reporting to the IRB 
 
Serious Adverse Events Reporting: 
Serious Adverse Events (SAEs) that are related and all deaths should be promptly (within 48 
hours) reported by the investigator to the WRAIR IRB. Immediate and prompt notifications 
should be in the form of email, phone, or fax. A written report for each reportable SAE and 
deaths will be provided to the WRAIR IRB within 10 working days of the initial notification. A 
summary of all Serious Adverse Events reported for the study will be provided in the continuing 
review reports. 
The investigator should not wait to receive additional information to fully document the event 
before notifying the HSPB/WRAIR IRB of a related serious adverse event. As new information 
is obtained, updated reports should be submitted, and if available, should include copies of 
relevant hospital case records, autopsy reports, and other documents where applicable. 
 
Unanticipated Problems Reporting: 
All unanticipated problems involving risk to subjects or others will be promptly (within 48 
hours) reported by phone, by email, or by fax to the WRAIR IRB. A complete written report will 
follow the initial notification within 10 working days. In addition to the methods above, the 
complete report may also be physically delivered to the address of the WRAIR HSPB. All 
unanticipated problems reported during the study will be summarized in the continuing review 
reports. 
Investigators are required to forward safety information provided by the sponsor’s representative 
to the WRAIR IRB. 
 
11.7.2. 
Reporting Additional Immediately Reportable Events to the Sponsor’s Safety 
Office and WRAIR IRB 
 
11.7.2.1. 
Pregnancy 
Each pregnancy must be reported within 24 hours of identification on the Pregnancy Report 
Form by email or fax to the sponsor’s safety office (ORA PSSO). Subject pregnancy will also be 
promptly reported to the WRAIR IRB within 48 hours by phone, fax, or email. 
Subjects who become pregnant after Day 0 will be followed to term, and the following 
information will be gathered for outcome: type and date of delivery, Apgar scores, health status 
of the mother and child including the child’s sex, height, and weight. Complications and or 
abnormalities should be reported including any premature terminations. A pregnancy is reported 
as an AE or SAE only when there is suspicion that the investigational product may have 
interfered with the effectiveness of contraception or there was a serious complication in the 
pregnancy including a spontaneous abortion or an elective termination for medical rationale. 
 
11.7.2.2. 
AE-related Withdrawal of Consent 
Any AE-related withdrawal of consent during the study must be reported within 24 hours of 
identification by email or fax to the sponsor’s representative via the safety mailbox. Report the 
withdrawal to the WRAIR IRB within 48 hours by phone, fax, or email. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
67 
US Government Proprietary 
 
 
11.7.2.3. 
Additional Immediately Reportable Event 
pIMDs must be reported to the WRAIR IRB and sponsor’s safety office (ORA PSSO) within 24 
hours after the diagnosis of a new pIMD or exacerbation of a pre-existing pIMD (serious or non- 
serious) are made known to the primary investigator or representative. 
 
11.7.2.4. 
Pending Inspections/Issuance of Reports 
The knowledge of any pending compliance inspection/visit by the FDA, Office for Human 
Research Protections (Department of Health and Human Services), or other government agency 
concerning clinical investigation or research, the issuance of Inspection Reports, FDA Form 483, 
warning letters, or actions taken by any regulatory agency including legal or medical actions and 
any instances of serious or continuing noncompliance with the regulations or requirements will 
be reported as soon as study investigators become aware to the WRAIR IRB and the 
USAMRMC ORP and the sponsor’s representative. 
 
11.7.3. 
IND Annual Report and Final Clinical Study Report 
 
11.7.3.1. 
IND Annual Report 
The PI will contribute to the preparation of a detailed annual synopsis of clinical activity, 
including adverse events, for submission to USAMRDC. Each annual report will summarize 
IND activity for 1 year beginning approximately 3 months before the IND FDA anniversary 
date. The sponsor’s representative will notify the PI of the due date with sufficient time for the 
PI to assemble the required information. 
 
11.7.3.2. 
Final Report 
A final study report will be prepared in accordance with “Guidance for Industry: Submission of 
Abbreviated Reports and Synopses in Support of Marketing Applications” and ICH E3 Guideline 
“Structure and Content of Clinical Study Reports” and provided to USAMRDC for review and 
approval. USAMRDC will use this report to prepare the final clinical study report for submission 
to the FDA. 
 
12. 
STATISTICS 
12.1. 
Description of Statistical Methods 
Detailed statistical procedures, listings, table shells and figures will be provided in a separate 
statistical analysis plan (SAP). The SAP will be finalized before database lock. The following 
key statistical components will be considered and a detailed description will be documented in 
the SAP: 
• Primary and secondary endpoints and how they will be measured, 
• Statistical methods and tests that will be used to analyze the endpoints, 
• Strategy that will be used if the statistical test assumptions are not satisfied, 
• Indication of whether the comparisons will be one-tailed or two-tailed (with justification 
of the choice) and the level of significance to be used, 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
68 
US Government Proprietary 
 
 
• Identification of whether any adjustments to the significance level or the overall p value 
will be made to account for any planned or unplanned subgroup analyses or multiple 
testing, 
• Specification of potential adjusted analyses and a statement with which covariates or 
factors will be included, 
• Planned exploratory analyses and justification of their importance, and 
• Any subgroup effects with biological justification and support from within and outside 
the study. 
 
12.1.1. 
Analysis Addressing the Primary Study Objective 
The primary objective is the safety and reactogenicity of the 2 vaccine candidates in a 
heterologous prime-boost strategy in the 27 days after each vaccination, inclusive of the day of 
vaccination. The percentage of subjects reporting any AE within 28 days (0-27) following 
vaccination and their 95% confidence interval (CI) will be computed using the Wilson score or 
exact (Clopper-Pearson) methods by groups according to the type of AE, intensity, and 
relationship to vaccination. The occurrence of related SAEs, PIMDs, and medically attended 
AEs will be reported descriptively.. 
 
12.1.2. 
Analysis Addressing the Secondary Study Objective 
Seropositivity rates for each DENV type along with trivalent and tetravalent antibody responses 
as determined by MN50 cutoff titer will be calculated with 95% CI using the Wilson score or 
exact (Clopper-Pearson) methods for each vaccination group. Antibody titers will be summarized 
as GMTs, with the 95% CI, and reverse cumulative curves. A DENV-primed subject is defined 
with a MN50 titer cutoff of ≥ 10. 
Additionally the cell-mediated immune (CMI) response to the 2 tetravalent dengue vaccine 
candidates in a heterologous prime-boost schedules will be assessed. CMI markers will be 
evaluated by, but not limited to, the following methods: ELISPOT, flow cytometry, CFSE dye- 
dilution proliferation, Luminex, transcriptomics, and proteomics. For adaptive B and T cell 
response assessments statistical analysis will be performed using GraphPad Prism 6, Stata, or 
other well established software packages. Non-parametric tests will be used as the default to 
compare effects between different test and control groups. Where appropriate parametric 
statistical tests (eg, T-tests, ANOVA) will be applied to normally distributed data. P values less 
than 0.05 will be considered significant. Standard deviations and 95% confidence intervals will 
also be measured. Corrections for multiple comparisons will be made where necessary and 
appropriate. For the multiplex bead-based assays and RNA expression studies proprietary and 
specialty software will be used for analysis. 
12.1.3. 
Analysis Addressing Exploratory Objective 
Analysis of type specific antibody levels rate of decay will be described and compared between 
the 0, 180 Day PIV/LAV and 0, 90 Day PIV/LAV regimens. The percentage of subjects with a 
measurable dengue RNAemia by DENV type will be tabulated after each dose of LAV for each 
study group and quantitative RNAemia levels will also be comparedSafety Analyses 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
69 
US Government Proprietary 
 
 
Safety analysis will include data collected from all subjects who received at least one 
vaccination. Adverse event data will be listed individually (including intervention and outcome) 
and summarized by body system and preferred terms within a body system for each treatment 
group. Serious and/or unexpected AEs will also be discussed on a case-by-case basis. For the 
tabulation of the AEs by body system, a subject will be counted only once in a given body 
system. For example, a subject reporting nausea and diarrhea will be reported as one subject, but 
the symptoms will be listed as 2 separate AEs within the class. Therefore the total number of 
AEs reported within a body system may exceed the number of subjects within the body system 
reporting AEs. The safety analysis will be included in the final study report. 
 
12.1.4. 
Clinical Laboratory Data Analyses 
For hematology and serum chemistry tests, the mean, mean change, median, median change 
along with standard deviations, and range of all values for each test for each treatment group at 
baseline and for the final ‘on therapy’ values will be printed in a summary table. Another table 
will be prepared displaying the numbers of subjects in each treatment group who had values 
below, within, and above the normal range at baseline and at the final visit. 
These tables will be reviewed by the PI or research monitor to evaluate whether any significant 
trends in laboratory values occurred. The PI or research monitor will also review the urinalysis 
data by inspecting the laboratory data tabulations, but no summary tables of these will be 
prepared. 
The cut-off value used to determine seropositivity for DENV neutralizing antibodies titers in a 
DENV-primed subject is defined by a MN50 titer cutoff of ≥ 10 or via flow cytometry based 
antibody neutralization assay NT50 ≥ 40. A seronegative subject for a specific DENV type is a 
subject whose titer for that DENV type is below the cut-off value while a seropositive subject is 
a subject whose titer for that DENV type is greater than or equal to the cut-off value. 
Tetravalent seropositivity rate is defined as the percentage of subjects who are seropositive to all 
4 DENV types. 
 
Trivalent seropositivity rate is defined as the percentage of subjects who are seropositive to 3 
DENV types. 
 
The GMT calculations are performed by taking the anti-log of the mean of the log 10 titer 
transformations. Antibody titers below the cut-off of the assay will be given an arbitrary value of 
half the cut-off for the purpose of GMT calculation. For a given subject and a given 
immunogenicity measurement, missing or non-evaluable measurements will not be replaced but 
will be noted. 
For the analysis of unsolicited adverse events/serious adverse event/concomitant medication, 
subjects who did not report an event will be considered as subjects without an event. In case of 
significant non-compliance of study procedures for reporting symptoms, the analysis plan will be 
reassessed to ensure more accurate reporting of study data by further analysis. 
 
For the analysis of solicited symptom, missing or non-evaluable measurements will not be 
replaced. Therefore the analysis of the solicited symptoms based on the total vaccinated cohort 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
70 
US Government Proprietary 
 
 
will include only subjects/doses with documented safety data (ie, symptom screen/sheet 
completed). 
 
12.1.5. 
Schedule of Analyses 
There will be 2 separate database locks that will occur at the visit level with analyses occurring 
after each one. 
Once all data for all subjects have been collected and entered into the database up to and 
including Visit 10 (28 days post final vaccination) the database will be locked/frozen for all data 
up to Visit 10 and these data will be analyzed as described above for both groups. 
After the database lock/freeze for data up to Visit 10, data will continue to accrue for only those 
events subsequent to Visit 10. The database will be locked/frozen for all events subsequent to 
Visit 10 after the remainder of the data have been collected and entered into the database. These 
data will be analyzed as described above for both groups after this final database lock. 
The analyses performed at these 2 time points will be used to generate the final clinical study 
report. 
 
12.2. 
Planned Enrollment and Reason for Sample Size 
The sample size of 40 subjects is appropriate to further define safety profile for PIV/LAV 
formulations in healthy adult population living in a non-endemic region and to perform further 
evaluation of immunogenicity in this population. No sample size calculation in relation to a 
specific objective was performed. 
Table 15 shows the precision of the 95% CI that a sample size of 20 subjects per group will 
provide for varying frequency rates of AEs using the Wilson score or exact (Clopper-Pearson) 
method. Major differences in terms of safety and reactogenicity between the vaccination 
schedules are not expected and this study is not powered to detect small differences in 
reactogenicity. Differences in immunogenicity in terms of GMTs and antibody level durability 
will be compared between the 0, 180 Day PIV/LAV and 0, 90 Day PIV/LAV and 95% CI will be 
compared for statistical significance. Given these represent continuous variables and up to 10 
fold differences in antibody titers were seen between some groups and timepoints in WRAIR 
2136, statistically significant differences in immunogenicity results are expected if such 
differences exist and are in fact substantial. Using all PIV-LAV antibody titers at six months for 
all dengue strains, the pooled standard deviation of the log-transformed antibody titers was 
0.049. Based on this standard deviation and a sample size of 20 subjects per group, the smallest 
detectable difference between groups that can be observed with 80% power and 95% confidence 
is 10.7% increase (a difference of 0.0444 in the log10-transformed means). At 90% power this 
increases to a 12.6% increase (a difference of 0.0444 in the log10-transformed means). Assuming 
a two-fold increase in antibody titers is the minimal potentially clinically significant difference, 
there is greater than 99.9% power to detect this difference under the current design. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
71 
US Government Proprietary 
 
 
Table 15: 
Precision for the 95% Confidence Interval for Varying Frequency Rates 
(N=20) 
 
Number of Subjects with Events 
/ Number of Vaccinated 
Subjects 
Frequency of Subjects 
with Event 
95% CIs for the Frequency 
of Subjects with Event 
1/20 
5.00% 
0% - 14.60% 
2/20 
10.00% 
0% - 23.10% 
5/20 
25.00% 
6.00% - 44.00% 
10/20 
50.00% 
28.10% - 71.90% 
 
12.3. 
Level of Significance to be Used 
Statistical significance will be defined with an alpha of 0.05 and/or lack of overlapping values 
when 95% CIs are calculated. 
 
12.4. 
Statistical Criteria for the Termination of the Trial 
12.4.1. 
Stopping Rules 
There are no statistical criteria for study termination in this uncontrolled clinical trial. Adverse 
event and laboratory assay study hold rules are presented in Section 9.2. 
 
12.5. 
Accounting for Missing, Unused, and Spurious Data 
Non-analyzable data will be documented in the deviations. 
 
12.6. 
Procedures for Reporting Deviations from the Original Statistical 
Plan 
Any planned changes from the original statistical plan will be described in an amendment to the 
protocol and/or the SAP as necessary. Deviations from the SAP will be documented in the final 
clinical study report. 
 
12.7. 
Selection of Subjects to be Included in Analyses 
12.7.1. 
Total Vaccinated Population 
The total vaccinated population will include all subjects who received at least one vaccination. 
While those who do not receive both vaccinations in this heterologous prime-boost study will be 
monitored for safety signals, only those who received 2 dengue vaccinations will be included in 
analysis of immunological endpoints. 
 
12.7.2. 
According to Protocol Population 
The according to protocol (ATP) population for analysis of immunogenicity will include all 
subjects who receive both doses of study vaccine according to protocol and: 
• Meet all eligibility criteria 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
72 
US Government Proprietary 
 
 
• Are seronegative to all 4 DENV types before vaccination 
• Comply with the procedures and intervals defined in the protocol 
• Did not present with a potentially confounding medical condition 
The ATP population will be defined at each analysis time point and subjects will be included 
based on their available data up to that time point. Subjects will not be retrospectively removed 
from the ATP population based on data from later time points. 
The following criteria should be checked at each visit subsequent to the first vaccination. If any 
become applicable during the study, it will not require withdrawal of the subject from the study 
but may determine a subject’s ability to be evaluated in the ATP analysis. 
• Use of any investigational or non-registered product (drug or vaccine) other than the 
study vaccine during the study period. 
• Chronic administration (defined as more than 14 days) of immunosuppressants or other 
immune-modifying drugs during the study period. For corticosteroids, this will mean 
prednisone ≥ 20 mg/d or equivalent. Inhaled and topical steroids are allowed. 
• Administration of a vaccine not foreseen by the study protocol during the period starting 
30 days before dose 1 and ending 30 days after dose 2. 
• Administration of immunoglobulins and/or any blood products during the study period. 
• Subject incurs a condition that has the capability of altering their immune response (ie, 
flavivirus vaccination) or is confirmed to have an immunodeficiency condition. 
A detailed, comprehensive list of reasons for elimination from ATP analyses will be established 
at the time of data cleaning. 
 
13. 
ETHICS 
13.1. 
Compliance Statement 
The study will be conducted according to the protocol and in accordance and in compliance with 
ICH GCP, Belmont Principles, DoD requirements (32 CFR 219, DoDI 3216.02), and 21 CFR 50, 
56, and 312.All identified study personnel will be trained to perform their roles and will carry out 
their responsibilities in accordance with ICH GCP guideline and clinic site SOPs. Roles and 
responsibilities of study staff are presented in Appendix A. 
 
13.2. 
Ethics Review 
The study is based on adequately performed laboratory and animal experimentation and will be 
conducted under a protocol reviewed by the WRAIR IRB. The study is to be conducted by 
scientifically and medically qualified persons. The IRB will determine whether the benefits of 
the study are in proportion to the risks. The rights and welfare of the subjects will be respected; 
the physicians conducting the study will ensure that the hazards do not outweigh the potential 
benefits; the results to be reported will be accurate; subjects will give their informed consent and 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
73 
US Government Proprietary 
 
 
will be competent to do so and not under duress; and all study staff will comply with the ethical 
principles in 21 CFR Part 50 and the Belmont Principles. 
 
13.2.1. 
Review/Approval of Study Protocol 
Before a clinical study can be initiated, the study protocol and other required documents will be 
submitted to the following departments in the order listed for review and/or approval, with the 
final review by the FDA: 
• Integrated Product Team 
• WRAIR Scientific Review Committee 
• Sponsor’s protocol review board 
• WRAIR Commander 
• WRAIR IRB 
• Sponsor’s Representative (acting for The Surgeon General of the Army) 
• USAMRMC Commanding General, if applicable 
Enrollment in this protocol may not begin until all approvals have been obtained, and the formal 
authorization letter is received by the PI from the sponsor’s representative. 
 
13.2.2. 
Confidentiality 
In this research, the subject’s health information will be collected and used to conduct the study; 
to monitor the subject’s health status; to measure effects of the investigational product; to 
determine research results, and possibly to develop new tests, procedures, and commercial 
products. Health information is used to report results of research to the sponsor’s representative 
and Federal regulators and may be reviewed during study audits for compliance with study plans, 
regulations, and research policies. After the study ends, each subject has the right to see and 
receive a copy of his/her information. 
Representatives of the TSG as the IND sponsor, USAMRDC as the sponsor’s representative, the 
WRAIR IRB and the ORP, the DoD, and the FDA are authorized to photocopy and review 
records related to this protocol as a part of their responsibility to protect the participants of this 
protocol. In addition, these representatives are authorized to witness the applicable study 
procedures to assure the safety of subjects. 
Each subject’s personally identifiable healthcare information will be kept confidential to the 
extent possible but confidentiality cannot be guaranteed. Subject identification numbers will be 
used to facilitate confidentiality in discussing subject specific information during and after the 
study. 
No personal identifier will be used in any publication or communication used to support this 
research study. The subject’s identification number will be used in the event it becomes 
necessary to identify data specific to a single subject. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
74 
US Government Proprietary 
 
 
13.2.3. 
Compensation for Participation 
Subjects will receive compensation for their participation in this study. Study volunteers will be 
provided a check, reloadable debit card, gift card or cash from the WRAIR CTC as 
compensation for visits. 
Subjects will receive $50 for the screening visit and $130 for each of the scheduled 14 study 
visits once enrolled. Blood draws are scheduled to occur at every visit. For non-active duty 
military and non-federal employees, the maximum compensation is $2210 if all visits are kept. 
Under 24 USC 30, active duty personnel and federal employees will receive $50 for the 
screening visit and $50 for each study visit involving blood draws during duty or work hours. If 
the visit occurs during off-duty hours or non-work hours, the subject will be eligible to receive 
the full compensation of $130. Other than medical care that may be provided and any other 
payment specifically stated in the consent form, there is no other compensation available to the 
subjects participating in this clinical trial. Subjects will not be compensated for any missed visits 
during the study. Subjects will be compensated $50 for each unscheduled visit if medically 
necessary and blood draw occurs. Subjects will be offered a $200 bonus on their last visit in 
addition to the $130 standard visit amount if they have made all of their study visits within the 
required time ranges. This is to encourage participants to stay in the study and complete all visits 
in this study which will run a total of 18 months. 
Compensation for non-military, non-federal employees and military or federal employee on 
leave or non-working hours: 
Screening ($50) + Visits 1-14 ($130 each x 12 = $1560) + Vaccination visits (2x $200) 
+completion bonus ($200) = $2210 total 
Compensation for military during duty or federal employees during work hours: 
Screening ($50) + Visits 1-14 ($50 each x 14 = $700) = $750 total 
There will be $25 referral bonus paid when recruiting another volunteer into the study. A subject 
is eligible for this referral bonus even if he or she is excluded at screening. Also note that active 
duty personnel or federal employees may only receive the completion bonus at their final visit if 
this visit occurs while on approved leave or outside duty hours.Medical Care for Research- 
Related Injury 
All non-exempt research involving human subjects shall, at a minimum, meet the requirement of 
32 CFR 219.116(a)(6). 
If a subject is injured because of participation in this research and is a DoD healthcare 
beneficiary (eg, active duty in the military, military spouse or dependent), the subject is entitled 
to medical care for that injury within the DoD healthcare system, as long as the subject remains a 
DoD healthcare beneficiary. This care includes, but is not limited to, free medical care at Army 
hospitals or clinics. 
If a subject is injured because of participation in this research and is not a DoD healthcare 
beneficiary, the subject is entitled to medical care for that injury at an Army hospital or clinic; 
medical care charges for care at an Army hospital or clinic will be waived. The subject is also 
entitled to care for that injury, but such care for that injury at other DoD (non-Army) hospitals or 
clinics may be limited by time, and the subject’s insurance may be billed. It cannot be 
determined in advance which Army or DoD hospital or clinic will provide care. If the subject 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
75 
US Government Proprietary 
 
 
obtains care for research-related injuries outside of an Army or DoD hospital or clinic, the 
subject or the subject’s insurance will be responsible for medical expenses. 
For DoD healthcare beneficiaries and non-DoD healthcare beneficiaries: Transportation to and 
from hospitals or clinics will not be provided. No reimbursement is available for incurred 
medical expenses to treat research-related injuries. No compensation is available for research- 
related injuries. 
Subjects are not waiving any legal rights. If a subject believes that they have sustained a 
research-related injury, they should contact the Principal Investigator (PI). If they have any other 
questions regarding research-related injuries, they should contact the PI. 
 
13.3. 
Biological Sample Handling and Analysis 
All sample testing will be done in line with the consent of the individual subject. 
Samples collected under this protocol will be used to conduct protocol-related safety and 
immunogenicity evaluations. 
Collected samples may also be used in other assays, for test improvement or test development of 
analytical methods related to the study vaccine and its constituents or the disease under study to 
allow a more reliable measurement of the vaccine response. Under these circumstances, 
additional testing on the samples may be performed by the sponsor outside the scope of this 
protocol. 
Genetic testing may be performed on these samples only after specific informed consent is 
acquired for such testing and IRB approval is obtained. 
In order to be enrolled in the study, the subject must agree to allow their samples to be used for 
future studies.. This is because we cannot predict what future studies will be most useful and 
cannot confidently enumerate all studies that need to be performed in this protocol. We 
anticipate that future laboratory analysis will be necessary and so we ask subjects to consent to 
this use as well. However, a subject may decide at any point during the duration of the study to 
withdraw consent for the future use of his/her samples. Should a subject withdraw consent for 
the use of his or her samples, the samples will be destroyed. 
Collected samples will be stored until exhausted through use in this or future laboratory studies, 
unless local rules, regulations or guidelines require different timeframes or different procedures, 
which will then be in line with the subject consent. Samples will not be sold for profit or used as 
a component in a commercial product being sold for profit. 
 
13.4. 
Specimen Storage and Labeling 
The Viral Diseases Branch (VDB), Compliance Management Unit (CMU) shall manage (log-in, 
track, and issue) specimens via the FreezerWorks® database. Specimens shall be physically 
stored in VDB CMU-controlled storage units based on temperature requirements [ie, -80±10°C, 
vapor-phase liquid nitrogen (-130°C or below), etc]. All storage units used to store specimens for 
this study shall be access-controlled and environmentally-monitored on a 24/7 basis. VDB CMU 
will also generate and manage specimen labels via a combination of the Zebra Designer Pro®, 
FreezerWorks®, and Microsoft® Access software. Specimens will be labeled in accordance with 
the template shown in Appendix B. Study primary investigator and sub-investigators along with 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
76 
US Government Proprietary 
 
 
VDB immunologists and microbiologists will have access to these specimens and may obtain 
these specimens for testing through request to the VDB CMU. 
 
14. 
DATA HANDLING AND RECORDKEEPING 
For this study, an electronic data capture (EDC) database system will be used for the collection 
of the study data in an electronic format. The EDC database system will be designed based on 
the protocol requirements, the approved eCRF layouts and specifications, and in accordance with 
21 CFR Part 11. The eCRF layouts and specifications define and identify the applicable source 
data that will be collected and captured into the EDC database system. The applicable source 
data will be electronically transcribed by the site designee onto the eCRF (data entry screens) in 
the EDC database system. The investigator is ultimately responsible for the accuracy of the data 
transcribed on the eCRF. Data monitoring and management will be performed in the EDC 
database system by the study clinical monitor and the designated Data Management group. 
A detailed data management plan will be written and approved by the study team and the PI prior 
to study start, with approval by the sponsor’s data manager in the USAMRDC ORA. All updates 
to the data management plan must be approved before study close-out and database lock. 
Any missing, unused or obviously spurious data will be rectified through review of the 
corresponding primary source documents. 
 
14.1. 
Inspection of Records 
The sponsor’s representative or designee will be allowed to conduct site visits at the 
investigation facilities for the purpose of monitoring or auditing any aspect of the study. The 
investigator agrees to allow the monitor to inspect the study product storage area, investigational 
product stocks, study product accountability records, subject charts, study source documents, and 
other records relative to study conduct. 
Subjects’ health information is used to report results of research to the sponsor’s representative 
and Federal regulators and may be reviewed during study audits for compliance with study plans, 
regulations, and research policies. The consent document indicates that by signature, the subject 
permits access to relevant medical records by the sponsor’s representative and by representatives 
of the FDA. 
Upon a subject’s termination from the trial, completed eCRFs will be ready and available for on- 
site review by the sponsor’s representative or the designated representative within 14 days after 
receipt of the subject’s data. 
 
14.2. 
Retention of Records 
The PI must maintain all documentation relating to the study for a period of 2 years after the last 
marketing application approval, or if not approved for 2 years following the discontinuance of 
the investigational product for investigation. If it becomes necessary for the sponsor’s 
representative or designee or the FDA to review any documentation relating to the study, the 
investigator must permit access to such records. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
77 
US Government Proprietary 
 
 
Completed, monitored eCRFs will be stored in a secure location by the sponsor’s representative 
or designee. A copy of each completed eCRF will be retained by the investigator. Subject charts 
will be maintained in a secure location in the CTC during the study with access permitted only to 
the PI and the project coordinator. Upon completion of the clinical phase, these records will be 
stored in a locked cabinet within the CTC, which also can be accessed by only the clinical 
coordinator or PI, until moved to the secure archives in Building 500 after study closeout. 
The PI will be responsible for retaining sufficient information about each subject, ie, name, 
address, telephone number, Social Security number, and subject identifier in the study, so that 
the sponsor’s representative, the WRAIR IRB, the FDA, employees of USAMRMC, or other 
regulatory authorities may have access to this information should the need arise. 
It is the policy of the USAMRMC that data sheets are to be completed for all subjects 
participating in research (Form 60-R, Volunteer Registry Data Sheet). The data sheets will be 
entered into this Command’s Volunteer Registry Database. The information to be entered into 
this confidential data base includes the subject’s name, address, and Social Security Number; 
study title; and dates of participation. The intent of this data base is twofold: first, to readily 
answer questions concerning an individual’s participation in research sponsored by USAMRMC; 
and second, to ensure that USAMRMC can exercise its obligation to ensure research subjects are 
adequately warned (duty to warn) of risks and to provide new information as it becomes 
available. The information will be stored at USAMRMC for a minimum of 75 years. The 
Volunteer Registry Database is a separate entity and is not linked to the study database. 
 
15. 
QUALITY CONTROL, QUALITY ASSURANCE, AND DIRECT 
ACCESS TO SOURCE DATA/DOCUMENTS 
Subjects will be identified on eCRFs by a unique subject identification number and on source 
documents by name and date of birth. No personal identifier will be used in any publication or 
communication used to support this research study. The subject identification number will be 
used if it becomes necessary to identify data specific to a single subject. Representatives of 
USAMRMC, the sponsor’s representative, the WRAIR IRB and/or the USAMRMC ORP, and 
the FDA are eligible to review medical and research records related to this study as a part of their 
responsibility to protect human subjects in clinical research. Personal identifiers will be removed 
from photocopied medical and research records. 
 
15.1. 
Study Monitoring 
Study monitoring will be the responsibility of the USAMRDC ORA. Upon successful approval 
of the protocol and establishment of the regulatory file, the clinical monitor will establish a 
clinical monitoring plan. To ensure that the investigator and the study staff understand and 
accept their defined responsibilities, the clinical monitor will maintain regular correspondence 
with the site and may be present during the course of the study to verify the acceptability of the 
facilities, compliance with the investigational plan and relevant regulations, and the maintenance 
of complete records. As needed, the clinical monitor may witness the informed consent process 
or other applicable study procedures to assure the safety of subjects and the investigators’ 
compliance with the protocol and GCPs. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
78 
US Government Proprietary 
 
 
Monitoring visits by a sponsor’s representative-designated clinical monitor will be scheduled to 
take place at the initiation of the study, during the study at appropriate intervals, and after the last 
subject has completed the study. A report of monitoring observations will be provided to the PI 
(for corrective actions) and members of the Sponsor’s Representative team. 
 
15.2. 
Audits and Inspections 
Authorized representatives of the sponsor, the FDA, the independent ethics committee, the 
WRAIR Institutional Review Board, or other regulatory agencies may visit the site to perform 
audits or inspections, including source data verification, and review the regulatory files. The 
purpose of the audit or inspection is to systematically and independently examine all study- 
related activities and documents to determine whether these activities were conducted, and data 
were recorded, analyzed, and accurately reported according to the protocol, GCP guideline of the 
ICH, and any applicable regulatory requirements. Auditing of the clinical trial may be conducted 
at any time during the study. 
The investigator should contact the sponsor’s representative and Office of Research Protections, 
Human Research Protection Office (ORP HRPO) immediately if contacted by a regulatory 
agency about an inspection. 
Auditing may also be performed by independent personnel designated by the Office of Quality 
Management, USAMRDC. Audit findings will be documented in a formal audit report that will 
detail the conduct of the audit and summarize the observations noted. 
 
15.3. 
Institutional Review Board 
As the IRB of record, the WRAIR IRB will serve as the responsible IRB and will review the 
protocol, informed consent, and progress reports on a continuing basis in accordance with all 
applicable regulations, including Title 21, Code of Federal Regulations (CFR), Parts 50 and 56. 
The PI must obtain IRB approval for the study. Initial IRB approval and all materials approved 
by the IRB for this protocol, including the subject consent form and recruitment materials, must 
be maintained by the PI or representative or designee and made available for inspection. The PI 
is responsible for preparing and submitting continuing review reports to the WRAIR IRB before 
the established continuing review date or at least on an annual basis, whichever comes first. All 
deviations, unanticipated problems, and SAEs occurring within the reporting period should be 
summarized in the continuing review reports submitted to the WRAIR IRB. The PI will submit a 
closeout report to the WRAIR IRB once the study is complete. 
 
15.4. 
Protocol Suspensions or Discontinuation 
The study may be suspended or discontinued at any time by the Sponsor, WRAIR IRB, US FDA, 
or other government agencies, as part of their duties to ensure that research participants are 
protected. Any suspensions (to include continuing review lapses), clinical holds (voluntary or 
involuntary), or terminations of this research by the IRB, the Sponsor, or Regulatory Agencies 
will be promptly (within 48 hours) reported to the WRAIR IRB by phone (301-319-9940), by 
email (usarmy.detrick.medcom-wrair.mbx.hspb@mail.mil), or by facsimile (301-319-9961). 
Such a change in study status should also be reported to the sponsor’s representative as well. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
79 
US Government Proprietary 
 
 
16. 
PROTOCOL MODIFICATIONS AND DEVIATIONS 
16.1. 
Protocol Modifications 
All amendments and modifications will be reviewed and approved by the WRAIR IRB prior to 
implementation. Any protocol amendment will be agreed upon and approved by the sponsor’s 
representative prior to submission to the WRAIR IRB and administrative review by NMRC. 
Any modification or amendment that could potentially increase risk to subjects must be 
submitted to the FDA prior to implementation. The informed consent document will be revised 
to concur with any significant amendment that directly affects volunteers, and will also be 
reviewed and approved with the amendment. New subjects enrolled in the study will be 
consented with the most recent approved consent documents. Subjects already enrolled in the 
study will be informed about the revision and, depending on the impact of the amendment, may 
be asked to re-consent. This will be accomplished by repeating the consent process with the 
revised consent document with attention given to the changes, or it may be done using an 
addendum consent that states the revision or new information. The new documents will be 
signed, placed in the study record, and signed copies will be given to the subject. 
 
16.2. 
Protocol Deviation Procedures 
A protocol deviation is defined as any occurrence involving a procedure that did not follow the 
study protocol, applicable procedures, and/or regulatory requirements. All staff involved in the 
conduct of a clinical trial will be aware of the specific protocol requirements for completing 
study visits and notify the PI in the event of any breach of protocol according to the following 
procedures. Deviations are recorded in the source documents and reported as per requirements. 
Notify the PI as soon as possible if they are not present when the deviation is discovered. 
Document in the volunteer’s study chart all protocol deviations directly involving or affecting 
the volunteer, and document a thorough explanation of the circumstances leading up to the 
deviation. The requirement and timeline for reporting protocol deviations to the WRAIR IRB is 
determined by the categorization of the deviation: major or minor. 
Major deviations are departures from protocol that have a potential to affect the rights of the 
research participant, to increase the risk to the participant, to change the willingness of the 
volunteer to continue participation, or to compromise the integrity of the study data in such a 
way that the study objectives may not be achieved. Major deviations that occur will be reported 
to the WRAIR IRB within 48 hours of identification and recorded in the deviation study log, and 
a written report submitted within 10 working days of the PI becoming aware of the deviation. 
All reports will be submitted with a cover memo naming the protocol, description of the event, 
summary of any harm to study participant(s) and steps to prevent further deviations. A summary 
of the major deviations occurring within the reporting period should also be included in the 
continuing review reports. Major deviations will be reported immediately to the sponsor’s 
representative as well. 
Minor deviations are routine departures that typically involve a volunteer’s failure to comply 
with the protocol (Example: missing scheduled visits). Minor deviations that occur will be 
reported to the WRAIR IRB in the Continuing Review Report(s). 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
80 
US Government Proprietary 
 
 
Knowledge of any instances of serious or continuing non-compliance with the regulations or 
requirements will be reported immediately to the WRAIR IRB. 
 
17. 
PUBLICATION POLICY 
All data collected during this study will be used to support this IND. All data may be published 
in the open medical or military literature with the identity of the subjects protected, once the final 
clinical study report has been submitted to the FDA. Anyone desiring to publish or present data 
obtained during the conduct of the study will conform to WRAIR/USAMRDC policies and then 
forward the publication for review to the Commander, USAMRDC or designee and 
usarmy.detrick.medcom-usamrmc.list.clearances@mail.mil prior to submission. 
17.1. 
Initial Analysis 
An initial analysis is planned to review data through to 28 days post vaccination for both Group 
1 and 2. These data will be analyzed following initial database lock/freeze as detailed in the 
statistics section above and will then be available for presentation and/or publication as needed 
following approval through the standard WRAIR/USAMRDC publication approval process. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
81 
US Government Proprietary 
 
 
18. 
LIST OF REFERENCES 
Bauer Kristen et al. A Phase II, Randomized, Safety and Immunogenicity Trial of a Re-Derived, 
Live-Attenuated Dengue Virus Vaccine in Healthy Children and Adults Living in Puerto Rico. 
Am J Trop Med Hyg. 93(3), 2015, pp. 441–453 
Bhatt S. Gething PW, Brady OJ, et al. The global distribution and burden of Dengue. Nature. 
2013;496(7446):504-7. 
Clapham et al. Within-host viral dynamics of Dengue serotype 1 infection. J Royal Soc Interface. 
2014 Jul 6;11(96). 
De Alwis et al. Identification of human neutralizing antibodies that bind to complex epitopes on 
dengue virions. PNAS. 2012; 109(19): 7439-7444. 
Gibbons RV, Streitz M, Babina T, Fried JR. Dengue and US military operations from the 
Spanish-American War through today. Emerg Infect Dis. 2012;18(4): 523-30. 
Halstead SB. Protective and immunologicalbehavior of chimeric yellow fever Dengue vaccine. 
Vaccine. 2016; 34:1643–7. 
Jansenius M, Han PV, Schlagenhauf P, et al. Acute and potentially life-threatening tropical 
disease in western travelers: a GenSentinel multicenter study. Am J Trop Med Hyg. 2013; 
88:397-404. 
Kraus et al. Comparison of Plaque- and Flow Cytometry- Based Methods for Measuring Dengue 
Virus Neutralization. J Clin Micro. 2007; 45(11): 3777-3780. 
Lu S. Heterologous prime-boost vaccination. Curr Opin Immunol . 2009;21:346-351. 
Pitisuttithum P, Bouckenooghe A. The first licensed Dengue vaccine: an important tool for 
integrated preventive strategies against Dengue virus infection. Expert Review of Vaccines. 
2016; 15(7):795–798 
Putnak et al. Compararive Evaluation of Three Assays for Dengue Virus Neutralizing 
Antibodies. AJTMH. 2008; 79(1): 115-122. 
Rothman AL. Dengue: defining protective versus pathological immunity. J Clin Invest. 2004; 
113(7):946-51. 
Sabchareon A, Wallace D, Sirivichayakul C, et al. Protective efficacy of the recombinant, live- 
attenuated, CYD tetravalent Dengue vaccine in Thai school children; a randomized, controlled, 
phase 2b trial. Lancet. 2012; 380:1559-67. 
Schmidt AC, Martinez LJ, Eckels KH et al. Phase I study of a tetravalent Dengue purified 
inactivated virus (DPIV) vaccine in healthy US adults. American Society of Tropical Medicine 
and Hygiene Meeting. LB Poster: 2454. Washington DC, Nov. 13-17, 2013. 
Simasathien, Sriluck et al. Safety and Immunogenicity of a Tetravalent Live-attenuated Dengue 
Vaccine in Flavivirus Naive Children. Am. J. Trop. Med. Hyg., 78(3), 2008, pp. 426–433 
Simmons M, Burgess T, Lynch J, Putnak R. Protection against Dengue virus by non-replicating 
and live-attenuated vaccines used together in a prime-boost vaccination strategy. Virology. 2010; 
396:280–288. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
82 
US Government Proprietary 
 
 
Thomas SJ, Eckels KH, Carletti I, et al. A phase II, randomized, safety and immunogenicity 
study of a re-derived, live-attenuated Dengue virus vaccine in healthy adults. Am J Trop Med 
Hyg. 2013; 88(1):73-88. 
Watanaveeradej, Veerachai et al. Safety and Immunogenicity of a Rederived, Live-Attenuated 
Dengue Virus Vaccine in Healthy Adults Living in Thailand: A Randomized Trial. Am J Trop 
Med Hyg. 91(1), 2014, pp. 119–128 
World Health Organization. Dengue and Severe Dengue Fact Sheet. Updated July 2016. 
http://www.who.int/mediacentre/factsheets/fs117/en/index.html 
Woodland D. Jump-starting the immune system: prime-boosting come of age. Trends Immunol. 
2004; 25(2):98-104. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
83 
US Government Proprietary 
 
 
19. 
APPENDICES 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
84 
US Government Proprietary 
 
 
APPENDIX A. ROLES AND RESPONSIBILITIES 
• Principal Investigator 
− Complies with protocol and all Federal and local regulations and policies and has 
ultimate responsibility for the conduct of the study and submission of the study 
report. 
− Complies with Good Clinical Practices and Belmont Principles 
− Qualified through training, education and experience 
− Supervises sub-investigators and protocol nurse coordinator 
− Trains study staff 
− Screens individuals and reviews all of the screening tests to determine eligibility 
of person to participate in the study. Investigator or designee makes final 
eligibility decision. 
− Permits auditing, institutional monitoring (US Army Medical Research and 
Development Command) and inspection by the US Food and Drug 
Administration 
− Assures adequate resources, time, and patient population to meet study 
requirements 
− Provides medical care for adverse events 
− Documents protocol deviations 
− Reports all serious adverse events to the regulatory bodies (WRAIR IRB) and 
sponsor’s representative (US Army Medical Research and Development 
Command) 
− Reports unanticipated problems and regulatory audits to the IRB 
− Delegates investigational product accountability and administration 
− Evaluates AEs for diagnosis, relationship, and severity, 
− Ensures documentation and safety reporting 
− Ensure that he/she has sufficient time to conduct and complete the study 
− Ensures he/she has adequate staff and appropriate facilities that are available for 
the duration of the study and to ensure that other studies do not divert essential 
subjects or facilities away from the study at hand 
− Submits an up-to-date curriculum vitae and other credentials (e.g. medical license 
number in the United States) to the Sponsor and, where required, to relevant 
authorities 
− Acquires the normal ranges for laboratory tests performed locally and, if required 
by local regulations, obtains the laboratory licenses or certifications 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
85 
US Government Proprietary 
 
 
− Informs the IRB of any non-protocol specified laboratory testing that may occur 
on stored serum samples left over after protocol specified laboratory testing is 
completed (e.g., clinical tests performed in the evaluation of a subject’s medical 
problem or additional specific immunologic evaluations as they become 
available) 
− Prepares and maintain adequate case histories designed to record observations and 
other data pertinent to the study 
− Conducts the study in compliance with the protocol and any amendments 
− Cooperates with representatives of USAMRDC in monitoring the study and in 
resolution of queries about the data 
• Sub-investigators 
− Comply with Good Clinical Practices and Belmont Principles 
− Qualified by training, education and experience 
− Assist Principal Investigator in conduct of study 
− Assist in supervising study staff 
− Review all of the screening tests to determine the eligibility of individuals to 
participate in the study (Principal investigator or designee makes final eligibility 
decision) 
− Evaluate adverse events 
− Screen individuals for protocol participation (physical examination and medical 
history) 
• Study Coordinator(s) 
− Complies with Good Clinical Practices and Belmont Principles 
− Qualified by training, education and experience, and holds a current, unrestricted 
nursing license 
− Delegates investigational vaccine accountability to licensed nursing personnel 
− Reviews all of the screening tests 
− Schedules follow-up appointments 
− Collects information on adverse events 
− Accurately and promptly completes case report forms 
− Trains staff on protocols/protocol changes 
− Documents protocol deviations 
− Stores IND product in temperature controlled and monitored units 
− Signs out IND product to the investigational product preparer 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
86 
US Government Proprietary 
 
 
− Verifies and records administration of investigational products to study 
participants 
• Other Study Staff 
− Comply with Good Clinical Practices and Belmont Principles 
− Qualified through training, education and experience 
− Recruit study participants with IRB approved materials 
− Collect pre- and post-vaccination vital signs 
− Contact study participants to collect adverse event data 
− Schedule follow-up appointments 
− Follow-up on non-compliant study participants 
− Ensure efficient clinic flow 
− Perform subject specific training 
• Study Administrative Support 
− Complies with Good Clinical Practices and Belmont Principles 
− Qualified through training, education and experience 
− Maintains essential clinical trial documents 
− Hosts visits of auditors, monitors and inspectors 
− Coordinates routing of all protocol activity 
− Maintains and updates all study staff curriculum vitae and Good Clinical Practices 
training certificates 
− Supports IND product accountability 
− Complies with Good Clinical Practices and Belmont Principles 
− Qualified through training, education and experience 
− Assists with vaccine accountability, storage and vial destruction 
− Issues vaccine to clinic 
• Research Monitor 
− Complies with Good Clinical Practices and Belmont Principles 
− Qualified through training, education and experience 
− Serves as a safety advocate for study participants 
− Reviews all severe adverse events, serious adverse events, unanticipated 
problems, protocol violations and annual reports 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
87 
US Government Proprietary 
 
 
− Discuss research progress with the PI, interview participants, consult on 
individual cases, or evaluate suspected adverse reaction reports on behalf of the 
IRB 
− Perform, at the direction of the IRB, oversight functions (e.g., observe 
recruitment, enrollment procedures, and the consent process for individuals, 
groups or units; oversee study interventions and interactions; review monitoring 
plans and UPIRTSO reports; and oversee data matching, data collection, and 
analysis) 
− Promptly report discrepancies or problems to the IRB 
− Have the authority to stop a research study in progress, remove individual 
participants from a study, and take whatever steps are necessary to protect the 
safety and well-being of research participants until the IRB can assess the 
research monitor's report. 
• Data Manager 
− Complies with Good Clinical Practices and Belmont Principles 
− Qualified through training, education and experience 
− Ensures the accuracy, quality, and integrity of the data: 
− Performs MedDRA and World Health Organization (WHO) coding 
− Reviews electronic case report forms (eCRF) for consistency and clarity, 
generates and resolves data queries and provides analysis and feedback to PI, 
protocol nurse coordinator and protocol team 
− Runs discrepancy report and quality control check reports and distributes to the 
protocol nurse coordinator to review for data accuracy against the eCRF 
− Oversees the data entry process for the study 
− Manages and maintains the database 
• Statistician 
− Complies with Good Clinical Practices and Belmont Principles 
− Qualified through training, education and experience 
− Statistically analyzes the verified data according to the protocol, statistical 
analysis plan and any amendments in place 
− Provides documentation of statistical findings to the principal investigator for 
incorporation into required reports 
• Internal Quality Assurance 
− Complies with Good Clinical Practices and Belmont Principles 
− Qualified through training, education and experience 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
88 
US Government Proprietary 
 
 
− Reviews study procedures, staff training and study documentation to ensure safety 
and accuracy in accordance with internal procedures 
• Regulated Studies Laboratory Staff and Research Investigators 
− Complies with Good Clinical Practices, Good Laboratory Practices and Belmont 
Principles 
− Qualified through training, education and experience 
− Analyzes study participants sera for immune response to vaccines 
• Clinical Laboratory Staff 
− Complies with Good Clinical Practices and Belmont Principles 
− Qualified through training, education and experience 
− Performs all phlebotomy for the study 
− Processes samples for biochemistry, hematology, urinalysis, hepatitis panel, 
human immunodeficiency virus, and β-HCG analysis on study participants 
− Performs urine pregnancy test 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
89 
US Government Proprietary 
 
 
APPENDIX B. LABELS 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
90 
US Government Proprietary 
 
 
APPENDIX C. RECRUITMENT SCRIPT 
When called by a potential subject, please make introductions and answer any questions that 
they may have concerning the study. If the potential subject is still interested, please read the 
following statement: 
THANK YOU FOR CALLING ABOUT INFORMATION 
ON VOLUNTEERING TO PARTICIPATE IN “A PHASE 
1, RANDOMIZED, OPEN-LABEL, SINGLE-CENTER, 
COMPARISON OF HETEROLOGOUS PRIME-BOOST 
VACCINATION SCHEDULES OF TETRAVALENT 
DENGUE VIRUS PURIFIED INACTIVATED VACCINE 
(PIV) AND TETRAVALENT DENGUE VIRUS LIVE 
ATTENUATED VACCINE (LAV) IN HEALTHY ADULTS 
IN A NONENDEMIC REGION”. 
We appreciate your interest. I will provide you some information that will help you 
determine if you can participate in the study. 
Alternatively, the information below can be emailed to potential subjects: 
I would like to share some information with you regarding an upcoming clinical trial entitled: 
“A Phase 1, Randomized, Open-label, Single-center, Comparison of Heterologous Prime- 
Boost Vaccination Schedules of Tetravalent Dengue Virus Purified Inactivated Vaccine 
(PIV) and Tetravalent Dengue Virus Live Attenuated Vaccine (LAV) in Healthy Adults in a 
Nonendemic Region”. 
Background: 
This study involves 2 experimental dengue vaccines. Dengue is a common infection affecting 
residents and travelers to many areas of the world, including Southeast Asia, Central 
America, South America, and the Caribbean. It is caused by a virus and is transmitted by a 
mosquito. Dengue can cause fever, tiredness, and even severe bleeding or death. It can pose a 
threat to military operations; therefore, the military is trying to develop a vaccine to protect 
against dengue. 
This study will look at the effects of combining 2 experimental dengue vaccines. This is not 
the first time that either of these dengue vaccines has been used in humans nor the first time 
that they have been used together. It will take place at a clinic-style research facility in Silver 
Spring, Maryland, and is sponsored by The Surgeon General, Department of the Army. The 
vaccines will be given in your upper arm using a needle, and blood samples will be collected 
to look at your body’s response. The goals of this study are to determine if the vaccines are 
safe when used together and to see how your body responds to the vaccines. One of the 
vaccines is a live-attenuated, or weakened, vaccine, just like the chickenpox or measles 
vaccines. The other is an inactivated or killed vaccine, like the flu vaccine or polio vaccine. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
91 
US Government Proprietary 
 
 
 
 
Duration: 
This study will last 18 months. One or 2 clinic visits are required to see if you qualify for the 
study. If you are accepted into the study, you will receive 1 dose of each of the vaccines All 
volunteers will get the killed vaccine first followed by the live vaccine. After each injection, 
there will be follow-up visits. These visits will continue for 12 months following the last 
vaccination in each group. There will be a total of 14 scheduled clinic visits (not including 
the initial screening). 
Requirements and Restrictions: 
You must meet ALL of the following requirements in order to participate in this study: 
• 
You MUST be 18 to 42 years of age (at the time of initial screening). 
• 
You MUST be in good health and have no significant medical conditions or diseases. 
• 
You CANNOT be pregnant, breastfeeding or planning to become pregnant during the study. For 
safety reasons, you must ALSO be willing to use a reliable form of birth control during the study. 
The effects of these vaccines have not yet been studied in infants or unborn children. 
• 
You must have NEGATIVE blood tests for hepatitis B, hepatitis C and HIV. 
• 
You CANNOT have ever had a dengue infection or received a dengue vaccine. 
• 
You MUST have a valid state or US government issued photo ID (such as a driver’s license, 
military ID or US Passport). 
• 
You MUST be willing to attend all of the required visits over the next 18 months. 
• 
You MUST be willing to refrain from participation in any other clinical studies involving 
investigational drugs or vaccines while participating in this study. 
• 
You CANNOT have donated or received blood, blood products, or plasma within 90 days prior to 
starting the study or plan on donating blood or plasma during the study. 
There may be other reasons why you cannot participate in this study. Those will be discussed at your 
initial screening visit. 
 
 
Possible Risks: 
There are risks associated with receiving these vaccines. 
Based on experience with these and similar vaccines, mild reactions are expected to occur in 
some subjects. These generally include tenderness, redness, and mild swelling at the injection 
site. These reactions will most likely resolve on their own within a few days. You may also 
experience other reactions, such as headache, nausea, a low fever, rash, or mild flu-like 
symptoms. There may be some risks that are unknown. There is always a chance that 
someone may experience a severe allergic reaction, just as some people do to other vaccines 
or drugs, like penicillin. After each vaccination you will be observed in the clinic for a short 
period of time to make sure you do not have an allergic reaction, or to treat you immediately 
if you do. 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
92 
US Government Proprietary 
 
 
After each vaccination you will see a physician in the clinic who will evaluate the number 
and type of reactions you may have. 
You will be paid for your participation in this study. Please refer to the study schedule or 
contact one of our recruiters for more detailed compensation information. 
 
 
 
Can I schedule an appointment for you to be seen by the research staff so that you can hear 
more about volunteering for this study? 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
93 
US Government Proprietary 
 
 
 
 
APPENDIX D. MODIFIED FDA LABORATORY TOXICITY SCALES 
 
Serum * 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially Life Threatening 
(Grade 4)** 
Sodium – Hyponatremia mEq/L 
132 – 134 
130 – 131 
125 – 129 
< 125 
Sodium – Hypernatremia mEq/L 
147-148 
149-150 
150-152 
> 152 
Potassium – Hyperkalemia mEq/L 
5.4-5.5 
5.6-5.7 
5.7-5.8 
> 5.8 
Potassium – Hypokalemia mEq/L 
3.3-3.4 
3.1-3.2 
3.0-3.1 
< 3.0 
Glucose – Hypoglycemia mg/dL 
60-64 
52-59 
45 – 51 
< 45 
Glucose – Hyperglycemia 
Fasting – mg/dL 
Random – mg/dL 
100 – 110 
110 – 125 
111 – 125 
126 – 200 
>125 
>200 
Insulin requirements or hyperosmolar 
coma 
Blood Urea Nitrogen 
BUN mg/dL 
26 – 28 
29 – 31 
> 31 
Requires dialysis 
Creatinine – mg/dL 
1.5 – 1.7 
1.8 – 2.0 
2.1 – 2.5 
> 2.5 or requires dialysis 
Calcium – hypocalcemia mg/dL 
8.0 – 8.4 
7.5 – 7.9 
7.0 – 7.4 
< 7.0 
Calcium – hypercalcemia mg/dL 
10.5 – 11.0 
11.1 – 11.5 
11.6 – 12.0 
> 12.0 
Magnesium – hypomagnesemia mg/dL 
1.3 – 1.4 
1.1 – 1.2 
0.9 – 1.0 
< 0.9 
Phosphorous – hypophosphatemia mg/dL 
2.3 – 2.4 
2.0 – 2.2 
1.6 – 1.9 
< 1.6 
CPK – mg/dL 
1.25 – 1.5 x 
ULN 
1.6 – 3.0 x ULN 
3.1 –10 x ULN 
> 10 x ULN 
Albumin – Hypoalbuminemia g/dL 
2.8 – 3.1 
2.5 – 2.7 
< 2.5 
-- 
Total Protein – Hypoproteinemia g/dL 
5.5 – 6.0 
5.0 – 5.4 
< 5.0 
-- 
Alkaline phosphate – 
increase by factor 
1.1 – 2.0 x ULN 
2.1 – 3.0 x ULN 
3.1 – 10 x ULN 
> 10 x ULN 
Liver Function Tests –ALT, AST 
increase by factor 
1.1 – 2.5 x ULN 
2.6 – 5.0 x ULN 
5.1 – 10 x ULN 
> 10 x ULN 
Bilirubin – when accompanied 
by any increase in Liver Function Test 
increase by factor 
1.1 – 1.25 x 
ULN 
1.26 – 1.5 x 
ULN 
1.51 – 1.75 x 
ULN 
> 1.75 x ULN 
Bilirubin – when Liver Function Test is normal; 
increase by factor 
1.1 – 1.5 x ULN 
1.6 – 2.0 x ULN 
2.0 – 3.0 x ULN 
> 3.0 x ULN 
Cholesterol 
201 – 210 
211 – 225 
> 226 
--- 
Pancreatic enzymes – amylase, lipase 
1.1 – 1.5 x ULN 
1.6 – 2.0 x ULN 
2.1 – 5.0 x ULN 
> 5.0 x ULN 
Hematology * 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially Life 
Threatening (Grade 4) 
Hemoglobin (Female) - gm/dL 
10.5-11.6 
9.5 – 10.4 
8.0 – 9.4 
< 8.0 

TDENV PIV and LAV Dengue Prime-boost Strategy, WRAIR #2453 
IND 16122, ADVP-004; S-16-23 
The Surgeon General 
Department of the Army 
Version 4 (06NOV2019) 
94 
US Government Proprietary 
 
 
 
Serum * 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially Life Threatening 
(Grade 4)** 
Hemoglobin (Male) - gm/dL 
12.5 – 13.0 
10.5 – 12.4 
8.5 – 10.4 
< 8.5 
3 
WBC Increase - cell/mm 
10,900 – 15,000 
15,001 – 20,000 
20,001 – 25, 
000 
> 25,000 
3 
WBC Decrease - cell/mm 
2,500 – 3,500 
1,500 – 2,499 
1,000 – 1,499 
< 1,000 
3 
Lymphocytes Decrease - cell/mm 
750 – 849 
500 – 749 
250 – 499 
< 250 
3 
Neutrophils Decrease - cell/mm 
1,499 – 2,000 
1,000 – 1,498 
500 – 999 
< 500 
3 
Eosinophils - cell/mm 
650 – 1500 
1501 - 5000 
> 5000 
Hypereosinophilic 
3 
Platelets Decreased - cell/mm 
125,000 – 
139,000 
100,000 – 
124,000 
25,000 – 99,000 
< 25,000 
PT – increase by factor 
(prothrombin time) 
1.0 – 1.10 x 
ULN 
1.11 – 1.20 x 
ULN 
1.21 – 1.25 x 
ULN 
> 1.25 ULN 
PTT – increase by factor 
(partial thromboplastin time) 
1.0 – 1.2 x ULN 
1.21 – 1.4 x 
ULN 
1.41 – 1.5 x 
ULN 
> 1.5 x ULN 
Fibrinogen increase - mg/dL 
426 – 500 
501 – 600 
> 600 
-- 
Fibrinogen decrease - mg/dL 
150 – 200 
125 – 149 
100 – 124 
< 100 or associated 
with gross bleeding 
or disseminated intravascular coagulation 
(DIC)