A Phase 1, Open-Label Clinical Trial With Dengue-1-Virus Live Virus Human Challenge (DENV-1-LVHC) Assessment of Healthy U.S. Adults Previously Primed With Tetravalent Dengue Virus Purified Inactivated Vaccine (TDEN-PIV) and Boosted With Tetravalent Dengue Virus Live Attenuated Vaccine Formulation (TDEN LAV)
Phase: PHASE1
Conditions: Dengue
Sponsor: University of Maryland, Baltimore

Clinical Protocol ADVP005 
The University of Maryland, Baltimore  
IND 016332; CVD Dengue 12000 
Center for Vaccine Development and Global Health 
V4.0, 04 Jan 2021 
1 
 
A Phase 1, Open-Label Clinical Trial with Dengue-1-Virus Live Virus Human Challenge (DENV-1-
LVHC) Assessment of Healthy U.S. Adults Previously Primed with Tetravalent Dengue Virus 
Purified Inactivated Vaccine (TDEN-PIV) and Boosted with Tetravalent Dengue Virus Live 
Attenuated Vaccine Formulation (TDEN LAV) 
  Abbreviated title: Study of the ADVP004 vaccinated volunteers with DENV-1-LVHC 
IND Sponsor: 
The University of Maryland Center for Vaccine Development 
and Global Health 
Principal Investigator: 
Kirsten E. Lyke, M.D. 
Professor of Medicine 
Director, Malaria Vaccine and Challenge Unit, 
Center for Vaccine Development and Global Health 
University of Maryland, Baltimore 
Telephone: 410-706-0462/7376 
Fax: 410-706-6205 
Email: KLyke@som.umaryland.edu 
 
Co-development Partners: U.S. Army Medical Materiel Development Activity 
(USAMMDA), Ft. Detrick, MD 
 
Funding Sponsor: 
Medical Technology Enterprise Consortium (MTEC) 
 
USAMMDA 
Representative 
 
Calli M. Rooney; MS, MBA, PMP 
Product Manager - Pharmaceutical Systems Project 
Management Office (PSPMO) U.S. Army Medical Materiel 
Development Activity (USAMMDA) 
1430 Veterans Drive 
Ft. Detrick, MD 21702-5009 
Office: 301-619-3751 
Cell: 240-315-8426 
Email:calli.m.rooney.civ@mail.mil 
  
MTEC and UMB Study 
Number and Abbreviated 
Title: 
 
MTEC-17-01; CVD Dengue 12000 (ADVP005 with DHIM-1) 
 
 
IND Number 
#26871 cross referencing #16332 

Clinical Protocol ADVP005 
The University of Maryland, Baltimore  
IND 016332; CVD Dengue 12000 
Center for Vaccine Development and Global Health 
V4.0, 04 Jan 2021 
2 
 
1. 
Contact Information 
Clinical Trial 
Sites 
Center for Vaccine Development 
University of Maryland  
685 W. Baltimore Street, HSF I, Room 480 
Baltimore, Maryland 21201 
Telephone: 410-706-0462/7376 
Principal 
Investigator  
Kirsten E. Lyke, M.D. 
Professor of Medicine 
Center for Vaccine Development and Global Health  (CVD) 
University of Maryland  School of Medicine 
685 West Baltimore Street 
HSF I, Room 480 
Baltimore, Maryland 21201 
Telephone: 410-706-0462/7376 / Fax: 410-706-6205 
Email: klyke@som.umaryland.edu 
Associate 
Investigator(s) 
Monica McArthur, MD, PhD. 
Assistant Professor of Pediatrics 
685 W. Baltimore Street, HSF I, Room 480 
Baltimore, MD 21201 
Telephone: 410-706-5328 
Fax: 410-706-6205 
E-mail: mmcarthu@som.umaryland.edu 
Joel V. Chua, MD 
Assistant Professor of Medicine  
Chief, Surgical Infectious Diseases Service 
725 W. Lombard Street, Rm. N146 
Baltimore, MD 21201 
Telephone: 410-706-5704 
Fax: 410-706-3423 
E-mail: jchua@ihv.umaryland.edu 
Institutional 
Review Boards 
University of Maryland Human Research Protections Office 
800 West Baltimore Street, Suite 100 
Baltimore, Maryland 21201 
Telephone: 410-706-5037 / Fax: 410-706-4189 
Email: HRPO@umaryland.edu 
Site 
Investigational 
Product 
Accountability 
UMMC Investigational Drug Service 
University of Maryland Medical Center 
IDS Pharmacy, Room N9E14 
22 South Greene Street 
Baltimore, MD 21201 
Telephone: 410-328-5344/5468 
Fax: 410-328-7326 
Laboratories 
Garcia Clinical Laboratories (Central) 
2195 Spring Arbor Road 
Jackson, MI 49203-2797 
Telephone: 517-787-9200 / Fax: 517-787-1249 

Clinical Protocol ADVP005 
The University of Maryland, Baltimore  
IND 016332; CVD Dengue 12000 
Center for Vaccine Development and Global Health 
V4.0, 04 Jan 2021 
3 
 
 
University of Maryland Medical Systems (Local) 
Laboratory of Pathology 
22 South Greene Room, N2W49 
Baltimore, Maryland 21201 
Telephone: 410-328-8378 / Fax: 410-328-2125 
 
WRAIR Pilot Bioproduction Facility 
503 Robert Grant Avenue 
Silver Spring, MD 20190 
POC: MAJ Jeffrey Froude  
Telephone: (301) 712-7391 
Email: jeffrey.w.froude2.mil@mail.mil 
 
WRAIR Viral Diseases Branch 
503 Robert Grant Avenue 
Silver Spring, MD 20190 
POC: MAJ Michael Koren  
Telephone: 301-3199904 
Email:  Michael.a.koren2.mil@mail.mil 
Independent 
Safety Monitor/ 
Research 
Monitors 
Justin R. Ortiz, MD, MS, FACP, FCCP 
Center for Vaccine Development and Global Health 
University of Maryland School of Medicine 
685 W. Baltimore Street, Room 480 
Baltimore, MD 21201 
Telephone: 410-706-6620 
Fax: 410-706-6205 
E-mail: jortiz@som.umaryland.edu 
And back-up: 
Wilbur Chen, MD  
Center for Vaccine Development and Global Health 
University of Maryland School of Medicine 
685 W. Baltimore Street, Room 480 
Baltimore, MD 21201 
Telephone: 410-706-1188 
Fax: 410-706-6205 
E-mail: wchen@som.umaryland.edu 
Data 
Management 
ICON Global and Public Health Solutions  
Senior Director, Business Development 
Telephone: 301-620-1987; 329  
Email: dtobiasz@icongphs.com 
Study 
Coordinator/ 
Protocol Nurse 
Center for Vaccine Development and Global Health, University of Maryland 
685 W. Baltimore St., HSF I, Rm. 480 
Baltimore, Maryland 21201 
(410) 706-6156 
Lchrisle@som.umaryland.edu 

Clinical Protocol ADVP005 
The University of Maryland, Baltimore  
IND 016332; CVD Dengue 12000 
Center for Vaccine Development and Global Health 
V4.0, 04 Jan 2021 
4 
 
IND Regulatory 
Affairs 
Specialist 
 
 
 
 
Clinical 
Regulatory 
Affairs 
Specialist 
 
Brian Roberts, MS, PMP 
Life Sciences Program Manager 
Leidos Inc.,  
5202 Presidents Court, Suite 110 
Frederick, MD  21703 
Tel:  240-529-0400 
Email: BRIAN.A.ROBERTS@leidos.com 
 
 
Aly Kwon, MS 
685 W. Baltimore St., HSF I, Rm. 480 
Baltimore, Maryland 21201 
(410) 706-6056 
AKwon@som.umaryland.edu 
 
Inpatient 
Facility 
Pharmaron Biomedical Laboratories  
Clinical Pharmacology Center  
University of Maryland BioPark Facility 
800 W. Baltimore St., 5th Floor 
Baltimore, MD 21201 
Phone: 410-760-8812 Contact: Bridget McMahon, Director 
 
Or 
 
General Clinical Research Center 
University of Maryland School of Medicine 
22 South Greene Street 
University of Maryland Medical Center  
Room S10D05 
Baltimore, Maryland 21201 
Email: mbanchero@som.umaryland.edu 
Phone: 410-328-7648 
Fax: 410-328-8749 
 
Sequestration 
Facility 
 
 
 
Funding Source 
Kimpton Hotel Monaco Baltimore Inner Harbor 
2 North Charles Street 
Baltimore, MD  21201 
POC:  Rebecca Gisriel 
Email: Rebecca.Gisriel@hotelmonaco.com  
 
MTEC 

Clinical Protocol ADVP005 
The University of Maryland, Baltimore  
IND 016332; CVD Dengue 12000 
Center for Vaccine Development and Global Health 
V4.0, 04 Jan 2021 
5 
 
Investigator’s Agreement 
Study Title: A Phase 1, Open-Label Clinical Trial with Dengue-1-Virus Live Virus Human 
Challenge (DENV-1-LVHC) Assessment of Healthy U.S. Adults Previously Primed with 
Tetravalent Dengue Virus Purified Inactivated Vaccine (TDEN-PIV) and Boosted with Tetravalent 
Dengue Virus Live Attenuated Vaccine Formulation (TDEN LAV) 
 
 
The signature below constitutes the approval of this protocol and the attachments and provides 
the necessary assurances that this trial will be conducted according to all stipulations of the 
protocol, including all statements regarding confidentiality, and according to local legal and 
regulatory requirements and applicable US federal regulations and ICH guidelines. 
 
 
 
 
Kirsten E. Lyke, MD 
Principal Investigator 
Center for Vaccine Development and Global Health 
University of Maryland School of Medicine 
 
Date 
 
 

Clinical Protocol ADVP005 
The University of Maryland, Baltimore  
IND 016332; CVD Dengue 12000 
Center for Vaccine Development and Global Health 
V4.0, 04 Jan 2021 
6 
 
2. 
Synopsis 
Name of IND Sponsor/Company: 
The University of Maryland Center for Vaccine Development and Global Health 
Co-development Partners: 
U.S. Army Medical Materiel Development Activity (USAMMDA), Ft. Detrick, MD 
Funding Partners: 
Medical Technology Enterprise Consortium (MTEC) 
Name of Investigational Product: 
Dengue-1-Virus-Live Virus Human Challenge - (DENV-1-LVHC) 
Previously vaccinated with: 
1. Tetravalent Dengue Virus (TDEN) Purified Inactivated Vaccine (PIV) with Alum adjuvant prime 
2. Tetravalent Dengue Virus Live Attenuated Vaccine Formulation 17 post transfection (LAV) boost 
Name of Active Ingredients: 
Dengue virus (DENV) type 1 strain 45AZ5  
Title of Study: A Phase 1, Open-Label Clinical Trial with Dengue-1-Virus Live Virus Human Challenge 
(DENV-1-LVHC) Assessment of Healthy U.S. Adults Previously Primed with Tetravalent Dengue Virus Purified 
Inactivated Vaccine (TDEN-PIV) and Boosted with Tetravalent Dengue Virus Live Attenuated Vaccine 
Formulation (TDEN LAV) 
Study Centers: Center for Vaccine Development and Global Health (CVD), University of Maryland Baltimore, 
Maryland 
Principal Investigators (PIs): Kirsten E. Lyke 
Study Period:  
Estimated date first volunteer enrolled: 4th quarter 2020 
Estimated date last volunteer completed:1st quarter 2021 
Phase of Development: 1 
Objectives:  
Primary: 
• 
To further evaluate the safety and reactogenicity of the DHIM-1 human challenge model 
• 
To determine the level of protection to dengue serotype 1 challenge related symptoms provided by 
previous vaccination with heterologous prime boost utilizing Tetravalent Dengue Virus Purified 
Inactivated Vaccine (PIV) and Tetravalent Dengue Virus Live Attenuated Vaccine (LAV) 
Secondary  
• 
To characterize the immunologic responses following dose exposure to the DENV-1-LVHC viral strain 
as a DHIM in vaccinated compared to unvaccinated volunteers 
• 
To compare viremia kinetics following dose exposure to DHIM-1 in vaccinated compared to 
unvaccinated groups 
Exploratory 
• 
To explore the immune response and host-virus interactions following exposure to DENV-1-LVHC 

Clinical Protocol ADVP005 
The University of Maryland, Baltimore  
IND 016332; CVD Dengue 12000 
Center for Vaccine Development and Global Health 
V4.0, 04 Jan 2021 
7 
 
Methodology:  
Design: Volunteers will be 18-50 years, inclusive, at enrollment. This study is a Phase 1 open-label, study with 3 
groups of 5 volunteers each. This will include 5 unvaccinated dengue naïve volunteers, 5 volunteers vaccinated 
with PIV followed by LAV at 90 day, and 5 volunteers vaccinated with PIV followed by LAV at 180 days. If funds 
allow and volunteers express interest, a fourth group of 5 previously vaccinated volunteers may be recruited. If an 
inadequate number of volunteers vaccinated on these schedules are available volunteers vaccinated with PIV 
followed by LAV 28 days later may also be included. 
Both vaccinated and unvaccinated volunteers will be challenged with the DHIM-1 challenge strain at University 
of Maryland’s CVD in at least two separate challenge events.  Volunteers who meet the criteria for inpatient 
analysis (i.e., develop fever, detectable viremia, and/or symptoms, signs or laboratory criteria concerning for 
development of severe dengue) will be hospitalized at a designated inpatient unit. 
After inoculation, volunteers will be seen and evaluated (including blood draw) closely (qd or qod per study 
schedule) until Day 28-post inoculation. If a volunteer develops viremia, symptoms or laboratory findings that 
meet sequestration (viremia) or hospital admission criteria (viremia plus symptoms and/or laboratory findings) he 
or she will be admitted. During hospitalization they will receive additional clinical and laboratory evaluations if 
determined necessary by treating physicians. Volunteers will be eligible for discharge when they have fever 
resolution, improvement in symptoms and absence of virus detection by polymerase chain reaction (PCR). 
Note: Due to the COVID-19 pandemic, additions have been added to the protocol in the event that local 
transmission of the SARS-CoV-2 virus continues concomitant to study operations. 
Estimated Number of Volunteers to be Screened:  
Approximately 31 volunteers will be screened  
Minimum and Maximum Number of Volunteers Planned for Enrollment:  
Minimum: 15 (10 previously vaccinated and 5 unvaccinated dengue naïve volunteers) 
Maximum: 20 volunteers (5 additional vaccinated if funds and interest allow) 
Volunteer Population for Inclusion/Exclusion:  
Healthy men and healthy, non-pregnant, non-breastfeeding women between the ages of 18 and 50 
Investigational Product Dosage, Schedule, and Mode of Administration:  
The investigational product, DENV-1-LVHC is an injection, powder, lyophilized for solution, reconstituted with 
0.7mL of water for injection (WFI) and diluted with formulated Eagle’s minimum essential medium (EMEM 
with human serum albumin, L-glutamine, and lactose monohydrate). 
• 
Dose - 0.5 mL of 6.5 x 103 PFU/mL  
All investigational products will be administered in a single inoculation subcutaneously (SC) in the triceps area 
of the arm 
Duration of Study:  
Approximately 6 months after inoculation (Day 0-180) per volunteer 

Clinical Protocol ADVP005 
The University of Maryland, Baltimore  
IND 016332; CVD Dengue 12000 
Center for Vaccine Development and Global Health 
V4.0, 04 Jan 2021 
8 
 
Criteria for Inclusion/Exclusion: 
Inclusion: 
• 
Male or non-pregnant, non-breastfeeding female between 18 and 50 years of age (inclusive) at the time 
of consent. 
• 
Tetravalent dengue antibody response at 28 days following final vaccination for vaccinated groups of 
volunteers. 
• 
Volunteers must be able and willing to provide written informed consent. 
• 
Volunteers must be healthy as established by medical history and clinical examination at study entry.  
• 
Volunteers must pass a comprehension test and be able to comply with all study requirements. 
• 
Female volunteers of non-childbearing potential (non-childbearing potential is defined as having had 
one of the following: a tubal ligation at least 3 months prior to enrollment, a hysterectomy, an 
oophorectomy, or is post-menopausal). 
• 
Female volunteers of childbearing potential may be enrolled in the study, if all of the following apply: 
-Practiced adequate contraception (see Definition of Terms, section 5.4.2.3.) for 30 days prior to 
challenge  
-Has a negative urine pregnancy test on the day of DHIM 
-Agrees to continue adequate contraception until two months after completion of the DHIM 
• 
Provide consent for release of medical history records from primary care physician, college or university 
medical center, urgent care, or emergency room visit  
Exclusion: 
• 
Planned travel during the study period (180 days) which would interfere with the ability to complete all 
study visits 
• 
Recent (in the past 4 weeks) travel to any dengue endemic area. These potential volunteers may be 
eligible for enrollment a minimum of 4 weeks later 
• 
Volunteer seropositive for hepatitis B surface antigen (HBsAg), hepatitis C virus antibodies (anti-HCV), 
or human immunodeficiency virus antibodies (anti-HIV)  
• 
Unvaccinated volunteers positive for antibodies to flaviviruses (FV) to include dengue virus, West Nile 
virus, Yellow Fever virus, Zika virus, and Japanese encephalitis virus. 
• 
Any history of FV infection or FV vaccination, except for participation in the ADVP003 or ADVP004 
dengue vaccination studies, during the study period  
• 
Medical history of, or current, diabetes, chronic obstructive pulmonary disease, peptic ulcer disease, 
coronary artery disease, cardiac arrhythmia, cardiomyopathy, pericarditis, or auto-immune disease 
• 
Known or suspected congenital or acquired immunodeficiency; or receipt of immunosuppressive 
therapy such as anti-cancer chemotherapy or radiation therapy within the preceding 6 months; or long-
term systemic corticosteroid therapy (prednisone or equivalent for more than 2 consecutive weeks 
within the past 3 months)  
• 
History of Guillain-Barré syndrome (GBS) 
• 
History of bipolar disorder, 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 volunteer from 
participating in the study 
• 
Safety laboratory test results at screening that are deemed clinically significant or more than Grade 1 
deviation from normal with the exception of PT/PTT, fibrinogen decrease, ALT/AST increase 
(acceptable to 1.1 ULN), platelet decrease which will be exclusionary at Grade 1 or higher 
• 
Significant screening physical examination abnormalities at the discretion of the investigator, including 
a BMI > 35 kg/m2 
• 
Women who intend to become pregnant or men who intend to father a child during the study period 
(approximately 6 months) 
• 
Female: pregnant, lactating or history of heavy menstrual bleeding menstrual periods defined as lasting 
consistently and regularly longer than 6 days, or consistently and regularly requiring 5 or more pads or 
tampons per day, and to the opinion and review of the investigator. Female volunteers with a history of 

Clinical Protocol ADVP005 
The University of Maryland, Baltimore  
IND 016332; CVD Dengue 12000 
Center for Vaccine Development and Global Health 
V4.0, 04 Jan 2021 
9 
 
clinically significant fibroids or uterine polyps, endometriosis, dysmenorrhea, adenomyosis, and uterine 
scarring (e.g. after D&C) accompanied by excessive bleeding, unless treated, with no active clinically 
significant disease 
• 
Allergy (hives, shortness of breath, swelling of the lips or throat), or hospitalization related to a previous 
vaccination, anaphylaxis of unknown etiology, or allergy to specific medications/animals for which 
antigens may be in the virus preparations to include: Shellfish allergy, Fetal Bovine Serum, L-
Glutamine, Neomycin and Streptomycin 
• 
Recent blood donation within prior 56 days of inoculation or planning to donate blood in the one 1 year 
following inoculation with dengue virus 
• 
Receipt of blood products or antibodies within 90 days of inoculation or during the study period 
• 
Any personal beliefs that bar the administration of blood products, transfusions, or serum albumin 
• 
Participation in the 4 weeks preceding inoculation, or planned participation during the present trial 
period, in another clinical trial investigating a vaccine except for participation in the ADVP004 study, 
drug, medical device, or medical procedure 
• 
Planned administration of a licensed or study vaccine not planned in the study protocol during the period 
starting 30 days prior to the DHIM for a live vaccine or 14 days prior to DHIM for inactivated vaccines 
and extending until 56 days after study completion  *Note:  An exception will be made for volunteers 
who have the option to obtain a COVID-19 vaccine.  In this case, we will allow a vaccination 14 days 
prior or 21 days following DHIM. 
• 
Planned or current administration of an HMG-CoA reductase inhibitor (i.e., lovastatin, simvastatin, 
atorvastatin, etc.) 
• 
Currently taking methadone or suboxone 
• 
Currently regularly taking anti-coagulant medication, aspirin or non-steroidal anti-inflammatory drugs 
(NSAIDs) 
• 
Chronic migraine headaches, defined as more than 15 headache days per month over a 3-month period 
of which more than 8 are migraines, in the absence of medication over use 
• 
Chronic or recent acute medical condition that, in the opinion of the investigator, impacts volunteer 
safety.  
Endpoint Analysis: 
Primary: 
The primary endpoints align with the safety and clinical objectives. The nature, frequency and severity of adverse 
events (AEs) associated with the attenuated DHIM-1 will be evaluated in the vaccinated ADVP004 volunteers 
and unvaccinated dengue-naïve control volunteers with statistical comparisons both within and between groups. 
• 
Occurrence, grade, and duration of solicited injection site symptoms until 7 days post virus inoculation  
• 
Occurrence, intensity, and duration of unsolicited injection site symptoms until 28 days post virus 
inoculation or 7 days post inpatient, whichever is later  
• 
Occurrence, intensity, and duration of solicited systemic symptoms until 28 days post virus inoculation 
or 7 days post inpatient, whichever is later  
• 
Number, intensity, and duration of abnormal laboratory measurements until 28 days post virus 
inoculation or 7 days post inpatient, whichever is later  
• 
Occurrence, intensity and duration of dengue like symptoms/adverse events until 28 days post virus 
inoculation or 7 days post inpatient whichever is later (See defined Dengue Illness Index) 
• 
Occurrence, intensity, and duration of unsolicited systemic symptoms until 28 days post virus 
inoculation or 7 days post inpatient, whichever is later  
• 
Number of SAEs until 28 days post virus inoculation or 7 days post hospitalization, whichever is later  
• 
Number of SAEs until 6 months post virus inoculation  
• 
The occurrence of fever defined as greater than or equal to 38°C (100.4° F) measured at least 2 times at 
least 4 hours apart 
Secondary: 

Clinical Protocol ADVP005 
The University of Maryland, Baltimore  
IND 016332; CVD Dengue 12000 
Center for Vaccine Development and Global Health 
V4.0, 04 Jan 2021 
10 
 
The secondary endpoints align with the viremia and immunogenicity objectives. Dengue viremia will be 
examined both as a binary endpoint (present or not present), peak, and as a function of area under the curve 
(AUC) 
• 
Viremia by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) up to 28 days post 
virus inoculation  
• 
MN50 antibody GMT titers at 28 days following inoculation in vaccinated and unvaccinated volunteers 
 
Statistical Methods of Dengue Illness Index (DII) of DHIM-1: 
The impact of the challenge virus up to 28 days after virus inoculation will be assessed descriptively using the 
following disease severity index (DSI) parameters and assessing differences between vaccinated and 
unvaccinated study volunteers: 
• Number and percentage of volunteers in each group (vaccinated vs. control) who develop signs and 
symptoms of dengue fever as described below 
• Time to onset of clinical signs and symptoms of dengue fever as described below 
• Magnitude of viremia, time to onset of viremia, duration of viremia, number and percentage of volunteers 
with measurable and sustainable viremia and area under the curve viremia analysis  
• Number and percentage of volunteers in each group with fever greater than or equal to 38°C (100.4°F) 
measured at least 2 times at least four hours apart in 24 hours  
• Number, percentage and severity score of volunteers in each group with each of the following clinical or 
laboratory symptoms as measured by the Dengue Illness Index: 
- Fever greater than or equal to 38°C (100.4°F) 
-  Headache/retro-orbital pain  
- Rash  
-  Fatigue and/or malaise 
-  Myalgia 
-  Arthralgia and/or bone pain  
- GI symptoms (nausea, vomiting, abdominal pain) 
- Liver function tests (ALT, AST)  
- Leukopenia  
- Thrombocytopenia  
*Note:  Post-hoc analysis can recategorize disease severity based upon WHO parameters 
Analysis of the secondary endpoints will be applied on per protocol population. Only those volunteers who 
receive inoculations will be included in the analysis.  Descriptive analysis will compare measurements from 
vaccinated and unvaccinated study volunteers.  
Statistical Methods for Exploratory Endpoints: 
Exploratory endpoints will be analyzed using the population of volunteers enrolled in the trial who receive 
dengue virus inoculation. Analysis will be done using the previously mentioned assays and appropriate statistical 
tests will be performed. The immune response to the challenge virus at each dose will be characterized 
descriptively by: 
• 
Geometric mean titer (GMT) and geometric mean titer rates (GMTRs) of neutralizing antibodies 
(measured by dengue neutralization titer (NT) at 0, 1, 3, and 6 months after virus inoculation (> 10 defined 
as response) 
• 
Cell mediated immunity (CMI) 
• 
Proteomics - microarray 
• 
Transcriptomics (to include single-cell analyses) 
• 
Evolutionary analysis of DHIM-1 strain whole genome sequence (consensus and quasi-species) 

Clinical Protocol ADVP005 
The University of Maryland, Baltimore  
IND 016332; CVD Dengue 12000 
Center for Vaccine Development and Global Health 
V4.0, 04 Jan 2021 
11 
 
3. 
Table of Contents, List of Tables, and List of Figures  
 
1. 
Contact Information .......................................................................................................... 2 
2. 
Synopsis ........................................................................................................................... 6 
3. 
Table of Contents, List of Tables, and List of Figures ...................................................... 11 
4. 
List of Abbreviations ...................................................................................................... 15 
5. 
Introduction .................................................................................................................... 18 
5.1. 
Rationale for the Study Design ........................................................................... 19 
5.2. 
Name and Description of the Investigational Product .......................................... 19 
5.3. 
Summary of Pre-clinical and Clinical Trials........................................................ 19 
Pre-clinical Studies ............................................................................................. 19 
Clinical Studies .................................................................................................. 20 
5.3.1.1. 
Early Human Challenge Studies with DENV-1 45AZ5 ........ 20 
5.3.1.2. 
Phase 2b Evaluation of Tetravalent Live-Attenuated Dengue 
Vaccines (WRAIR #877) (A-11014, IND 8796) .................. 21 
5.3.1.3. 
Attenuated Candidate Dengue 1 Vaccine (45AZ5) in Human 
Volunteers (IND 1952)........................................................ 21 
5.3.1.4. 
A Phase 1 Evaluation of the Protective Efficacy of a Single 
Dose of the Live Attenuated Tetravalent Dengue Vaccine 
TV003 to Protect Against Infection with Attenuated DENV-2, 
rDEN2Δ30-7169 (NIAID, NCT02021968) .......................... 22 
5.3.1.5. 
Past Human Studies with Any Dengue Serotypes ................ 22 
5.3.1.6. 
Heterologous Prime Boost using tetravalent dengue PIV and 
tetravalent dengue LAV ...................................................... 23 
5.4. 
Known and Potential Risks and Benefits to Human Volunteers ........................... 24 
5.4.1.   Dengue Classification ............................................................................. 24 
5.4.2.  Risks/Discomfort to Volunteers and Precautions to Minimize Risk........... 25 
5.4.2.1.      Local Reactions .................................................................... 25 
5.4.2.2.      Systemic Reactions ............................................................... 25 
5.4.2.3.      Pregnancy ............................................................................. 25 
5.4.2.4.      Lactation............................................................................... 26 
5.4.2.5.      Venipuncture ........................................................................ 26 
5.4.2.6.      Allergic Reaction .................................................................. 26 
5.4.2.7.      Guillain-Barré Syndrome (GBS) or adverse reactions to  
vaccines .................................................................................... 26 
5.4.2.8.       Observations from Natural Dengue Infection ....................... 26 
5.4.2.9.      Unknown Risks .................................................................... 29 
5.4.2.10.     SARS-CoV-2 Precautions .................................................... 29 
5.4.3.  Alternatives to This IND Product or Study ............................................... 29 
5.4.4.  Intended Benefit for Volunteers ............................................................... 29 
5.4.5.  Risks to the Study Personnel and the Environment ................................... 29 
5.5. 
Route of Administration, Dosage Regimen, and Experimental Vaccination ......... 30 
5.6. 
Compliance Statement ........................................................................................ 30 
5.7. 
Study Population ................................................................................................ 30 
5.8. 
Study Sites ......................................................................................................... 30 
6. 
Trial Objectives .............................................................................................................. 31 
6.1. 
Primary Objectives ............................................................................................. 31 

Clinical Protocol ADVP005 
The University of Maryland, Baltimore  
IND 016332; CVD Dengue 12000 
Center for Vaccine Development and Global Health 
V4.0, 04 Jan 2021 
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6.2. 
Secondary Objectives ......................................................................................... 31 
6.3. 
Exploratory Objectives ....................................................................................... 31 
7. 
Trial Design .................................................................................................................... 31 
7.1. 
Study Endpoints ................................................................................................. 31 
7.1.1.   Primary Endpoints .................................................................................. 31 
7.1.2.   Secondary Endpoints............................................................................... 32 
7.1.3.   Exploratory Endpoints ............................................................................ 32 
7.2. 
Overall Study Design.......................................................................................... 32 
7.3. 
Investigational Product ....................................................................................... 33 
7.3.1.  Investigational Product Packaging and Labeling....................................... 34 
7.3.2.  Investigational Product Shipment ............................................................. 34 
7.3.3.  Investigational Product Storage ................................................................ 34 
7.3.4.  Investigational Product Accountability ..................................................... 35 
7.3.5.  Investigational Product Preparation .......................................................... 35 
7.3.6.  Investigational Product Administration..................................................... 36 
7.3.7.  Investigational Product Dose Selection..................................................... 36 
7.3.8.  Investigational Product Replacement Doses ............................................. 36 
7.4. 
Identification of Data to be Recorded on the Case Report Forms ......................... 36 
8. 
SELECTION AND WITHDRAWAL OF VOLUNTEERS.............................................. 37 
8.1. 
Recruitment of Volunteers .................................................................................. 37 
8.2. 
Eligibility Screening ........................................................................................... 37 
8.2.1.   Volunteer Inclusion Criteria .................................................................... 37 
8.2.2.   Volunteer Exclusion Criteria ................................................................... 38 
8.3. 
Temporary Exclusion Criteria ............................................................................. 40 
8.4. 
Volunteer Withdrawal Criteria ............................................................................ 40 
8.4.1.  When and How to Withdraw Volunteers .................................................. 40 
8.4.2.  Data Collected for Withdrawn Volunteers ................................................ 40 
8.4.3.  Lost to Follow-up Procedure .................................................................... 40 
8.4.4.  Replacement of Volunteers ...................................................................... 41 
8.4.5.  Follow-up for Withdrawn Volunteers ....................................................... 41 
9. 
TREATMENT OF VOLUNTEERS ................................................................................ 42 
9.1. 
Informed Consent Process .................................................................................. 47 
9.2. 
Screening ........................................................................................................... 47 
9.2.1.   Screening Visit 1 (Day -90 to -1)............................................................. 47 
9.2.2.   Screening Visit 2..................................................................................... 48 
9.3. 
Inoculation and Follow-up Visits ........................................................................ 49 
9.3.1.  Day 0 Inoculation..................................................................................... 49 
9.3.2.  Day 2 (+/- 1) ............................................................................................ 50 
9.3.3.  Days 4 to 16 Daily Follow-up .................................................................. 50 
9.3.4.  Days 19, 22, and 25(+/- 1), and 28 (+/- 2) Post-Daily Follow-up .............. 51 
9.3.5.  Days 56 (± 3 days), 90 and 180 (± 7 days) Safety Follow-ups................... 51 
9.4. 
Inpatient Admission............................................................................................ 52 
9.4.1. Criteria for Admission to Inpatient Service............................................. 52 
9.4.2. Procedures During Inpatient Admission ................................................. 53 
9.4.3. Criteria for Discharge/Release from Sequestration or Inpatient Admission
 .............................................................................................................. 54 
9.5. 
Biological Samples ............................................................................................. 54 
9.5.1.  Biological Sample Handling.................................................................. 54 
9.5.1.  Future Use of Human Samples .............................................................. 55 

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9.6.       Concomitant Medications ................................................................................... 55 
9.7. 
Research Assessments ........................................................................................ 55 
9.8. 
Serum Antibodies ............................................................................................... 56 
9.9. 
Polymerase Chain Reaction (PCR) Detection of Viremia .................................... 56 
9.10. 
Immune Responses ............................................................................................. 56 
9.11. 
Transcriptomics .................................................................................................. 57 
9.12. 
Single cell RNA Sequencing (scRNAseq) ........................................................... 57 
10.0. 
 SAFETY ASSESSMENT .............................................................................................. 57 
10.1. 
Consortium Data Safety Committee .................................................................... 57 
10.2.     Clinical Laboratory Assessment.......................................................................... 57 
10.3. 
IND Safety Reporting Definitions ....................................................................... 58 
10.3.1. Adverse Event/Suspected Adverse Reaction/Unexpected Adverse Reaction
 .............................................................................................................. 58 
10.3.2. Solicited Adverse Event ......................................................................... 59 
10.3.3. Serious Adverse Event/Serious Suspected Adverse Reaction .................. 59 
10.3.4. Unanticipated Problems Involving Risks To Volunteers Or Others 
(UPIRTSOs) .......................................................................................... 60 
10.3.5. Pregnancy .............................................................................................. 60 
10.4. 
Relationship to Investigational Product ............................................................... 61 
10.5. 
Severity Assessment ........................................................................................... 61 
10.6. 
Recording Adverse Events.................................................................................. 62 
10.6.1. Methods/Timing for Assessing, Recording, and Analyzing Safety 
Endpoints............................................................................................... 62 
10.6.2. Duration of Follow-Up of Volunteers after Serious Adverse Events ....... 63 
10.7. 
Study Halting Criteria......................................................................................... 63 
10.8. 
Termination Rules .............................................................................................. 64 
10.9. 
Reporting Requirements ..................................................................................... 64 
10.9.1. Reporting Adverse Events ...................................................................... 64 
10.9.2. Reporting Serious Adverse Events/Unanticipated Problems Involving Risk 
to Volunteers or Others .......................................................................... 64 
10.9.3. Reporting to the UMB IRB .................................................................... 66 
10.9.4. Sponsor Reporting Requirements to FDA............................................... 66 
10.10. Reporting Additional Immediately Reportable Events to the Sponsors Safety 
Office and HRPO ............................................................................................... 66 
10.10.1.  Pregnancy ............................................................................................ 66 
10.10.2.  AE-related Withdrawal of Consent ....................................................... 67 
10.10.3.  Pending Inspections/Issuance of Reports .............................................. 67 
10.10.4.  Final Report ......................................................................................... 67 
11.0. 
STATISTICS .................................................................................................................. 67 
11.1. 
Description of Statistical Methods ...................................................................... 68 
11.1.1. Analysis Addressing the Primary Endpoints ........................................... 68 
11.1.2. Multiple Comparison Endpoint .............................................................. 72 
11.1.3. Analysis Addressing the Secondary Endpoints ....................................... 72 
11.1.4. Analysis Addressing the Exploratory Endpoints ..................................... 72 
11.2. 
Planned Enrollment and Reason for Sample Size ................................................ 72 
11.3. 
Procedures for Reporting Deviations from the Original Statistical Plan ............... 73 
11.4. 
Selection of Volunteers to be Included in Analyses ............................................. 73 
11.4.1. Safety Analysis Set ................................................................................ 73 
11.4.2. Full Analysis Set .................................................................................... 73 
11.4.3. Per-Protocol Analysis Set....................................................................... 73 

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11.4.4. Populations Used in Analyses ................................................................ 73 
11.5. 
Handling of Missing Data and Outliers ............................................................... 74 
12.0. 
 Direct Access to Source Data/Documents ....................................................................... 74 
13.0. 
 QUALITY CONTROL AND QUALITY ASSURANCE ............................................... 75 
13.1. 
Study Monitoring ............................................................................................... 75 
13.2. 
Audits and Inspections........................................................................................ 75 
14.0. 
 ETHICS ......................................................................................................................... 75 
14.1. 
Ethical Conduct of the Study .............................................................................. 75 
14.1.1. Confidentiality ....................................................................................... 76 
14.1.2. Compensation ........................................................................................ 76 
14.1.3. Written Informed Consent ...................................................................... 76 
14.1.4. Medical Care for Research-Related Injury .............................................. 77 
14.2. 
Ethics Review .................................................................................................... 77 
14.2.1. Review/Approval of Study Protocol ....................................................... 78 
14.2.2. Institutional Review Board ..................................................................... 78 
14.2.3. Protocol Modifications ........................................................................... 78 
15.0. 
 DATA HANDLING AND RECORD KEEPING ........................................................... 79 
15.1. 
Inspection of Records ......................................................................................... 79 
15.2.   Retention of Records ............................................................................................ 80 
16.0. 
PUBLICATION POLICY ............................................................................................... 80 
16.1. 
IND Annual Reports and Final Clinical Study Reports ........................................ 80 
16.1.1. IND Annual Report to the FDA.............................................................. 80 
16.1.2. Study Results and Clinical Trial Registries .............................................. 80 
17.0. 
 LIST OF REFERENCES ............................................................................................... 81 
18.0 APPENDIX A: .............................................................................................................................. 84 
 
 

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4. 
List of Abbreviations  
AE 
Adverse event, adverse experience 
ALT 
Alanine aminotransferase 
AST 
Aspartate aminotransferase 
βhCG 
Beta human chorionic gonadotropin 
BS 
Blood sample 
BSE 
Bovine spongiform encephalopathy 
C 
Celsius 
CBER 
Center for Biologics Evaluation and Research (United States Food and Drug 
Administration) 
CFR 
Code of Federal Regulation 
CI 
Confidence interval 
CLIA 
Clinical laboratory improvement amendments (42 CFR 493) 
CMI  
Cell-mediated immunity 
CMP 
COVID-19 
CRADA 
Comprehensive metabolic panel 
Coronavirus disease 2019 
Cooperative Research and Development Agreement 
CSSD 
CTA 
Clinical Services Support Division 
Clinical Trials Agreement 
CTC 
Clinical Trials Center 
CVD 
Center for Vaccine Development and Global Health (Maryland) 
DENV 
Dengue virus 
DENV-1, -2, -3, -4 
Dengue virus serotype 1, 2, 3 or 4 
DENV-4-LVHC 
Dengue-4 Virus – Live Virus Human Challenge 
DHF 
Dengue hemorrhagic fever 
DHIM 
Dengue Human Infection Model 
DoD 
Department of Defense 
DPIV 
dengue virus purified inactivated vaccine 
DSMB 
Data Safety Monitoring Board 
eCRF 
electronic case report form 
ELISA 
Enzyme-linked immunosorbent assay 
ELISpot 
Enzyme-linked immunospot assay 
F 
Fahrenheit 
FDA 
FV 
US Food and Drug Administration 
Flavivirus 
GBS 
Guillain-Barrè syndrome 
GCP 
Good clinical practice 
GCRC 
General Clinical Research Center 
GLP 
Good laboratory practice 
GMT 
H 
Geometric mean titer 
Hour 
HBsAg 
Hepatitis b surface antigen 

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HCV 
Hepatitis c virus 
HIPAA 
Health Insurance Portability Accountability Act 
HIV 
human immunodeficiency virus 
HSPB 
Human Subjects Protection Branch 
HSPO 
Human Volunteers Protection Office 
IB 
Investigator brochure 
ICF 
Informed consent form 
ICH 
International Conference on Harmonization 
ICS 
Intracellular cytokine staining 
IDS 
Investigational drug services (pharmacy) 
IFN 
Interferon 
IND 
Investigational new drug 
IRB 
Institutional review board 
LAV 
Live attenuated virus (Tetravalent Dengue Virus) 
mAb 
Monoclonal antibody  
MD 
Medical doctor 
MedDRA 
Medical Dictionary for Regulatory Activities 
Mg 
Milligram 
mL 
Milliliter 
Mm 
MOP 
Millimeter 
Manual of Procedures 
NIAID 
National Institute of Allergy and Infectious Diseases 
NCS 
Not clinically significant 
NP 
Nasopharyngeal 
NSAID 
Non-steroidal anti-inflammatory drug 
ORA 
Office of Regulated Activity 
ORP 
Office of Research Protections 
PPE 
Personal Protective Equipment 
PBF 
Pilot Bioproduction Facility  
PBMC 
Peripheral blood mononuclear cells 
PDMP 
Protocol Deviation Management Plan 
PI  
PIV 
Principal investigator 
Purified Inactivated Vaccine 
PFU 
Plaque forming units 
PPAS 
Per-protocol analysis set 
PSSB 
Product Safety Surveillance Branch 
PVG 
PharmacoVigilance Physician 
RBC 
Red blood cell 
RDE 
Remote data entry 
RN 
Registered nurse 
RNA 
Ribonucleic acid 
RT-PCR 
Reverse transcription – polymerase chain reaction 

Clinical Protocol ADVP005 
The University of Maryland, Baltimore  
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SAE 
Serious adverse event 
SAP 
Statistical analysis plan 
SARS-CoV-2 
Severe acute respiratory syndrome coronavirus 2 
SC 
Subcutaneous 
SOP 
Standard operating procedure 
SUNY-UMU 
State University of New York, Upstate Medical University 
TDEN 
Tetravalent Dengue Virus 
ULN 
Upper limit of normal 
UMB 
University of Maryland, Baltimore 
UMB HRPP 
University of Maryland, Baltimore Human Research Protection Program 
UMMC 
University of Maryland Medical System 
US 
United States 
USAMMDA 
United States Army Medical Materiel Development Activity 
USAMRDC 
United States Army Medical Research and Development Command 
WRAIR 
Walter Reed Army Institute of Research 
WBC 
White blood cell 
WHO 
World Health Organization 

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5. 
Introduction 
Dengue, a mosquito-borne disease caused by serologically related but antigenically distinct viruses 
grouped into 4 types (DENV-1 to DENV-4), occurs in more than 128 countries in the tropical and 
subtropical regions of Asia-Pacific, the Americas, the Middle East, and Africa (estimated 2.5 
billion people at-risk). Dengue is the most common arboviral (mosquito-borne) infection of 
humans and a major cause of acute febrile illness affecting an estimated 100 million people 
worldwide annually, with 500,000 cases of dengue hemorrhagic fever (DHF) and 22,000 deaths 
annually (WHO-2009).1 Dengue is associated with explosive urban epidemics and has become a 
major public health problem, with significant economic, political, and social impact. Dengue is a 
significant cause of febrile illness in returning travelers, especially those from the Caribbean and 
Southeast Asia.2 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.3 
The annual proportionate morbidity due to dengue in Southeast Asia during an epidemic is 
estimated to be 159 cases per 1,000 travelers.4 The risk is likely higher for deployed military 
personnel due to longer exposure periods.  
Dengue ranks third on the US DoD infectious disease threat list, after malaria and infectious 
diarrhea. US military operations over the past century have experienced numerous disruptions due 
to dengue. It has particularly affected US troops in the Pacific Theater, with over 91,000 cases 
observed in WWII. Dengue infection accounted for approximately 30% of febrile viral illnesses 
in Vietnam, affecting 40/1000 troops. More recently it was the cause of 289 hospitalizations in 
Somalia and 406 hospital admissions among US troops in Haiti. Soldiers who are hospitalized due 
to dengue take 14-21 days to recover to the point where they are fully mission capable. As the 
United States shifts its foreign policy and military focus to the Pacific region, the number of 
military and civilian personnel traveling to dengue endemic regions is also expected to rise 
significantly.5 Once infected with dengue virus (DENV), the incubation period typically lasts 4 to 
7 days, ranging from 3 to 14 days.6 Infection with dengue virus may cause an undifferentiated 
febrile illness with a maculopapular rash. The majority of infections, especially in children under 
age 15, are asymptomatic or minimally symptomatic.7 Older children and adults sustain either a 
mild febrile syndrome or the classical, self-limited, but incapacitating illness dengue fever 
consisting of fever (often 102°F-105°F), myalgias, rash, frontal headache, retro-orbital pain, 
nausea, vomiting, anorexia, altered taste and olfactory perception, and malaise. The fever typically 
lasts 2-7 days and viremia lasts an average of 5 days.8 Following the painful “breakbone” and 
febrile phase most patients slowly improve. Dengue virus disappears from the bloodstream at 
approximately the same time that the fever dissipates. Leukopenia and thrombocytopenia are 
common findings in mild dengue infection.  
A small proportion of individuals will progress to a more severe form of dengue infection. 
Manifestations of severe dengue (DHF or dengue shock syndrome [DSS]) include severe 
hemorrhage leading to shock through blood loss, sudden increased vascular permeability leading 
to shock with or without hemorrhage, and severe encephalopathy with hepatitis. There is no 
pathognomonic sign or symptom for DHF during the acute stage, but as fever remits, characteristic 
manifestations of plasma leakage appear, making accurate clinical diagnosis possible in many 
cases. An individual who has been infected with a specific DENV serotype, develops serotype 
specific antibodies and is generally considered to be protected from future infection from the same 
serotype. However, they are not protected against infection from a heterologous serotype, and 
approximately 10% are at increased risk for progressing to DHF or DSS during the second 

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infection.9 This is theorized to be due to antibody-enhanced entry of dengue virus into cells.10 No 
specific treatment is currently available but symptomatic and supportive measures (e.g., 
intravenous [IV] fluid management) significantly reduce the morbidity and mortality rates. Less 
well verified has been the impact of exposure to prior flavivirus infections.11  Additionally, host 
characteristics such as pediatric obesity may play a role in disease severity.12 Currently, vector 
control is the only method with a demonstrated although limited efficacy, but it is costly. Mosquito 
control does not offer satisfactory control of dengue and additional prevention methods are 
required. There are no antivirals registered, but several candidates are under evaluation at 
preclinical stages. Several vaccine candidates are at various stages of development. There is one 
FDA licensed dengue vaccine but is only indicated in pediatric populations with prior dengue 
exposure.13 This labeling is in part due to the concern of enhanced dengue disease in vaccine 
recipients with no previous dengue.14  
5.1. 
Rationale for the Study Design 
To evaluate the effectiveness of candidate dengue vaccine formulations, and to refine our 
understanding of correlates of dengue protection, it is prudent to develop an appropriate challenge 
model. To this end, this study will examine the level of protection following Dengue 1 Live Virus 
Human Challenge (DENV-1-LVHC) product in volunteers previously vaccinated with 
heterologous prime boost utilizing Tetravalent Dengue Virus (TDEN) Purified Inactivated 
Vaccine (PIV) and Tetravalent Dengue Virus Live Attenuated Vaccine (LAV) as compared to 
unvaccinated, healthy control volunteers. We will further examine the safety and effectiveness of 
the DENV-1-LVHC and assess the ability of this virus strain to elicit an uncomplicated dengue-
like illness. 
5.2. 
Name and Description of the Investigational Product 
The product used in this study, the Walter Reed Army Institute of Research (WRAIR) challenge 
lot 1806, DENV-1-LVHC, is a lyophilized powder for injection consisting of: DENV-1 strain 
45AZ5 virus, Eagle’s minimal essential medium (EMEM, modified) culture medium, human 
plasma albumin-USP, L-glutamine, streptomycin, neomycin, and lactose. The freeze-dried 
DENV-1-LVHC is rehydrated with 0.7 mL of water for injection (WFI). The route of 
administration is subcutaneous (SC) injection for the proposed indication of infection of volunteers 
to produce an uncomplicated dengue-like illness in order to support development of vaccines and 
drugs. 
5.3. 
Summary of Pre-clinical and Clinical Trials 
Pre-clinical Studies 
In 2013, the new DENV-1-LVHC was used in a non-Good Laboratory Practices (GLP) preclinical 
study entitled “Pre-clinical evaluation of dengue virus type 1 (DENV-1) pre- and post-transfection 
human challenge viruses in the rhesus macaque (Macaca mulatta) infection model.” The purpose 
of the study was to test the re-derived (post-transfection) DENV-1 challenge virus in rhesus 
macaques and compare it with its pre-transfection parent (Salk vaccine, lot 1-82). The DENV-1 
challenge virus was administered SC (0.5 mL) at an infectivity titer of 10 5 plaque forming units 
(PFU)/mL to 10 healthy adult rhesus macaques. Five of the animals were administered DENV-1 
45AZ5 (DENV-1-LVHC), Salk lot 1-82, Run 2, pre-transfection seed; and 5 of the animals were 

Clinical Protocol ADVP005 
The University of Maryland, Baltimore  
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administered DENV-1 45AZ5, lot 1806, post-transfection seed. Blood samples collected daily for 
14 days and on Days 16, 18, and 21, were used to measure serum virus (viremia) by virus 
amplification in Vero cells (qualitative viremia), 50% cell culture infective dose (CCID50) assay 
(quantitative viremia), and viral RNA (RNAemia) by quantitative reverse transcription polymerase 
chain reaction (RT-PCR) assay. A blood sample collected 28 days post-challenge was used for 
measuring virus neutralizing antibody titers by PRNT50. The animals were also observed for local 
changes or possible systemic side effects. 
The results of this study showed no significant differences between the pre- and post-transfection 
virus seeds in terms of their ability to induce viremia or RNAemia in rhesus macaques, nor were 
there significant differences in viremia onset, duration, or mean virus titers between groups that 
received the different challenge viruses. Furthermore, both groups exhibited similar 
seroconversion rates and neutralizing antibody geometric mean titers (GMTs) approximately 
1 month after virus challenge. These results support the original hypothesis that re-derivation of 
DENV-1 by transfection of viral RNA in FRhL cell culture has no effect on the infectivity 
phenotype in an accepted nonhuman primate model. Please refer to the Investigator’s brochure for 
additional information. 
Clinical Studies 
This protocol is aligned with a previous protocol (S-14-09) optimizing the DENV-1-LVHC (IND 
016332) and performed by the State University of New York, Upstate Medical University (SUNY-
UMU). 
Refer to the IB for additional safety and immunogenicity data.  
5.3.1.1. 
Early Human Challenge Studies with DENV-1 45AZ5 
In 2001, study A-9211, “Clinical and Immunological Evaluations of Four Dengue Viruses as 
Challenge Strains in Immune and Susceptible Volunteers,” was completed under BB-IND-8796. 
The clinical study report (CSR) was submitted on 14 June 2002; serial number (SN) 0018 and a 
second iteration (for site number 2) was submitted on 02 January 2003 (SN 0020). This was an 
exploratory study to evaluate possible dengue challenge viruses that will reproducibly cause 
uncomplicated dengue fever lasting 3-7 days.  
Out of 15 volunteers total; two volunteers received DENV-1, three volunteers received DENV-2, 
three volunteers received DENV-3, four volunteers received DENV-4, and three volunteers 
received placebo. DENV-1 45AZ5 was administered SC to two flavivirus (FV) naïve volunteers 
at a dose of 0.5 mL containing 1.6x104 PFU.  
Of the 12 volunteers in this study who were challenged with one of 7 different attenuated strains 
of dengue, 7 volunteers developed asymptomatic fluid collections around the liver, spleen, lungs, 
or heart.15 An analysis of the clinical results can be found in Mammen, 2014, and a summary of 
sonographic findings for this study was published in Staler et al., 2008.15,16 This DENV-1 
challenge strain was further characterized by Endy et al. who inoculated 12 volunteers at two 
different concentrations.17 All except one subject developed detectable viremia and there were no 
statistically significant differences in mean symptom severity between the low and mid dose 
groups. There were no SAEs and all adverse events resolved without complications or sequelae. 

Clinical Protocol ADVP005 
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5.3.1.2. 
Phase 2b Evaluation of Tetravalent Live-Attenuated Dengue Vaccines (WRAIR 
#877) (A-11014, IND 8796) 
Study A-11014, “Phase 2b Evaluation of Tetravalent Live-Attenuated Dengue Vaccines (WRAIR 
#877),” was completed under BB-IND-8796, and the Quality (Chemistry, Manufacturing, 
Controls) information was included in Master File 8797. The final CSR was submitted to the US 
Food and Drug Administration (FDA) on 04 March 2008 (IND 8796, SN0029). This clinical study 
was performed using both previously vaccinated and non-vaccinated (FV-naïve) volunteers. 
In regard to DENV-1, all 5 volunteers previously vaccinated with tetravalent live-attenuated 
dengue vaccine (volunteers 1, 4, 6, 7, and 12) were protected against DENV-1 virus challenge. 
One volunteer who was previously vaccinated with tetravalent vaccine did not have measurable 
neutralizing antibody at the time of challenge. DENV-1 in control volunteers (volunteers 17 and 
18) caused febrile illness lasting 2-6 days after incubation periods of 17 and 21 days, respectively. 
Only one of these control recipients was viremic. All volunteers recovered without sequelae.18,19  
5.3.1.3. 
Attenuated Candidate Dengue 1 Vaccine (45AZ5) in Human Volunteers (IND 
1952) 
DENV-1 45AZ5 was administered under BB-IND-1952 as SC injection of 0.5 mL containing 3.0 x 
104 PFU to 2 adult volunteers with pre-existing neutralization titers >1:10 against yellow fever 
virus from prior yellow fever virus vaccination but no pre-existing hemagglutination inhibition 
(HI) or neutralizing antibody to any dengue serotype. (Additionally, one volunteer had received 
an inactivated tick-borne encephalitis vaccine.) Neither volunteer reported significant discomfort 
at the injection site, and no redness, swelling, or regional adenopathy were seen. After an 
incubation period of 8-9 days, both volunteers developed characteristic dengue fever; headache, 
myalgia, fever (lasting for 3 and 5 days, respectively), rash, progressive leukopenia (reaching 
nadirs of 2,700/mm3 and 2,300/mm3, respectively) and mild platelet count depression (below 
200,000/mm3, the lower count of the two was 123,000/mm3). They experienced neither 
hemorrhage nor hypotension. Proteinuria and mild pyuria were noted incidentally in one volunteer. 
One volunteer had resolution of his fever and symptoms by the 6th day of illness. The other 
volunteer achieved resolution of fever and overall symptomatic improvement by the fifth day of 
illness; however, he experienced malaise, easy fatigability, and a sense of depression until the 
eleventh day of illness. 
Viremia, using both direct plaque assays of plasma and amplification of viremia isolates by 
passage in LLC-MK2 and C6/36 cell cultures, began on the day after vaccination, lasted for 19 and 
12 days, respectively. Both volunteers developed neutralizing and HI antibodies to DENV-1 virus. 
The earliest day when HI antibody could be detected at titer >1:10 in either volunteer was Day 15 
post-vaccination. Additionally, broadly reactive HI antibodies developed against yellow fever 
virus and the remaining 3 dengue serotypes. DENV-1-specific IgM antibody was detected in sera 
from both volunteers beginning 13 days post-vaccination and increased through Day 19, and then 
leveled off to remain relatively stable through Day 30. The values at Day 60 were inconsistent 
between the two volunteers. DENV-1-specific IgG antibody appeared initially on Day 15 in both 
volunteers, increased through Days 17 and 19 respectively, and remained stable through Day 30 
but decreased significantly by Day 60. The phenotypic characteristics of the virus obtained from 
these volunteers differed with respect to colony size and temperature sensitivity.20 

Clinical Protocol ADVP005 
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5.3.1.4. 
A Phase 1 Evaluation of the Protective Efficacy of a Single Dose of the Live 
Attenuated Tetravalent Dengue Vaccine TV003 to Protect Against Infection with 
Attenuated DENV-2, rDEN2Δ30-7169 (NIAID, NCT02021968) 
The National Institute of Allergy and Infectious Diseases (NIAID) initiated a dengue challenge to 
evaluate the protective effectiveness of a dengue virus vaccine in healthy adults. This was a phase 
1, randomized, double-blind, parallel, placebo controlled, challenge study. Volunteers were 
randomized to either TetraVax-DV-TV003 vaccine, a live attenuated, tetravalent DENV vaccine 
or placebo. On Day 180, volunteers received a single injection of the "challenge" virus, 
rDEN2∆30, an attenuated DENV-2 vaccine.  Of the 21 volunteers were received the TV003 and 
were challenged, all were protected from infection. No rash, viremia or laboratory abnormality 
was noted.  All 20 placebo recipients who were challenged, developed viremia, with 80% 
developing a rash and a minority exhibiting neutropenia (20%), indicative of an attenuated 
challenge strain.21   
5.3.1.5. 
Past Human Studies with Any Dengue Serotypes 
Other previous clinical studies with any dengue serotype are listed in Table 1 
Table 1: Additional Past Clinical Studies Including Challenge with Any Dengue Serotype 

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Investigator(s) 
Publication 
Year 
Volunteers 
Inoculated 
Dengue 
Serotype 
Comment 
Graham22 
1902 
6 
Unknown 
Infection by mosquito inoculation 
Ashburn and 
Craig23 
1907 
22 
Unknown 
Injection of blood and mosquito inoculation 
Cleland24 
1919 
21 
Unknown 
Injection of human and animal blood; mosquito 
inoculation 
Siler25 
1926 
64  
DENV-4 
Infection by mosquito inoculation 
Simmons26 
1931 
81 
DENV-1 
infection by mosquito inoculation 
Misao27 
1944 
120 
DENV-1 
Injection of blood and mosquito inoculation 
Hotta28 
1952 
>10 
Unknown 
Injection of blood 
Sabin and 
Schlesinger29 
1945 
16 
DENV-1, 
DENV-2 
Injection of mouse-adapted dengue 
Sabin and 
Schlesingera 
1944-6  
>125, >75 
DENV-1, 
DENV-2 
Injection of mouse-adapted dengue 
Taniguchi30 
1951 
35 
Unknown 
Injection of blood; mouse-, rabbit-, and chick 
embryo-adapted dengue 
Schlesinger31 
1956 
17 
DENV-1, 
DENV-2 
Injection of mouse-adapted dengue vs. sterile 
saline; then unmodified dengue challenge 
Wisseman32 
1966 
15 
DENV-1 
Injection of unmodified virus 
Eckels33 
1984 
2 
DENV-4 
Vaccine trial: injection of virus from cell 
culture 
McKee20 
1987 
2 
DENV-1 
Vaccine trial: injection of virus from cell 
culture 
Innis34 
1988 
2 
DENV-3 
Vaccine trial: Injection of virus from cell 
culture 
Edelman35 
1994 
29 
DENV-1 
Vaccine trial: SC injection of under-attenuated 
DENV-1 45AZ5 
Sun18 
2013 
14 
DENV-1, 
DENV-3 
Injection of virus from cell culture 
Mammen16 
2014 
15 
DENV-1, 
DENV-2, 
DENV-3, 
DENV-4 
Injection of virus from cell culture 
Kirkpatrick21 
2016 
41 
DENV-2 
Vaccine trial: SC injection of attenuated 
rDEN2∆30 virus 
a Telephone communication between Dr. Schlesinger and COL Bruce Innis 
5.3.1.6. 
Heterologous Prime Boost using tetravalent dengue PIV and tetravalent dengue 
LAV 
Two dengue vaccine trials have been conducted at WRAIR utilizing a heterologous prime boost strategy. 
The first study entitled, “A Phase 1, Randomized, Open-label, Single-center, Study of TDENV-PIV and 
LAV Dengue Vaccine Platforms as part of a Heterologous Prime-boost Strategy in Healthy Adults in a 
Nonendemic Region” (ADVP003) involved 4 groups of 20 volunteers who received PIV followed by LAV 
at either 28 or 180 day or LAV followed by PIV at 28 or 180 days. This study demonstrated a substantial 
boost in immune response as measured by dengue neutralizing antibody in the groups that received PIV 

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prior to LAV. Those volunteers who received LAV followed by PIV were not observed to produce a 
substantial boost in antibody production following receipt of PIV boosting. The overall antibody titers were 
noted to be substantially higher in the PIV/LAV 0,180 group than the PIV/LAV 0,28 with persistently 
higher titers through 6 months post final vaccination and persistent tetravalent seroconversion status for all 
volunteers in this group. (ASTMH Annual Meeting presentation Nov 2016). 
A second study of prime boost was subsequently initiated to further evaluate the optimal schedule for this 
vaccination regimen. The second study 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”(ADVP004)  enrolled 40 volunteers in two groups of 20 who received 
either PIV/LAV 0,90 day or PIV/LAV 0,180 days. This study demonstrated similarly high rates of antibody 
production at 28 days post boost in both the PIV/LAV 0,90 and PIV/LAV 0,180 groups along with 95% 
tetravalent seroconversion in the 0,90-group compared to 100% in the 0,180 group overall suggesting 
comparability of these schedules in terms of preliminary immunogenicity readouts. This trial has completed 
all late time point study visits and final analysis is currently underway.  
5.4. 
Known and Potential Risks and Benefits to Human Volunteers 
 
5.4.1.   Dengue Classification 
According to the World Health Organization (WHO) classification, dengue severity is divided 
into the following: dengue with or without warning signs, and severe dengue 36 
Dengue without warning signs is defined as fever and two (2) of the following: 
• 
Nausea 
• 
Rash 
• 
Aches and pains 
• 
Leukopenia 
• 
Positive tourniquet test 
Dengue with warning signs is dengue defined above with any of the following: 
• 
Abdominal pain or tenderness 
• 
Persistent vomiting 
• 
Clinical fluid accumulation (ascites, pleural effusion) 
• 
Mucosal bleeding 
• 
Lethargy, restlessness 
• 
Liver enlargement > 2 cm 
• 
Laboratory increase in HCT concurrent with rapid decrease in platelet count 
Dengue with warning signs requires strict observation and medical intervention. Severe dengue 
is defined by severe plasma leakage, severe hemorrhage, and severe organ impairment. Severe 
dengue also requires strict observation and medical intervention. We do not anticipate dengue 
with warning signs, based upon prior experience with this attenuated DHIM-1 strain, however, 
we will categorize volunteers into this WHO categorization on a post-hoc basis if symptoms 
dictate. 

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5.4.2.  Risks/Discomfort to Volunteers and Precautions to Minimize Risk 
Outlined below are anticipated and unexpected/unanticipated 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. Of note, the challenge strain is an 
attenuated strain of dengue virus. 
5.4.2.1.      Local Reactions 
Local reactions may include pain, redness, and swelling at the injection site. 
5.4.2.2.      Systemic Reactions 
The symptoms of classic dengue infection range from asymptomatic infection, to a mild febrile 
syndrome, or the classical, self-limited, but incapacitating illness dengue fever consisting of fever 
(often 102°F-105°F), joint pain, myalgias, frontal headache, retro-orbital pain, nausea, vomiting, 
anorexia, rash, increase in liver enzymes, leukopenia, thrombocytopenia, and mild bleeding 
manifestation (e.g., nose or gum bleed), petechiae, or easy bruising. Any or all of these clinical 
manifestations may occur. While the incubation period for natural occurring dengue is typically 7 
days (range 4 – 10 days) with manifestations of complicated dengue likely to appear by Day 10 
(range 3-7 days post symptom onset), the incubation and signs of complicated dengue infection 
may be different for the attenuated study product. A recent trial conducted at SUNY Upstate 
utilizing the DHIM-1 study product demonstrated that 5 of 6 volunteers developed viremia 
between Days 4-16 (range 9-12 days) accompanied by rash (5 of 5 volunteers) between Days 6-
24 (mean of 9 days), and fever (>100.4º F) in only on volunteer (Day 12). Other reported symptoms 
included headache, eye pain, weakness, bone, muscle, and/or joint pain, indicative of an 
uncomplicated dengue infection.  
In-patient patients will receive symptomatic and supportive care as needed. Most will only require 
oral hydration and acetaminophen for fever and myalgias. Volunteers with more severe symptoms 
may require IV fluid hydration. The risks for this are pain and bruising at the IV site, 
thrombophlebitis, fluid extravasation, or volume overload.  Local infection or systemic infection 
(bacteremia) are extremely uncommon risks related to IV insertion. 
Severe dengue infection (DHF) with evidence of plasma leakage and hemorrhage is extremely rare 
in first time infections. Only previously vaccinated or dengue naïve volunteers will be eligible to 
participate in this study. No volunteers who have received live attenuated virus strains in prior 
vaccination or challenge studies (including other DENV challenge studies of those who have 
received dengue vaccines) have developed severe dengue symptoms.21  
5.4.2.3.     Pregnancy 
Risks to unborn babies are not well characterized at this time, with a recent meta-analysis 
indicating a possible association with dengue and pre-term delivery and/or lower birthweight.37 
Pregnancy is an exclusion criterion for enrollment in this study and contraception should be used 
by female and male volunteers for the duration of the study. Female volunteers who become 
pregnant following inoculation will not be discontinued from the trial and will be followed for 
safety assessment for the duration of the pregnancy. Female partners of male volunteers who father 
a child during the study will be asked to be followed for the duration of the pregnancy. 
A sexually active man is defined as one whose partner is a woman of childbearing potential (see 
definition below) and has not had a vasectomy performed > 1 year prior to screening. Dengue virus 

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has not been detected in human semen.38 However, out of an abundance of caution, they must 
agree not to father a child until 6 months after the inoculation. These volunteers must agree to use 
a barrier method of birth control (e.g., either condom with spermicidal foam/gel/film/cream or 
partner usage of occlusive cap [diaphragm or cervical/vault caps] with spermicidal 
foam/gel/film/cream/suppository). 
Women of childbearing potential are defined as those who have not been sterilized via tubal 
ligation, bilateral oophorectomy, bilateral salpingectomy, hysterectomy, or successful Essure® 
placement (permanent, non-surgical, non-hormonal sterilization) with history of documented 
radiological confirmation test at least 90 days after the procedure (or with use of another birth 
control method if history of confirmation test not confirmed), still menstruating or < 1 year of the 
last menses if menopausal. For this study, an effective contraceptive method is defined as one that 
results in a failure rate of less than 1% per year when it is used consistently. Adequate contraceptive 
precautions include- intrauterine contraceptive device, oral contraceptives, diaphragm, or condom 
in combination with contraceptive jelly, cream, or foam; Norplant® or Depo-Provera®, through 56 
days after the dengue challenge injection to minimize any potential risk. 
5.4.2.4.      Lactation 
Risks to nursing infants are unknown at this time. Females who are breastfeeding a child will be 
excluded from enrollment into the study. Females will be instructed not to become pregnant for 
the duration of the study. 
5.4.2.5.      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 at the needle puncture site 
and/or nerve damage. Occasionally, the act of blood sampling or the injection of study product 
induces a vasovagal reaction.  Volunteers will be queried as to their propensity for this reaction 
and will be encouraged to remain lying down during procedures. 
5.4.2.6.       Allergic Reaction 
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.  
5.4.2.7.       Guillain-Barré Syndrome (GBS) or adverse reactions to vaccines 
A serious but rare, neurologic disorder called Guillain-Barré syndrome can occur after certain 
infections, due to immune activation which damages the body’s peripheral nerves, resulting in 
muscle weakness and some degree of paralysis. GBS can last for weeks to months. There is no 
known causal association of dengue and GBS. Approximately 85% of people eventually recover 
completely or nearly completely, but some people have permanent nerve damage, and between 
3% and 4% of people who develop GBS die. To decrease this risk, individuals with a history of 
GBS or other significant reaction to vaccines will be excluded from participating in the study.  
5.4.2.8.       Observations from Natural Dengue Infection 
Patients who have antibody from an earlier dengue virus infection and who are subsequently 
infected by a dengue virus strain of another serotype, have been shown to be at higher risk for 

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DHF and/or dengue shock syndrome. The virus to be used in this study is designed to induce 
Dengue-1 virus sero-responsiveness. There is the possibility that a volunteer exposed in the future 
to serotypes 2, 3, or 4 may develop a more severe case of dengue infection (i.e., DHF/DSS) than 
if they had been dengue naïve. To date, there is no evidence that attenuated dengue virus vaccines 
(monovalent and tetravalent) place adults at increased risk for severe dengue.39 However,  a 
chimeric dengue vaccine (Dengvaxia®) produced by Sanofi, has demonstrated a safety signal 
suggesting increased risk in seronegative pediatric patients less than nine years of age.40 
Volunteers from the ADVP-004 study, included in this protocol, will have received a tetravalent 
dengue virus (TDEN) purified inactivated vaccine (PIV) followed by TDEN live attenuated virus 
(LAV) at 90 or 180 days post TDEN-PIV. Other than one volunteer in the 0, 90-day vaccination 
group, volunteers developed high titer tetravalent antibody responses at 28 days following boost 
dose. The exact correlate of protection from dengue infection remains unknown but neutralizing 
antibody is believed to be important.41 The potential risk of enhanced dengue infection upon 
natural infection following vaccination and subsequent inoculation with this challenge strain is 
unknown. Tetravalent responders (defined as Day 28 titers following final vaccination) from the 
ADVP-004 study will be selected, and titers repeated at screening. Volunteers who have sustained 
tetravalent responses will be preferentially asked to participate in order to mitigate risk.  
Theoretical Risks 
Theoretical risks based on symptoms/outcomes typically associated with wild-type dengue 
infection, but not seen in prior attenuated DHIM studies may include: 
• 
Volunteer(s) may develop dengue fever with one or more of the following events 
− Hemorrhage 
− Evidence of end organ involvement defined by clinical or lab findings greater 
than grade 2 severity 
− Intravenous (IV) fluid requirement for hemodynamic instability (not for comfort 
or the inability to take oral fluids) 
• 
Volunteer(s) may develop dengue hemorrhagic fever (DHF) with symptoms similar 
to dengue fever plus, any one of the following 
− Severe and continuous pain in abdomen 
− Ascites and or/pleural effusions  
− Bleeding from the nose, mouth and gums or skin bruising 
− Frequent vomiting with or without blood 
− Black stools, like coal tar 
− Excessive thirst (dry mouth) 
− Pale, cold skin 
− Restlessness, or sleepiness 
• 
Volunteer(s) may develop dengue shock syndrome (DSS), which can be defined as 
DHF plus the following symptoms 

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− Weak rapid pulse 
− Hypotension 
− Respiratory distress 
− Altered level of consciousness 
− Narrow pulse pressure (less than 20 mm Hg)  
− Cold, clammy skin 
− Restlessness 
• 
A volunteer who receives this viral challenge could be primed for more severe 
disease, if exposed to another dengue virus following this study.  Post hoc analysis 
may re-categorize volunteers according to the WHO grading system in the unlikely 
event that non-severe cases with warning signs or severe cases occur.1 
There is a very low risk of acquiring dengue in the greater Baltimore-Washington area, and 
although the mosquito vector exists in the southern United States and along the mid-
Atlantic coastal regions, transmission from one individual with imported dengue to another 
individual is still very rare in the continental US. However, if a volunteer traveled to a part 
of the world where dengue is present, they have the potential to contract a second infection. 
There are four distinct, but closely related, serotypes of the virus that cause dengue 
(DENV-1, DENV-2, DENV-3, and DENV-4). Recovery from infection by one provides 
lifelong immunity against that particular serotype. However, cross-immunity to the other 
serotypes after recovery is only partial and temporary. Subsequent infections by other 
serotypes increase the risk of developing severe dengue. In primary wild type dengue 
infection, there is a less than 1% risk of developing DHF or DSS. With a secondary 
infection, this risk may increase to as high as  5-10%.9 
Globally, there are an estimated 50 to 100 million cases of dengue fever (DF) and several 
hundred thousand cases of dengue hemorrhagic fever (DHF) per year. There are 
approximately 30,000 deaths per year related to severe dengue. There is no specific 
treatment for dengue/ severe dengue, but early detection and access to proper medical care 
lowers fatality rates below 1%.36 The patients who succumb to dengue fever are typically 
very young, very old, or have underlying immune issues present. 
• 
Mosquitos may theoretically transmit the DENV-1-LVHC challenge strain to people 
in the community surrounding the clinical site.  DENV LVHC (rDEN230)  has been 
conducted within the greater Baltimore-Washington region.21  To mitigate the risk of 
challenge strain transmission, challenges will be conducted in the autumnal/winter 
months (November-March) when mosquitoes cannot replicate/transmit or volunteers 
will be sequestered during periods of viremia (April – October). 
• 
Female volunteers with excessive menstrual bleeding and hypermenorrhea have been 
associated with both severe and uncomplicated dengue.42  

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5.4.2.9.      Unknown Risks 
As with all research there is the remote possibility of risks that are unknown or that cannot be 
foreseen based on current information. This would include late effects that have been seen with 
some vaccines.  
5.4.2.10.     SARS-CoV-2 Precautions 
On December 31, 2019, a novel coronavirus termed SARS-CoV-2, was described.  Since that time, 
the virus has spread and has been termed a global pandemic by the World Health Organization. As 
such, special attention must be paid to volunteers participating in human clinical trials.  The Center 
for Vaccine Development and Global Health has been on the leading edge of implementing safety 
guidelines for the safe conduct of clinical studies during this outbreak (while conducting Covid-
19 vaccine studies). The center follows a policy of strict pre-screening of all volunteers admitted 
to the University campus. This includes mandatory mask use on campus, the completion of a pre-
screening questionnaire assessing for the presence of COVID-19 symptoms and a temperature 
check. All clinical staff will don appropriate personal protective equipment during study 
operations. Additionally, all rooms and equipment are wiped down between each volunteer and 
strict social distancing practices conducted.  Upper respiratory, either nasopharyngeal (NP) or 
nasal, swab for the presence of the SARS-CoV-2 virus by PCR, will be performed prior to dengue 
inoculation and if a temperature develops following this inoculation. Only those volunteers testing 
negative will be inoculated with the DHIM-1 challenge product. Volunteers will be asked to 
maintain self-quarantine conditions or sequester within a local hotel (e.g., The Lord Baltimore or 
Hotel Monaco) from the day of challenge inoculation until completion of daily follow-up. 
 
As of 12 December, mRNA vaccines have been issued EUAs for prevention of COVID-19.  Both 
the BioNTech/Pfizer and Moderna products are mRNA vaccines.  We will provide an vaccine 
exception for volunteers who have the opportunity to receive the vaccine, to receive one as early 
as 21 days following the DHIM, out of an abundance of caution and safety during the pandemic 
(defined in the Exclusion Criteria).   
 
5.4.3.  Alternatives to This IND Product or Study 
An alternative is to not participate in this study.  
 
5.4.4.  Intended Benefit for Volunteers 
There is no direct medical benefit for volunteers participating in this study.  
 
5.4.5.  Risks to the Study Personnel and the Environment 
The principal risk in the clinical setting is handling needles that may be contaminated with the 
challenge strain or human pathogens.  Adherence to universal precautions will reduce the risk of 
exposure. If pandemic conditions continue, strict attention to COVID-19 precautions will be 
adhered to including the use of masks and gloves for both volunteers and staff and appropriate 
PPE, pre-screening assessments prior to visits, and the sequestration of volunteers to the hotel to 
prevent the acquisition of the SARS-CoV-2 virus during the post-dengue inoculation phase.  Social 

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distancing practices will be maintained. All volunteers will be provided single hotel rooms with 
restricted access to any guests. 
There are no known risks to the environment other than those associated with the generation of 
biohazardous waste attendant to inoculation of humans. All biohazardous waste will be disposed 
of as stipulated by local, state, and federal regulations and in accordance with study site SOPs. 
5.5. 
Route of Administration, Dosage Regimen, and Experimental Vaccination  
The investigational product, DENV-1-LVHC will be administered in a single inoculation 
subcutaneously (SC) over the triceps muscle of the arm.  
The volume of a single dose is 0.5 mL for each challenge. See Section 7.3.7. for details regarding 
the challenge strain.  
5.6. 
Compliance Statement 
The study will be conducted according to the protocol and in compliance with International 
Conference on Harmonization (ICH) Good Clinical Practice (GCP), Belmont Principles, and other 
applicable regulatory and Department of Defense (DoD) requirements. All identified study 
personnel will be trained to perform their roles and will carry out their responsibilities in 
accordance with the ICH GCP guidelines and clinic site SOPs. A list of roles and responsibilities 
is included in the sponsor’s information sheet. 
5.7. 
Study Population 
This study will enroll healthy male and non-pregnant, non-breastfeeding female volunteers 
between the ages of 18 and 50 (inclusive) for the naïve volunteers as well as volunteers that were 
previous participants of a TDEN vaccine study (ADVP-004) conducted at Clinical Trials Center, 
Walter Reed Army Institute of Research (CTC, WRAIR). The study will include 5 unvaccinated 
dengue naïve volunteers (recruited from the greater Baltimore-Washington area), 5 volunteers 
vaccinated with TDEN PIV followed by TDEN LAV at 90 day, and 5 volunteers vaccinated with 
PIV followed by LAV at 180 days. If funds allow and volunteers express interest, a fourth group 
of 5 previously vaccinated volunteers may be recruited. If an inadequate number of volunteers 
vaccinated on these schedules are available volunteers vaccinated with PIV followed by LAV 28 
days later may also be included (ADVP-003 trial participants). WRAIR and WRAIR staff will not 
be directly involved in the clinical conduct of this study. Contact information from previous 
WRAIR CTC vaccine trial participants will be provided, and University of Maryland will perform 
recruitment and screening activities from this available list. 
5.8. 
Study Sites 
This study will be conducted at the Center for Vaccine Development and Global Health (CVD) at 
the University of Maryland, Baltimore (UMB). The UMB CVD is a clinic-based research center 
affiliated with the UMB School of Medicine (SOM) and is located on the 3rd and 4th floors of the 
Health Sciences Facility (HSF) Building. The outpatient phase will occur at the UMB CVD. A 
sequestration phase, precipitated upon detection of asymptomatic dengue viremia, will occur at a 
local hotel within a few blocks of the CVD.  The inpatient phase will occur at one of two sites.  
The first is Pharmaron, a biopark facility located across the street from the CVD and within two 
blocks of the University of Maryland Medical Center (UMMC). The General Clinical Research 

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Center (GCRC) is an alternative site located on the 10th floor of University Hospital itself. Only 
one inpatient site will be used per challenge period and the site will be chosen based upon inpatient 
site schedule availability and volunteer numbers. The CVD is staffed with Infectious Disease 
physicians, Internists and Pediatricians, with robust nursing and laboratory support. It is physically 
attached to UMMC, an 800-bed hospital with access to its tertiary care inpatient facilities, 
including radiology, cardiology and an Intensive Care Unit (ICU).  
 
6. 
Trial Objectives  
6.1. 
Primary Objectives  
• To further evaluate the safety and reactogenicity of the DHIM-1 human challenge model 
• To determine the level of protection to dengue serotype 1 challenge related symptoms 
provided by previous vaccination with heterologous prime boost utilizing TDEN PIV and 
TDEN LAV. 
6.2. 
Secondary Objectives 
• To characterize the immunologic responses following dose exposure to the DENV-1-
LVHC viral strain as a DHIM in vaccinated compared to unvaccinated volunteers 
• To compare virologic responses following dose exposure to DHIM-1 in vaccinated 
compared to unvaccinated groups 
6.3. 
Exploratory Objectives  
• To explore the immune response and host-virus interactions following exposure to 
manufactured, attenuated DENV-1-LVHC 
7. 
Trial Design 
7.1. 
Study Endpoints 
 
7.1.1.    Primary Endpoints  
The primary endpoints align with the safety and clinical objectives. The nature, frequency and 
severity of adverse events (AEs) associated with the attenuated DHIM-1 will be evaluated in the 
vaccinated ADVP004 volunteers and unvaccinated dengue-naïve control volunteers with 
statistical comparisons both within and between groups. 
The primary endpoints to address safety are as follows: 
• 
Occurrence, intensity, and duration of solicited injection site symptoms until 7 days post 
virus inoculation  
• 
Occurrence, intensity, and duration of unsolicited injection site symptoms until 28 days 
post virus inoculation or 7 days post hospitalization, whichever is later  
• 
Occurrence, intensity, and duration of solicited systemic symptoms until 28 days post 
virus inoculation or 7 days post hospitalization, whichever is later  

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• 
Number, intensity, and duration of abnormal laboratory measurements until 28 days post 
virus inoculation or 7 days post hospitalization, whichever is later  
• 
Occurrence, intensity and duration of dengue like symptoms/adverse events until 28 days 
post virus inoculation or 7 days post inpatient whichever is later (See defined Dengue 
Illness Index) 
• 
Occurrence, intensity, and duration of unsolicited systemic symptoms until 28 days post 
virus inoculation or 7 days post hospitalization, whichever is later  
• 
Number of SAEs until 28 days post virus inoculation or 7 days post hospitalization, 
whichever is later  
• 
Number of SAEs until 6 months post virus inoculation  
• 
The occurrence of fever defined as greater than or equal to 38°C (100.4° F) measured at 
least 2 times at least 4 hours apart 
7.1.2.   Secondary Endpoints  
The secondary endpoints align with the viremia and immunogenicity objectives. Dengue viremia 
will be examined both as a binary endpoint (present or not present) and as a function of area under 
the curve (AUC): 
• 
Viremia by reverse transcriptase-polymerase chain reaction (RT-PCR) up to 28 days post 
virus inoculation  
• 
MN50 antibody titers following inoculation in vaccinated and unvaccinated volunteers at 
28 days post inoculation 
 
7.1.3.   Exploratory Endpoints 
The endpoints for the characterization of response to the challenge virus may include the 
following: 
• 
Proteomics - microarray 
• 
Cell mediated immunity (CMI) 
• 
Transcriptomics (including single-cell transcriptomic assays) 
• 
Evolutionary analysis of DHIM-1 strain whole genome sequence (consensus and 
quasi-species)  
7.2. 
Overall Study Design 
The goal of the study will be to compare the level of protection (as determined by symptoms and 
viremia) against dengue serotype 1 challenge following previous vaccination with heterologous 
prime boost utilizing TDEN PIV and TDEN LAV and compared to unvaccinated controls. A 
Dengue Illness Index (DII) will assess the following parameters descriptively and as a function of 
a numerical calculated value between vaccinated and unvaccinated controls. 
The clinical characteristics (the level of uncomplicated dengue measured) are defined as: 
1. Measurable viremia by RT-PCR and duration in days 
2. Viremia by quantitative RT-PCR (pfu/mL) and calculated AUC 

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3. Number, percentage and severity score of volunteers in each group with each of the 
following clinical or laboratory symptoms as measured by the Dengue Illness Index: 
• 
Fever greater than or equal to 38°C (100.4°F) 
• 
Headache/retro-orbital pain  
• 
Rash 
• 
Fatigue and/or malaise 
• 
Myalgia  
• 
Arthralgia and/or bone pain 
• 
GI symptoms (nausea, vomiting, abdominal pain) 
• 
Liver function tests (ALT, AST)  
• 
Leukopenia 
• 
Thrombocytopenia  
*Note:  Disease severity can be re-classified into the WHO categorizations (Section 
5.4.1) in the unlikely event that complicated symptoms develop  
Volunteers will be followed with consideration of both the safety and performance profile 
following challenge and differences noted between previously vaccinated volunteers compared to 
unvaccinated controls. All volunteers will receive a single SC inoculation of DENV-1-LVHC (the 
inoculation). After the inoculation, volunteers will be seen and evaluated in the clinic beginning 
on Day 4 and then every day until Day 16 in the absence of viremia, upon which volunteers will 
be seen every other day until Day 28 post virus inoculation. If a volunteer develops asymptomatic 
viremia, they will enter the sequestration phase of the study. If a volunteer meets inpatient criteria 
listed in Section 9.4.1, they will be admitted to the inpatient facility (GCRC or Pharmaron) and 
will follow the inpatient phase schedule. Data will be collected and compiled during the 28 days 
after inoculation or through 7 days post inpatient discharge, whichever is later. 
7.3. 
Investigational Product 
A summary description of the investigational product is presented in Table 2. The DENV-1-
LVHC investigational challenge material consists of the dengue-1 virus strain 45AZ5 as a powder, 
lyophilized for reconstitution. The product is reconstituted with 0.7 mL of water for injection 
(WFI, Lot 6007223, manufactured by APP Pharmaceuticals, Grand Island, NY) for injection at a 
concentration of 6.5 x 105 PFU/mL. The reconstituted DENV-1 LVHC will be diluted in EMEM 
(BPR No.: BPR-1162-00 Lot 1889, 4.5 mL), which is provided as a sterile, vialed product. The 
WRAIR Pilot Bioproduction facility will be responsible for providing the investigational product 
to the study clinical site and maintaining adequate viable stocks of product on hand for the conduct 
of this study. 
 
Table 2: 
Investigational Product 
Product Name 
Dengue 1 Live Virus Human Challenge (DENV-1-LVHC) 
Diluent Name 
Diluent Eagle’s minimal essential medium (EMEM, modified)  
Dosage Form  
Liquid injectable 
Unit Dose  
0.5 mL of 6.5 x 103 PFU/mL 
 

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Product Name 
Dengue 1 Live Virus Human Challenge (DENV-1-LVHC) 
Route of Administration  
Subcutaneous in triceps region of arm 
Physical Description  
Yellow dense cake with no particulates (clear solution when 
reconstituted with WFI) 
Manufacturer  
WRAIR Pilot Bioproduction Facility 
Lot Number  
1806 
7.3.1.   Investigational Product Packaging and Labeling 
Sample investigational product labels are as follows: 
Dengue Virus Type 1 (45AZ5) (Live Virus) Vial # XXXX 
for Human Challenge 
BPR No.:  BPR-1090-01          Lot No.:  1806 
Contents: 0.7 ml (Freeze Dried) Storage: -15 to -30 °C 
Caution: New Drug – Limited by Federal (or United 
               States) law to investigational use. 
Date of Mfg.:  21 May 2013 
Manufactured By: WRAIR, Silver Spring, MD 20910 
 
EMEM Diluent                     Vial # XXXX 
BPR No.: BPR-1169-00        Lot No. 1898 (or equivalent Lot) 
Contents: 4.5 mL                   Storage 2 – 8oC 
Caution:  New Drug – Limited by Federal (or United States) 
                law to investigational use. 
Date of Mfg:  10 Oct 14 
Manufactured By:  WRAIR, Silver Spring, MD 20910 
 
7.3.2.  Investigational Product Shipment 
DENV-1-LVHC and the EMEM diluent will be shipped under controlled temperature conditions; 
-15°C to -30°C for the DENV-1-LVHC and 2ºC and 8ºC for the EMEM. DENV-1-LVHC and the 
EMEM diluent will be quarantined upon receipt by the study site, and temperature records will be 
downloaded and submitted to the designated representative for review. Upon verification that the 
cold chain was maintained during transit, WRAIR will provide assurance of the product’s integrity 
and authorization for use. Any temperature excursion, (i.e., temperature outside the defined range of 
storage) must be reported to the sponsor’s representative as soon as detected. After exposure to a 
temperature deviation, the product will not be used until written approval has been given by the 
sponsor’s representative. 
7.3.3.  Investigational Product Storage 
At the study site, the investigator or his or her designee (e.g. Investigational Drug Pharmacy) will be 
responsible for product management. The lyophilized DENV-1-LVHC to be administered to the 
volunteers must be stored at -15°C to -30°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 WFI used for reconstitution will be stored at 20°C to 
25°C. The EMEM used for diluting the investigational product will be stored under controlled 
refrigerator temperature at 2°C to 8°C. Any temperature excursion (i.e., temperature outside the 

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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. 
 
 
7.3.4.  Investigational Product Accountability 
The sponsor’s representative is responsible for receipt of the investigational product at the study 
site. The sponsor’s representative has delegated drug accountability responsibility for this product 
to PI; however, the sponsor’s representative has ultimate responsibility for product accountability. 
After the investigational product is distributed, the PI is responsible for and will maintain logs of 
investigational product receipt, storage, reconstitution, accountability by volunteer, and 
investigational product remaining before final disposition. The 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.  
All vials (unused, partially used, and spent) will be retained by the study staff for accountability. 
No vials should be disposed of or destroyed without sponsor’s representative’s approval. The 
disposition records will account for all remaining investigational products.  
7.3.5.  Investigational Product Preparation 
Detailed instructions for handling and diluting DENV-1-LVHC are provided in the Pharmacy 
guidelines and a summary is provided below. 
The reagents for preparing the investigational product reconstitution and dilutions are: 
• Investigational Product:  Dengue Virus Type 1 (45AZ5) (Live Virus) for Human 
Challenge, BPR No. 1090-01, Lot No. 1806, and volume: 0.7 mL upon rehydration with 
WFI 
• WFI: Sterile Water for Injection USP, Preservative Free, APP Pharmaceuticals, LLC, 5 
mL 
• Diluent: EMEM diluent BPR No.: BPR-1169-00 Lot 1898 (or equivalent lot), 4.5 mL  
Prior to preparation, all study products must be inspected visually for cracks, broken seals, correct 
label content, and extraneous particulate matter and/or discoloration, whenever solution and 
container permit. If any of these conditions exist, the product will not be used. A replacement dose 
will be used, and the event will be reported to the sponsor’s representative.  
The investigational products are removed from the freezer and refrigerator and prepared at room 
temperature. All drug product reconstitutions and dilutions will be conducted in a certified cleaned 
biological safety cabinet by trained personnel wearing appropriate personal protective equipment 
(PPE). 
The DENV-1-LVHC will be reconstituted with WFI prior to injection. The reconstituted DENV-
1-LVHC will be further diluted in EMEM. Per Pharmacy guideline, 0.5mL of diluted DENV-1-

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LVHC will be drawn into a syringe, labeled and stored at 2°C to 8°C. Once the product is 
reconstituted it is stable at 2°C to 8°C for up to 6 hours. 
7.3.6.  Investigational Product Administration 
Prior to administration, study product must be inspected visually for correct label content, and 
extraneous particulate matter and/or discoloration, whenever solution and container permit. If any 
of these conditions exist, the product will not be administered. A replacement dose will be used, 
and the event will be reported to the sponsor’s representative. 
Volunteer must be inoculated within 30 minutes of the investigational product being removed from 
2°C-8°C. DENV-1-LVHC will be administered SC in the triceps of the upper arm in a volume of 
0.5 mL. 
Volunteers will be kept under observation for at least 30 minutes after each inoculation to ensure 
their safety, and any reactions during this period will be documented. Appropriate medical 
equipment and emergency medications, including epinephrine (1:1000), will be available on site 
in the event of an anaphylactic or other immediate allergic reaction. 
If a vial or syringe is accidentally broken and the DENV-1-LVHC spills out, appropriate 
disinfection procedures must be used. 
The pharmacy label from the vaccine syringe will be placed in the source document.  
7.3.7.  Investigational Product Dose Selection 
The dose of DENV-1-LVHC results from optimization under protocol 5.3.5.2 S-14-09 conducted 
by the State University of New York (SUNY), Upstate Medical University, sponsored by the 
Surgeon General, Department of the Army under IND 016332. 
• 0.5 mL of 6.5 x 103 PFU/mL 
This study is intended to be a proof of concept study to further characterize the safety and 
immunogenicity of the DHIM-1 challenge product as well as to assess the protective efficacy of 
the TDEN PIV followed by LAV prime-boost vaccination. Based on the data from previous 
clinical studies using DENV-1, 45AZ5, we will inoculate at 6.5 x 103 PFU/mL). We will further 
characterize the DENV-1 attenuated strain to assess performance parameters and immunogenicity. 
7.3.8.  Investigational Product Replacement Doses 
There is no blinding or randomization code for this study, so if a dose needs to be replaced, 
another vial of product from the site’s supply can be used. 
7.4. 
Identification of Data to be Recorded on the Case Report Forms 
The electronic case report form (eCRF) data will be transcribed from source documentation. No 
source data will be recorded directly in the eCRF (i.e., without prior written or electronic record 
of data). The transcribed data will be consistent with the source documents or the discrepancies 
will be explained. For more information on data handling, refer to Section 15.0. 

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8. 
SELECTION AND WITHDRAWAL OF VOLUNTEERS 
8.1. 
Recruitment of Volunteers 
Volunteers will be recruited from the greater Baltimore-Washington area. Volunteers will be 
recruited via IRB approved flyers, email, advertisements, word of mouth, site database and social 
media. Volunteers previously vaccinated in ADVP003 (WRAIR#2136) and ADVP004 
(WRAIR#2453) have already been contacted to solicit interest in participating in this study. 
Personal protected information will be shared between investigators at USAMMDA and the study 
site at UMB CVD, under a cooperative trial agreement (CTA) and a cooperative research and 
development agreement (CRADA). Those who expressed interest have provided signed consent 
for their contact information to be provided to UMB to be contacted to screen for this study. This 
pool of volunteers will be utilized to recruit vaccinated volunteers for the vaccinated groups of 
volunteers in this study. Only those with day 28 post vaccination tetravalent seroconversion will 
be eligible for this study.  Preference toward those with detectable tetravalent neutralizing 
antibodies at the time of ADVP005 screening will be made if possible. As part of the ADVP005 
consent process, volunteers will be asked to provide consent for UMB CVD investigators to access 
their prior clinical and immune read-out data from the ADVP003 or ADVP004 studies.  Late time 
point serology from ADVP003 and -004 can be tested in parallel with ADVP005 screening sera. 
Refer to Section 5.7. for a detailed description of the study population. 
8.2. 
Eligibility Screening 
Each volunteer must meet all inclusion and no exclusion criteria. The PI or designee will make 
the final decision of the eligibility. Only eligible volunteers will be given the investigational 
product. 
8.2.1.    Volunteer Inclusion Criteria 
Volunteers must meet all the following criteria to be included in the study: 
1. Male or non-pregnant, non-breastfeeding female between 18 and 50 years of age 
(inclusive) at the time of consent. 
2. Tetravalent dengue antibody response at 28 days following final vaccination for 
vaccinated groups of volunteers. 
3. Volunteers must be able and willing to provide written informed consent. 
4. Volunteers must be healthy as established by medical history and clinical examination at 
study entry.  
5. Volunteers must pass a comprehension test and be able to comply with all study 
requirements. 
6. Female volunteers of non-childbearing potential (non-childbearing potential is defined as 
having had one of the following: a tubal ligation at least 3 months prior to enrollment, a 
hysterectomy, an oophorectomy, or is post-menopausal). 
7. Female volunteers of childbearing potential may be enrolled in the study, if all of the 
following apply: 

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-Practiced adequate contraception (see Definition of Terms, section 5.4.2.3.) for 30 
days prior to challenge 
-Has a negative urine pregnancy test on the day of DHIM 
-Agrees to continue adequate contraception until two months after completion of the      
DHIM 
8. Provide consent for release of medical history records from primary care physician, 
college or university medical center, urgent care, or emergency room visit  
 
8.2.2.   Volunteer Exclusion Criteria 
Volunteers meeting any of the following criteria will be excluded from the study: 
1. Planned travel during the study period (180 days) which would interfere with the ability 
to complete all study visits 
2. Recent (in the past 4 weeks) travel to any dengue endemic area. These potential 
volunteers may be eligible for enrollment a minimum of 4 weeks later 
3. Volunteer seropositive for hepatitis B surface antigen (HBsAg), hepatitis C virus 
antibodies (anti-HCV), or human immunodeficiency virus antibodies (anti-HIV)  
4. Unvaccinated volunteers positive for antibodies to flaviviruses (FV) to include dengue 
virus, West Nile virus, Yellow Fever virus, Zika virus, and Japanese encephalitis virus. 
5. Any history of FV infection or FV vaccination except for participation in the ADVP003 
or ADVP004 dengue vaccination studies; during the study period (Note: Late time point 
serology from the trials can be tested concomitant to screening serology to clarify if 
incident FV infection has occurred between vaccination and challenge)  
6. Medical history of, or current, diabetes, chronic obstructive pulmonary disease, peptic 
ulcer disease, coronary artery disease, cardiac arrhythmia, cardiomyopathy, pericarditis, 
or auto-immune disease 
7. Known or suspected congenital or acquired immunodeficiency; or receipt of 
immunosuppressive therapy such as anti-cancer chemotherapy or radiation therapy within 
the preceding 6 months; or long-term systemic corticosteroid therapy (prednisone or 
equivalent for more than 2 consecutive weeks within the past 3 months)  
8. History of Guillain-Barré syndrome (GBS) 
9. History of bipolar disorder, 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 volunteer from participating in the study 
10. Safety laboratory test results at screening that are deemed clinically significant or more 
than Grade 1 deviation from normal with the exception of PT/PTT, fibrinogen decrease, 
ALT/AST increase (<1.1 x ULN acceptable), platelet decrease which will be 
exclusionary at Grade 1 or higher 
11. Significant screening physical examination abnormalities at the discretion of the 
investigator, including a BMI > 35 kg/m2 

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12. Women who intend to become pregnant or men who intend to father a child during the 
study period (approximately 6 months) 
13. Female: pregnant, lactating or history of heavy menstrual bleeding menstrual periods 
lasting consistently and regularly longer than 6 days, or consistently and regularly 
requiring 5 or more pads or tampons per day, and volunteer to the opinion and review of 
the investigator. 
14. Female volunteers using an intrauterine device (IUD) or Mirena® 
15. Female volunteers with a history of clinically significant fibroids or uterine polyps, 
endometriosis, dysmenorrhea, adenomyosis, and uterine scarring (e.g. after D&C), unless 
treated, with no active clinically significant disease 
16. Allergy (hives, shortness of breath, swelling of the lips or throat), or hospitalization 
related to a previous vaccination, anaphylaxis of unknown etiology, or allergy to specific 
medications/animals for which antigens may be in the virus preparations to include: 
Shellfish allergy, Fetal Bovine Serum, L-Glutamine, Neomycin and Streptomycin 
17. Recent blood donation within prior 56 days of inoculation or planning to donate blood in 
the one 1 year following inoculation with dengue virus 
18. Receipt of blood products or antibodies within 90 days of inoculation or during the study 
period 
19. Any personal beliefs that bar the administration of blood products, transfusions, or serum 
albumin 
20. Participation in the 4 weeks preceding inoculation, or planned participation during the 
present trial period, in another clinical trial investigating a vaccine except for 
participation in the ADVP003 or ADVP004 study, drug, medical device, or medical 
procedure 
21. Planned administration of a licensed or study vaccine not planned in the study protocol 
during the period starting 30 days prior to the DHIM for a live vaccine or 14 days prior to 
DHIM for inactivated vaccines and extending until 56 days after study completion.  
*Note:  An exception will be made for volunteers who have the option to obtain a 
COVID-19 vaccine.  In this case, we will allow a vaccination 14 days prior or 21 days 
following DHIM. 
22. Planned or current administration of an HMG-CoA reductase inhibitor (i.e., lovastatin, 
simvastatin, atorvastatin, etc.) 
23. Currently taking methadone or suboxone 
24. Currently regularly taking anti-coagulant medication, aspirin or non-steroidal anti-
inflammatory drugs (NSAIDs) 
25. Chronic migraine headaches, defined as more than 15 headache days per month over a 3-
month period of which more than 8 are migraines, in the absence of medication over use 
26. Chronic or recent acute medical condition that, in the opinion of the investigator, impacts 
volunteer safety. 

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8.3. 
Temporary Exclusion Criteria 
Volunteers meeting any of the following criteria may be temporarily excluded from the study until 
the condition for exclusion resolves or is no longer applicable: 
1. Acute disease and/or fever (> 38°C/100.4°F oral body temperature) at the time or within 
6 hours of challenge inoculation:  note that a volunteer with a minor illness such as mild 
upper respiratory infection, etc., without fever, and with a negative SARS-CoV-2 upper 
respiratory (NP or nasal) swab PCR on the day of inoculation, may be enrolled at the 
discretion of the investigator 
2. Recent blood donation (within prior 56 days) 
3. Recent or scheduled receipt of any live vaccine 30 days and/or inactivated or sub-unit 
vaccine 14 days prior to inoculation 
4. Safety labs may be repeated once.  If outside the 56-day screening window, the volunteer 
may be rescreened with the exception of FV, hepatitis, and HIV viral screens. 
8.4. 
Volunteer Withdrawal Criteria 
Each volunteer may withdraw consent at any time during the study without penalty. Counseling 
about the volunteer’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 volunteer will be provided. 
The PI may discontinue the volunteer’s activity without the volunteer’s consent if any of these 
criteria is met: 
• 
A volunteer fails to comply with study procedures 
• 
A volunteer’s safety or health may be compromised by further participation 
 
8.4.1.   When and How to Withdraw Volunteers 
If a volunteer withdraws, the PI will make a reasonable effort to determine the reason for the 
withdrawal from the study and to complete termination procedures as described in Section 8.4.3. 
and Section 8.4.5. For volunteers leaving the study, a targeted examination may be performed, if 
medically indicated and if permitted by the volunteer. 
If a volunteer meets withdrawal conditions for a concomitant medication violation or 
noncompliance, this should clearly be stated in the source document and the study termination 
electronic CRF. 
8.4.2.  Data Collected for Withdrawn Volunteers 
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. 
8.4.3.  Lost to Follow-up Procedure 
In the case of volunteers who fail to return for a follow-up examination, documented reasonable 
effort (i.e. telephone calls and certified mail) should be undertaken to locate or recall them, or at 
least to determine their health status while fully respecting their rights. These efforts should be 
documented in the source documents. 

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8.4.4.  Replacement of Volunteers 
Volunteers will not be replaced during the study if they are unable to complete the study procedures 
required during the first 28 days of the study.  However, if the budget allows, an additional cohort 
of 5 individuals may be recruited from the previously vaccinated volunteer lists, under protocol 
ADVP 003.  Back-up, alternate volunteers will be on hand to replace unvaccinated controls on the 
day of DHIM-1 inoculation, in the event that circumstances prevent the original designee from 
participating (e.g., illness, personal circumstances, etc.) 
8.4.5.  Follow-up for Withdrawn Volunteers 
If a volunteer withdraws from the study, the study team will attempt to schedule at least one follow 
up visit before or on Day 56. If a volunteer drops out in the first 30 days before symptoms manifest, 
the risks as discussed during the consent process will be discussed with the volunteer. Volunteers 
will be asked to return for a close out visit at which time safety and immunologic samples will be 
drawn depending on the volunteer’s preference. For volunteers where the reason for early 
termination was lost to follow-up or if the volunteer withdrew informed consent and specified that 
they do not want to be contacted again and it is documented in the source document, the site will 
not attempt to obtain further safety information. No follow up for withdrawn volunteers will occur 
once the study termination eCRF is completed. 

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9. 
TREATMENT OF VOLUNTEERS 
The schedule of study procedures is provided in Table 3. More detailed descriptions of the study 
procedures are provided in the following sections. The procedures in Table 3 will be followed 
until a volunteer meets the criteria for sequestration or inpatient admission (Section 9.4). At that 
point, the procedures in Table 4 will be followed. Protocol deviations will not occur for visits that 
do not commence (i.e., if a volunteer is hospitalized and discharged on Day 4, there is no protocol 
deviation for assessments not performed on Day 5 of hospitalization). This will also be true for 
procedures not done on the non-hospitalized schedule while the volunteer is hospitalized. The 
blood volumes collected at each visit and for specific assessments for non-hospitalized and 
hospitalized volunteers are provided in Table 3 and Table 4, respectively. 

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9.1. 
Informed Consent Process 
A written informed consent document will be signed by the volunteer before any study-related 
procedures are initiated for that volunteer. 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 provided ample time to read the informed 
consent form and ask questions. Volunteers recruited from the previous ADVP003 or -004 trials 
will also be informed of the intention to share data acquired from participation in the prior trial 
including but not limited to contact information and immunologic data.  If a volunteer decides to 
participate, he/she will sign and date the informed consent form (ICF). A signed copy of the ICF 
will be provided to the volunteer before any study procedure is performed.  
Volunteers will be given a test on their understanding of the information from this informed 
consent and they must score at least 75% correct in order to continue with the screening process. 
In case they do not pass the test on the first try with a score of 75%, they will be given 2 additional 
opportunities to pass the test (3 total). All questions and answers will be reviewed for all candidate 
volunteers. 
9.2. 
Screening 
 
9.2.1.   Screening Visit 1 (Day -90 to -1) 
After signing and dating the ICF, previously vaccinated and unvaccinated volunteers will be 
sequentially assigned a unique volunteer identifier number starting with -001. The screening 
process is a two-step process occurring over a minimum of two visits. The procedures and 
assessments listed in Table 3, Day -90 and Day -28, respectively, will be carried out for each 
volunteer to determine their eligibility for participation in the study.  
• 
The informed consent process will be performed 
• 
Inclusion and exclusion criteria will be assessed 
• 
Demographic information will be collected 
• 
A complete medical history will be collected 
• 
Concomitant medications will be recorded 
• 
A complete physical examination will be performed including the collection of vital signs 
(Heart rate and blood pressure) and temperature  
• 
Urine will be collected for pregnancy test, females only 
• 
Blood will be drawn for screening serology tests (valid for 56 days prior to enrollment after 
which tests will be repeated)  
- 
Blood will be drawn for safety labs which consist of complete blood count and 
differential (WBC, hemoglobin, platelets, neutrophil and lymphocyte counts), liver 
function tests (AST/ALT, serum chemistry (Cr, glucose), prothrombin time/partial 
prothrombin time, albumin, and fibrinogen 

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- 
Screening serologies for antibodies to HIV-1/2, hepatitis C, and hepatitis B surface 
antigen. 
- 
Vaccinated volunteers from ADVP 004 will have screening serology tests to include 
neutralizing antibodies to dengue, Zika and West Nile Virus. Preference towards those 
with tetravalent neutralizing antibodies will be made but it is possible that these values 
will have waned by screening and will not serve to be exclusionary. 
 
9.2.2.   Screening Visit 2 
After the initial screening visit, available information will be reviewed to assess eligibility. If the 
unvaccinated volunteer is eligible to continue, a second screening visit will be completed 
(volunteers from the ADVP003 or-004 trial will have procedures completed in a single screening 
visit to minimize inconvenience and travel to the greater Baltimore-Washington area): 
• 
Inclusion and exclusion criteria will be re-assessed 
• 
Medical history will be updated 
• 
Concomitant medications will be reviewed and updated 
• 
Vital signs (Heart rate and blood pressure) and temperature will be performed 
• 
Unvaccinated control volunteers will have blood drawn for screening serology test to 
include neutralizing antibodies to dengue, West Nile, Yellow Fever, Japanese B 
encephalitis and Zika.  
• 
Safety labs that were exclusionary grade 1 in abnormality may be repeated if the 
investigator believes that there is a laboratory error or reasonable transient explanation for 
the abnormality (e.g., elevated AST after vigorous exercise), or is the result of a normal 
variant of a healthy state.   
• 
Blood will be drawn for research assays which consist of serum for microarray and 
ELISAs, PBMC for core cellular assays and scRNAseq and transcriptome analysis  
All screening related procedures and assessments should be completed no more than 90 days prior 
to inoculation. If volunteer is not inoculated within 56 days of screening, they will be re-screened 
before inoculation with the possible exception of FV, HIV and hepatitis serologies (if < 6 months). 
Laboratory evaluation for FVs will be considered evaluable for inclusion/exclusion for up to 12 
months as long as the volunteer has not traveled to a dengue endemic area in that time.  
If volunteers test positive for Zika antibodies, they will be referred to their healthcare provider for 
additional follow-up and retesting. Close observation will be made between pre-vaccination 
(ADVP003 or ADVP004) titers and pre-challenge titers to ascertain if this represents a new 
infection, antibody decay or potential cross-reactions with the previous dengue vaccination. 
Although most antibody positive cases are asymptomatic, there is a chance for sexual transmission. 
We will counsel the volunteer to abstain from sexual activity, reduce the exposure to mosquito 
bites and see their doctor immediately. 
When a volunteer has completed the screening visits and is eligible and willing to continue 
participation, he/she will be scheduled for inoculation (Day 0). Information on volunteers that fail 
the screening visit(s) will not be recorded in the data management system.  

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9.3. 
Inoculation and Follow-up Visits 
Volunteers will follow the procedures listed in Table 3.  This schedule will be followed until the 
volunteer meets the criteria for sequestration or inpatient admission (Section 9.4.).  Upon 
admission to the hospital, the procedures for hospitalized volunteers (Table 4) will be followed. 
9.3.1.  Day 0 Inoculation 
To confirm a volunteer is still eligible the following procedures will be completed prior to 
inoculation: 
• 
Inclusion and exclusion criteria will be reviewed 
• 
Medical history will be reviewed 
• 
A targeted physical examination, including vital signs (HR and BP) and temperature, will be 
conducted  
• 
Concomitant medications will be reviewed  
• 
Screening safety lab results will be re-reviewed  
• 
In the event of ongoing local transmission of the COVID-19 virus, an upper respiratory (e.g. 
NP or nasal) swab sample will be collected for same day testing for the SARS-CoV-2 virus. 
Volunteers with positive results will be referred to their personal physician or local public 
health department for definitive testing and examination. Results will not be available prior to 
the challenge inoculation. A positive test in an asymptomatic individual would result in 
quarantining but will not result in study removal following challenge if DHIM had occurred. 
• 
Urine will be collected; pregnancy test (females only) will be performed, and results will be 
reviewed  
• 
Blood will be drawn for complete blood count and differential (WBC, hemoglobin, platelets, 
neutrophil and lymphocyte counts), liver function tests (AST/ALT, serum Cr, prothrombin 
time/partial prothrombin time, albumin, and fibrinogen (henceforth termed ‘safety labs’). 
• 
Blood will be drawn for a research assay which consists of single cell RNA sequencing 
(scRNAseq). 
• 
If the volunteer is still eligible, DENV-1-LVHC will be administered as a single SC 
inoculation in the triceps area of the arm.  
• 
Post-injection observation period/reactogenicity assessment (Volunteers will be kept under 
observation for at least 30 minutes after each inoculation to ensure their safety, and any 
reactions during this period will be documented. Appropriate medical equipment and 
emergency medications, including epinephrine [1:1000], will be available on site in the event 
of an anaphylactic or other immediate allergic reaction.) 
- 
Give volunteer thermometer and explain how to use (Day 0 only) 
- 
Distribute 28-day memory card and instructions for use 
- 
Repeat examination of injection site 
- 
Do vital signs prior to discharge from clinic 

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- 
Evaluate volunteer for occurrence of any immediate local and systemic 
reactogenicity and record AE(s) on appropriate form prior to discharge 
- 
Volunteers will also be provided with a wallet card to carry with them during the 
study that provides 24-hour contact information for the study team with language to 
carry with them in the future to alert potential treating physician of their involvement 
in this study, should they become ill while traveling in or after travelling to dengue 
endemic areas. 
The thermometers will be checked prior to distribution to ensure they are functional. Volunteers 
will be reminded before they leave to complete the memory card as instructed and to bring the 
completed memory card to all clinic visits. Volunteers will also be told to notify the site of any 
SAE.  
ADVP 004/ADVP003 vaccinated volunteers may reside as far as Frederick County and will be 
offered the opportunity to remain in Baltimore (e.g. The Lord Baltimore or Hotel Monaco) during 
the 28-day post-inoculation follow-up phase. 
9.3.2.  Day 2 (+/- 1) 
The following procedures will be performed at the Day 2 visit: 
• 
Medical history will be reviewed. 
• 
A symptom-driven physical examination will be performed, vital signs will be obtained, and 
temperature will be measured 
• 
Concomitant medications will be reviewed 
• 
The injection site will be evaluated 
• 
Solicited and unsolicited AEs and SAEs will be assessed 
• 
Memory cards will be reviewed 
• 
Blood will be drawn for research transcriptome labs 
• 
The volunteer will be reminded to notify the site in the event of an SAE 
 
9.3.3.  Days 4 to 16 Daily Follow-up 
Beginning on Day 4, volunteers will be followed daily to evaluate for symptoms and viremia. 
Optimization studies for DHIM-1 revealed that 0/6 volunteers developed viremia before Day 6 
(however Day 5 was not tested) and symptoms were minor. 
• 
A symptom-driven physical examination will be performed, vital signs will be obtained, and 
the volunteer’s temperature will be measured.  Note:  If local pandemic conditions continue, 
a repeat upper respiratory (e.g. NP or nasal swab) for SARS-CoV-2 detection via PCR will 
be performed on the day of the first temperature (defined as > 100.4F).  
• 
Concomitant medication will be reviewed 
• 
The injection site will be evaluated through Day 7  
• 
Solicited and unsolicited AEs and SAEs will be assessed  

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• 
Memory cards will be reviewed  
• 
Blood will be drawn for RT-qPCR 
• 
Blood will be drawn for research labs on D(ays) 4, 8, 10, 14, and 16  
• 
Blood will be drawn for safety labs on D 4, 6, 8, 10, 12, 14, and 16 
 
9.3.4.  Days 19, 22, and 25(+/- 1), and 28 (+/- 2) Post-Daily Follow-up 
Following the period of daily follow-up when viremia is expected to occur (note: 0/6 volunteers 
in the DHIM-1 optimization experienced viremia after Day 16), volunteers who are viremia 
negative will complete a visit every 3 days until Day 28.  The following procedures will be 
performed at each visit: 
• 
A symptom-driven physical examination will be performed, vital signs will be obtained, and 
the volunteer’s temperature will be measured  
• 
Concomitant medication will be reviewed 
• 
Solicited and unsolicited AEs and SAEs will be assessed  
• 
Memory cards will be reviewed  
• 
Blood will be drawn for research labs which consists of scRNAseq and transcriptome analysis 
• 
Blood will be drawn for safety labs (Day 28 only) 
• 
Blood will be drawn for RT-qPCR (for volunteers who remain aviremic or with ongoing 
viremia).  If viremia continues past D28, volunteers will be followed until aviremic. 
• 
The volunteer will be reminded to notify the site in the event of an SAE 
• 
Memory card collected (Day 28 only) 
 
9.3.5.  Days 56 (± 3 days), 90 and 180 (± 7 days) Safety Follow-ups 
The following procedures will be performed: 
• 
A symptom-driven physical examination will be performed, vital signs will be obtained, and 
temperature will be measured 
• 
Concomitant medication will be reviewed 
• 
AEs and SAEs will be assessed 
• 
The volunteer will be reminded to notify the site in case of an SAE 
• 
Blood will be drawn for research assays on Days 90 and 180 
• 
AEs and SAEs will be assessed 

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9.4. 
Inpatient Admission 
9.4.1. 
Criteria for Admission to Inpatient Service 
Due to the possibility of signs and symptoms associated with dengue fever, and the presence of 
mosquitoes theoretically competent for dengue virus, volunteers will be admitted to an inpatient 
service for observation if defined criteria are met. Viremia is expected, on average, to occur 
between days 5-16 after challenge (D0). Aedes aegypti and Aedes albopictus, mosquito dengue 
vectors found in the United States, are present in the mid-Atlantic region of Maryland, and have 
the potential to transmit Zika, dengue, chikungunya, and other viruses. Infected mosquitoes are 
not present in this area, but their presence requires vigilance by sequestration to ensure that viremic 
volunteers are not able to infect local mosquito vectors. The CDC website notes 12 imported cases, 
and no locally transmitted cases of dengue were detected in the state of Maryland and reported 
through ArboNET in 2019 (as of October 2, 2019).36 Volunteers will be sequestered within a local 
hotel (e.g. the Lord Baltimore or Hotel Monaco) if asymptomatic save for viremia or admitted to 
the inpatient facility (Pharmaron or the GCRC) for closer observation if any of the following 
additional criteria are met (or arise during the sequestration phase). If volunteers remain viremic 
for periods beyond Day 16, they will remain sequestered or an inpatient until documented 
clearance. If a DHIM occurs in late autumn/winter months in which there are no circulating Aedes 
spp., viremia will not result in sequestration (sustained temperatures above 59° F required for 
Aedes albopictus).43 This would conservatively range from November to April.  Note:  If local 
pandemic conditions continue, volunteers will be encouraged to sequester at the local hotel from 
the time of challenge onwards. If local, they will be asked to self-quarantine at home and apply 
social distancing standards to minimize any inadvertent exposure to the SARS-CoV-2 virus.  
Temperatures and vital signs will be monitored daily and an upper respiratory (e.g. NP or nasal) 
swab obtained for SARS-CoV-2 PCR at the first temperature (>100.4F) to distinguish the fever 
from that related to COVID-19. 
Sequester Criteria: 
− 
Viremia (D+24h) as detected by RT-qPCR during the months of May through October 
 
Inpatient Criteria:   
− 
Viremia (D+24h) as detected by RT-qPCR and two or more of the following symptoms: 
Symptoms/lab parameters: 
− 
Fever (> 100.4°F) at 2 time points at least 4 hours apart in the absence of antipyretic 
medication or any two or more of the following (per FDA Guidance for Industry) Note:  
If local pandemic conditions continue, a repeat upper respiratory (e.g. NP or nasal) swab 
for SARS-CoV-2 detection via PCR will be performed on the day of the first temperature 
(defined as > 100.4F). 
− 
Headache ≥ grade 2 
− 
Eye pain ≥ grade 2 
− 
Bone pain > grade 2 

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− 
Joint pain > grade 2 
− 
Abdominal pain > grade 2 
− 
Muscle pain > grade 2 
− 
Nausea and/or Vomiting > grade 2 
− 
Liver function tests (ALT, AST) > grade 3 
− 
Thrombocytopenia ≥ grade 3 
− 
Weakness/malaise ≥ grade 2 
− 
Any signs or symptoms determined by the PI to warrant hospital admission  
The volunteers will remain sequestered/inpatient for observation until the criteria for their 
admission (i.e., viremia and/or > grade 2 symptoms) resolve and meet the discharge criteria 
(Section 9.4.3.).  
Volunteers will not be admitted if, in the opinion of the PI, an unrelated medical condition is 
responsible for the volunteer meeting hospitalization criteria. If required, the volunteer will be 
referred to their primary care physician for evaluation. Of special concern is the underlying risk of 
exposure to SARS-CoV-2.  Close attention will be paid to symptoms consistent with COVID-19 
illness to include upper respiratory symptoms (new or increased nasal discharge, sore throat, 
shortness of breath, wheezing, or the onset of loss of taste or smell).  If necessary, additional testing 
or referrals for nasopharyngeal swabs for COVID-19 PCR may be done. 
9.4.2. 
Procedures During Inpatient Admission 
Once a volunteer is admitted to the hospital, the Dengue Human Infection Model Clinical 
Observation and Inpatient Management Phase Algorithm, will be followed. The procedures are 
listed in Table 4 and summarized below. 
During inpatient admission and sequestration, the following procedures will be performed daily: 
A symptom-driven physical exam, AE/SAE assessment, concomitant medication review, and 
Memory card review if applicable. The presence of viremia (RT-qPCR) will be checked daily from 
Days 4-16 (or extended until clearance if viremia continues past Day 16). Research sample 
collection will be performed according to Tables 3 and 4. Safety labs will be performed every 
other day (Table 4) and consist of complete blood count, liver function tests, Cr, prothrombin 
time/partial prothrombin time, albumin and fibrinogen. In addition to the previously described 
procedures, the following daily additional procedures will be followed for the inpatient phase of 
the study. The volunteer’s temperature and other vital signs will be measured at least 3 times per 
day (q 8 hours +/- 1 hour), or more if needed for volunteer care, and fluid intake and output 
measurement will be recorded on the volunteer’s medical record. The standard of care is at the 
discretion of the PI and may include medication for management of pain, antipyretics 
(acetaminophen), the management of fluid loss through oral or IV hydration, monitoring and 
periodic clinical assessment, and judicious fluid replacement. 

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Although past clinical trials with the DENV-1, -3, or -4 strains have not produced any cases with 
severe symptoms, dengue with warning signs or severe symptoms may occur. Experienced 
clinicians will be expected to treat patients in accordance with the protocol and using the CDC and 
WHO guidelines as reference.  
9.4.3. 
Criteria for Discharge/Release from Sequestration or Inpatient Admission 
The actual discharge day will vary by volunteer. For volunteers to be discharged from observation, 
the following criteria must be met:  
Sequestration: 
• 
Two negative qualitative RT-PCR tests for dengue run on samples obtained via 2 different 
blood samples obtained at least 8 hours apart 
Inpatient admission: 
• 
Amelioration of symptoms to < grade 2 admission criterion* and: 
• 
No fever (< 100.4°F) 
• 
Laboratory parameters resolving and are  admission grade criteria, at clinician discretion  
• 
Clinical symptoms are resolving and are  grade 2, at clinician discretion 
*Note:  Will continue daily follow-up until two negative qualitative RT-PCR tests for dengue 
obtained on 2 difference blood samples at least 8 hours apart, as per protocol. 
All volunteers discharged from sequestration or inpatient admission will be contacted via 
telephone the day after discharge and will return for follow up 3 days post discharge and 7 days 
post discharge. Volunteers will be seen at 28 days post inoculation, only if discharge occurs prior 
to Day 28. All volunteers will be seen on Days 56, 90, and 180 (2, 3, and 6 months post inoculation) 
according to Table 3 and Table 4.  
9.5. 
Biological Samples 
9.5.1.  
Biological Sample Handling    
Samples will be coded with information including, but not limited to, volunteer study number, 
study time-point, sample type and date/time. Samples will not be labeled with information that 
directly identifies the volunteer. WRAIR investigators with access to study data from 
ADVP003 and ADVP004 may have access to subject identifiers from these studies as 
information will be shared to facilitate research. However, laboratory investigators will not 
have access to this level of information. Samples will be held in storage until exhausted 
through use in research assays or until directed to be destroyed by overseeing IRB or other 
oversight authority (e.g. FDA, USAMRDC etc.). Samples sent to WRAIR will follow these 
same guidelines and will be dispositioned and tracked by the WRAIR VDB quality 
management unit and held in WRAIR VDB cold storage facilities. 

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9.5.1.  
Future Use of Human Samples 
Samples collected under this protocol will be used for protocol mandated and future research, 
research related to the development, maintenance, quality assurance, and improvement of the 
laboratory tests described in this protocol, the development of new test methods, and making sure 
that new tests work reliably and are comparable to previous methods.  
It may be desirable to use the samples acquired in this study for future research, not described in 
this protocol. Therefore, all volunteers will be asked to give a specific consent to allow the 
sponsor’s representative to use the samples for future research.  
Volunteers not authorizing future use of samples will be tracked, and their samples will be 
destroyed upon completion of the study (note: samples intended to be run as part of the study 
endpoints and/or exploratory analyses will continue until completion). Samples stored for future 
use will be stored indefinitely, unless local rules, regulations or guidelines require different 
timeframes or different procedures.  
A volunteer may decide at any point during the study to withdraw consent for the future use of 
his/her samples. Should a volunteer withdraw consent for the future use of his/her samples, 
remaining samples will be destroyed after the conclusion of testing for this clinical study. Testing 
sites will provide verification of destruction in writing to the clinical site. 
9.6. Concomitant Medications 
Volunteers are not being asked to discontinue current medications. In the event that medical 
conditions not related to the study arise after inoculation and dictate use of medications, volunteers 
are encouraged to obtain appropriate care, comply with the course of therapy as prescribed by their 
physician, and inform the investigators as soon as practicable. Details of all medications taken 
during the course of this study must be recorded on the volunteer’s record. Some medications will 
be exclusionary (e.g. HMG-CoA reductase inhibitors which have been shown to ameliorate dengue 
viremia).  
Volunteers should not use any aspirin or nonsteroidal anti-inflammatory drugs (NSAIDs), 
including Advil, Motrin, Nuprin, Aleve, Naproxen, and Celebrex, during the 28 days after 
inoculation or through 7 days post hospital discharge, whichever is later. Aspirin and NSAIDs can 
interfere with the ability of the blood to clot. It is not anticipated that any volunteers will have 
severe dengue infection with bleeding problems; however, it is still recommended to avoid these 
drugs. The investigators may recommend Tylenol or Paracetamol, which can be used safely for 
fevers and body aches. 
9.7. 
Research Assessments   
A summary of research specimen assays is summarized below in Table 5: 
 

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Table 5: 
Assessment Summary 
Assessment 
Method 
Serum antibodies 
ELISA 
Antibody quantification 
MN50 (microneutralization)  
Viremia (RNAemia) 
Quantitative RT-PCR  
Viremia (RNAemia)for discharge 
Qualitative RT-PCR 
PBMC cellular immune response 
ELISPOT/Flow cytometry 
PBMC (fresh if logistically feasible) 
Single Cell RNA sequencing 
Transcriptomics 
PAXgene (TBD) 
9.8. 
Serum Antibodies 
Blood samples will be obtained for the measurement of antibodies against the four dengue 
serotypes, DENV-1-4 and quantification of neutralizing antibodies via the MN50 
microneutralization assay to be performed at WRAIR. Anti-dengue IgG, IgA and IgM may be 
measured using research lab (WRAIR) or commercial ELISA kits. Other functional assays, such 
as in vitro antibody-dependent enhancement (ADE) assays or antibody depletion assays may also 
be used to characterize serum antibodies. Screening flavivirus neutralization assays will be 
performed by the WRAIR Pilot Bioproduction facility using their microneutralization assay. 
 
9.9. 
Polymerase Chain Reaction (PCR) Detection of Viremia 
Blood samples will be obtained for measurement of levels of viremia using qualitative and/or 
quantitative (qPCR).  Samples will be analyzed in real time (RT) in house using nucleic acid-
based methods for the detection and quantification of dengue virus. WRAIR laboratories may be 
involved in the performance of this testing if needed. 
 
9.10.  Immune Responses 
Cryopreserved peripheral blood mononuclear cells (PBMCs) will be tested for innate, T and B cell 
profiling and/or cellular immune responses, antigen-specific cytokine responses, chemokines and 
cell surface markers in order to examine the acquisition of T and B cell memory responses 
following challenge. Conventional flow cytometry (CFCM) and CyTOF® and Helios® mass 
cytometers may be utilized for novel examinations of the memory response. Panels of 30-38 
parameters are designed to identify innate mechanisms of immune response, effector/memory and 
peripheral T follicular helper cell subsets, pro-inflammatory cytokine/chemokine production, T 
cell homing patterns, B cell profiling, and the activation status of various T cell subsets. The use 
of a human challenge model will allow for investigation of predominant anti-DENV immune 
responses and the kinetics to examine early responses, and identify immune signatures, which 
differ in susceptible and resistant volunteers that may elucidate important correlates of protection. 
If funds allow, studies to investigate antigen-specific T cell functional responses against various 
DENV-derived antigens (e.g., whole cell DENV homogenate, overlapping peptide pools, etc.) will 
be performed. WRAIR laboratories will be involved in the performance of some of this testing. 

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9.11. Transcriptomics  
A global approach to transcriptional analyses will be performed for gene expression analysis 
during primary infection using the HTA 2.0 array and/or total ribonucleic acid sequencing (RNA-
Seq). This approach allows for unbiased analysis of genes regulated in response to dengue 
infection and has the potential to identify key pathways modulated. Initial analysis will focus on 
activation of innate immune pathways, as this type of analysis has not been possible in the natural 
infection setting. Given that the innate immune system is key to generating an effective acquired 
immune response, identification of key innate pathways regulated during infection may provide 
rational targets for enhancing a protective immune response. Subsequent confirmation of select 
targets will be done by quantitative PCR to confirm expression. Blood will be collected directly 
into PAXgene® sample preparation tubes optimized for stabilizing RNA for long-term storage 
prior to transcriptional profiling. Genetic testing may be performed on these samples only after 
specific informed consent is acquired for such testing and IRB approval is obtained. Additionally, 
minor variant sequence analysis may be performed utilizing RNA. WRAIR laboratories will be 
involved in the performance of some of this testing. 
9.12. Single cell RNA Sequencing (scRNAseq) 
A global approach to characterizing cell populations will be pursued using the 10X Genomics 
Single Cell Immune Profiling technique which allows for a kinetic examination of the adaptive 
immune response in a subset of lymphocytes collected over the course of the DHIM.  Barcoding 
technology allows for enhanced immune cell phenotyping to study the dynamics of adaptive 
immunity in vaccinated vs. unvaccinated controls. WRAIR laboratories will be involved in the 
performance of some of this testing. 
10.0.  
SAFETY ASSESSMENT 
10.1. Consortium Data Safety Committee 
The core Consortium Data Safety Committee (CDSC) includes, at a minimum, each site Principal 
Investigator (or designate), the protocol Independent Safety Monitor/Research Monitor 
(ISM/RM), and the IND sponsor Medical Officer.  Participants in the CDSC reviews may also 
include site Assistant/Associate Investigators, or designated team representatives including but not 
limited to study coordinators, study clinicians, or protocol specialists. The CDSC performs the 
safety reviews for the DHIM’s conducted as part of the Dengue Consortium operations and will 
perform safety reviews and review the summary of study safety data reports on a weekly basis 
through 4 weeks after the last volunteer receives the last study injection in order to ensure that the 
DHIM has an acceptable safety profile. Otherwise, if there have been no DHIM, in the prior 4 
weeks, the CDSC will monitor safety data on a monthly basis through completion of the last study 
visit.  At each site, an ISM/RM (advanced practitioner or research physician) will participate in 
study safety meetings and conduct a safety review of all UPIRTSOs and SAEs as well as submit 
a short summary of the event. The CDSC will evaluate and respond to safety concerns in a timely 
manner. 
10.2.    Clinical Laboratory Assessment 
Safety laboratory assays will be performed at the study site-designated Clinical Laboratory 
Improvement Amendments (CLIA)-certified laboratory. All safety-related clinical laboratory 

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values will be reviewed, and all abnormal values will be assessed by the investigators as clinically 
significant or not, with respect to safety. See Table 6 for further details. 
Urinalysis testing for pregnancy (urine β-hCG) will be assessed at screening, on the day of 
challenge and at Day 28 following challenge. Volunteers with a positive pregnancy test result 
will be discontinued from the study treatment but will be followed as explained in Section 
10.9.1. 
The laboratory safety assessments at specified study time points are summarized in Tables 3 and 
6. 
Table 6: 
Clinical Laboratory Evaluations 
Volunteers 
Assessments 
Laboratory  
UMB 
WBC count 
Hemoglobin (hematocrit will be reviewed) 
Platelet count  
Total neutrophils and lymphocytes within 
manual differential 
Biochemistry [creatinine, (glucose for screen 
only)]  
Liver Panel (AST, ALT) 
Fibrinogen, Albumin, PT/PTT 
Garcia Clinical Laboratories 
2195 Spring Arbor Road 
Jackson, MI 49203-2797 
 
and/or 
 
University of Maryland Medical Systems 
Laboratory of Pathology 
22 South Greene Room, N2W49 
Baltimore, Maryland 21201  
 
10.3. IND Safety Reporting Definitions  
The following terms, as defined by 21 CFR 312.32, apply to IND safety reporting. 
 
10.3.1. 
Adverse Event/Suspected Adverse Reaction/Unexpected Adverse Reaction 
Adverse event means any untoward medical occurrence associated with the use of a drug in 
humans, whether or not considered drug related. 
Suspected adverse reaction means any adverse event for which there is a reasonable possibility 
that the drug caused the adverse event. For the purposes of IND safety reporting, “reasonable 
possibility” means there is evidence to suggest a causal relationship between the drug 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 drug. 
An adverse event or suspected adverse reaction is considered “unexpected” if it is not listed in the 
investigator brochure or is not listed at the specificity or severity that has been observed. 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 drugs or as anticipated from 

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the pharmacological properties of the drug but are not specifically mentioned as occurring with 
the particular drug under investigation. 
See Section 10.6 for Adverse Event Reporting 
10.3.2.  
Solicited Adverse Event 
A solicited AE is a predetermined event, identified in the investigator’s brochure (IB), which may 
reflect safety concerns related to the investigational product. The solicited AEs for this study 
include: 
• 
Fever ≥ 38°C (100.4°F) 
• 
Rash 
• 
Headache 
• 
Retro-orbital (eye) pain 
• 
Muscle pain (Myalgia) 
• 
Arthralgia (Joint) pain and/or bone pain 
• 
Fatigue and/or malaise (weakness) 
• 
Pain at injection site 
• 
Swelling at injection site 
• 
Erythema at injection site 
• 
GI Symptoms (Abdominal pain, nausea, vomiting) 
Solicited AEs will be captured during all clinical visits. 
Refer to Section 10.9.1 for adverse event reporting. 
10.3.3. 
Serious Adverse Event/Serious Suspected Adverse Reaction 
An adverse event or suspected adverse reaction is considered “serious” if, in the view of either the 
PI or Sponsor’s Representative, it results in any of the following outcomes:  
• 
Death 
• 
Life-threatening adverse event 
• 
Unanticipated hospitalization or prolongation of existing hospitalization 
• 
Persistent or significant incapacity or substantial disruption of the ability to conduct normal 
life functions 
• 
Congenital anomaly/birth defect  
An adverse event or suspected adverse reaction is considered “life-threatening” if, in the view of 
either the PI or Sponsor’s Representative, its occurrence places the patient or volunteer 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. 

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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 or volunteer and may require medical or surgical intervention to prevent one of the 
outcomes listed in this definition. Examples of such medical events include allergic bronchospasm 
requiring intensive treatment in an emergency room or at home, blood dyscrasias or convulsions 
that do not result in inpatient hospitalization, or the development of drug dependency or drug 
abuse. 
For the purposes of this study, planned inpatient admission for dengue symptoms without serious 
deterioration in health, will not be considered an SAE as it is an expected outcome. Inpatient 
admission (defined as admission resulting in an overnight stay of > 24h) not related to dengue 
symptoms will be considered an SAE.  
See Section 10.9.2 for SAE reporting. 
10.3.4. 
Unanticipated Problems Involving Risks To Volunteers Or Others 
(UPIRTSOs) 
Federal regulations require that unanticipated problems involving risks to volunteers or others be 
promptly reported to the IRB. These events encompass a broader category of events than SAEs 
and may include issues such as problems with loss of control of volunteer data or the 
investigational product, adverse psychological reactions, or breach of confidentiality. Risks to 
others (e.g., program personnel) must also be reported. 
Unanticipated problems involving risks to volunteers or others (UPIRTSOs) 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 volunteer population; 
• 
Related or possibly related to a volunteer’s participation in the study; and 
• 
Suggests that the study places volunteers or others at a greater risk of harm than was 
previously known or recognized. 
The IRB will evaluate the PI’s and safety monitor’s reports to determine whether a given incident, 
experience or outcome constitutes an unanticipated problem involving risk to volunteers or others 
and, in coordination with the Sponsor’s Representative, ensure upward reporting of the 
unanticipated problems involving risk to volunteers or others to the appropriate regulatory offices. 
See Section 10.9.2. for UPIRTSO reporting. 
10.3.5. 
Pregnancy 
Volunteers who become pregnant after Day 0 will be followed to term, and the following 
information will be gathered for outcome, date of delivery, health status of the mother and child 
including the child’s gender, height/length, and weight. Complications and or abnormalities should 

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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.  
See Section 10.10.1. for pregnancy reporting. 
10.4. 
Relationship to Investigational Product 
The PI 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 including underlying 
disease, concurrent illness or concomitant medications. The guidelines shown in Table 7 should 
be used by investigators to assess the relationship of an AE to study product administration. Only 
a physician can make this determination. 
Table 7: 
Adverse Event Relationship to Investigational Product Categories 
Category 
Description 
Not related 
No relationship to investigational product. Applies to those events for which evidence 
exists that there is an alternate etiology. 
Unlikely 
Likely unrelated to the investigational product. Likely to be related to factors other than 
investigational product but cannot be ruled out with certainty.  
Possible 
An association between the event and the administration of investigational product 
cannot be ruled out. There is a reasonable temporal association, but there may also be an 
alternative etiology such as the volunteer’s clinical status or underlying factors including 
other therapy.  
Probable 
There is a high degree of certainty that a relationship to the investigational product exists. 
There is a reasonable temporal association, and the event cannot be explained by known 
characteristics of the volunteer’s clinical state or factors including other therapy. 
Definite 
An association exists between the receipt of investigational product and the event. An 
association to other factors has been ruled out 
To align consortium protocols, AE relationship uses 5 categories of relatedness (defined above), 
which are to be used for this clinical trial. The category of “Not Related” maps to the dual 
category of “Not Related,” while the categories of “Unlikely,” “Possible,” “Probable,” and 
“Definite” map to the dual category of “Related.” 
10.5. 
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. Each event will be assigned 1 of the following categories: mild, moderate, 
severe, potentially life-threatening, or fatal using the criteria in the FDA’s Guidance for Industry: 
Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventative 
Vaccine Trials (2014) summarized in Appendix A. Any AE not included in the scale will be graded 
according to Table 8. For laboratory results, any event identified as abnormal according to the 

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local laboratory normal ranges will also be graded according to the FDA’s Toxicity Grading Scale 
(Tables 12 and 13) in Appendix A.  
FDA guidelines for toxicity will be followed; however, if a volunteer is evaluated in an emergency 
room for nonlife threatening illness or symptoms (i.e., 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 volunteer’s clinical 
signs and symptoms. 
As defined by the ICH guideline for 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 volunteer/event outcome or action criteria usually 
associated with events that pose a threat to a volunteer’s life or functioning. Seriousness (not 
severity) serves as a guide for defining regulatory reporting obligations.  
Table 8: 
Adverse Event Severity Categories  
Category 
Grade 
Description 
Mild 
1 
Does not interfere with routine activities 
Minimal level of discomfort  
Moderate 
2 
Interferes with routine activities 
Moderate level of discomfort  
Severe 
3 
Unable to perform routine activities 
Significant level of discomfort  
An AE that is assessed as Grade 3 (severe) should not be confused with a SAE. Grade 3 is a 
category utilized for rating the intensity of an event; and both AEs and SAEs can be assessed as 
Grade 3.  
10.6. 
Recording Adverse Events 
The PI will report all AEs to the UMB CVD IRB in the appropriate safety, annual, and/or final 
reports. The study site will provide data files for preparation of annual and final reports to the 
FDA. 
10.6.1. 
Methods/Timing for Assessing, Recording, and Analyzing Safety Endpoints 
AEs and SAEs will be assessed at all study visits, documented in the source records, and recorded 
on the eCRF’s using accepted medical terms and/or the diagnoses that accurately characterize the 
event. When a diagnosis is known, the AE term recorded on the eCRF will be the diagnosis rather 
than a constellation of symptoms. The PI will assess all AEs for seriousness, relationship to 
investigational product, severity, and other possible etiologies. When an event has not resolved by 
the end of the volunteer’s participation (6 months), it will be documented on the AE eCRF as “not 
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The timeframe for the collection of AEs begins at time of inoculation (Day 0) through end of 
volunteer’s participation (6 months). SAEs will also be collected from Day 0 through end of 
volunteer’s study participation (6 months).  
The timeframe for the collection of solicited AEs begins at the administration of investigational 
product through Day 28 (Local injection site solicited AEs will be collected through Day 7). All 
solicited AEs collected in the first 28 days will be considered ‘definitely related’ to the 
investigational product unless they can be attributed to a known cause unrelated to the 
investigational agent. 
10.6.2. 
Duration of Follow-Up of Volunteers after Serious 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 
representative using the Serious Adverse Event Report Form. 
Investigators are not obligated to actively seek SAEs in former volunteers; 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 
representative’s safety office as defined in Section 9. 
10.7. 
Study Halting Criteria 
The PI, study site safety office, or the sponsor’s representative will place the study on hold for any 
of the following criteria: 
Further enrollment and study inoculation will be halted for safety review if any of the following 
are reported: 
1. Any volunteer experiences a study product-related SAE from the time of the study 
product administration through the volunteer’s last study visit. 
2. Any volunteer experiences laryngospasm, bronchospasm, generalized urticaria or 
anaphylaxis within 1 day after administration of study product that is considered 
related to study product. 
This trial will also be halted for safety review if any of the following occurs: 
1. Two or more volunteers experience a Grade 3 unsolicited AE in the same 
MedDRA system organ class after administration of challenge inoculation that is 
considered anything other than not related to study product and not resolved or 
improved to lower grade within 2 days. 
2. Two or more volunteers experience a Grade 3 solicited local adverse event that is 
considered anything other than not related to study product and not resolved or 
improved to lower grade within 2 days. 
3. One or more volunteers experience a Grade 3 solicited systemic adverse event 
that is considered anything other than not related to study product and not 
resolved or improved to lower grade within 2 days. 

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4. Two or more volunteers experience a clinically significant Grade 3 laboratory 
adverse event (excluding leukopenia that would be expected with a viral 
infection) that is considered anything other than not related to study product. 
In addition, the PI, sponsor’s representative, UMB IRB or the FDA may place this study on hold 
at any time. 
Grading scales for solicited local (application site) and systemic AEs are included in Appendix 
A. 
Grading scales for clinical safety laboratory adverse events are included in Appendix A. 
10.8. Termination Rules 
If the AE/SAE profile is not acceptable to the PI, sponsor’s safety office, sponsor’s representative, 
Consortium Data Safety Committee (CDSA) or FDA, the trial may be terminated.  
If the trial is terminated, a notification (via memo) indicating the reasons for suspending the study 
will be provided by the sponsor’s representative to the PI for submission to the IRB. 
10.9. 
Reporting Requirements 
10.9.1. 
Reporting Adverse Events 
Adverse Events (including Solicited Adverse Events and Unsolicited Adverse Events) will be 
reported in the volunteer source document and in the eCRF. 
The PI will report all AEs to the sponsor’s representative’s safety office and the UMB IRB in the 
appropriate safety, annual, and/or final reports. 
10.9.2. 
Reporting Serious Adverse Events/Unanticipated Problems Involving Risk to 
Volunteers or Others 
Initial Report: All SAEs, and UPIRTSOs, whether or not the event is considered related to the 
study product, must be reported promptly (within 24 hours of site awareness of the event) to the 
sponsor’s representative’s office and the sponsor’s safety office (Table 9).  
Follow-up report: The site will provide a follow up SAE report at any point additional information 
related to the event is available to the sponsor’s safety office and the safety monitor. 
Contact information for reporting SAEs/UPIRTSOs are provided in the table below.  

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Table 9: 
Study Contacts for Reporting Serious Adverse Events, Unanticipated Problems Involving 
Risk to Volunteers or Others 
Sponsor’s Safety Office 
 
 
 
Research Monitor/Independent Safety 
Monitor 
Wilbur Chen 
Center for Vaccine Development and Global Health 
University of Maryland School of Medicine 
685 W. Baltimore Street, Room 480 
Baltimore, MD 21201 
Telephone: 410-706-1188 
Fax: 410-706-6205 
E-mail: WChen@som.umaryland.edu 
 
and/or 
 
Justin Ortiz (Back-up) 
Center for Vaccine Development and Global Health 
University of Maryland School of Medicine 
685 W. Baltimore Street, Room 480 
Baltimore, MD 21201 
Telephone: 410-706-3502 
Fax: 410-706-6205 
Email:  JOrtiz@som.umaryland.edu 
 
Institutional Review Board 
Human Research Protections Office 
University of Maryland, Baltimore 
800 W. Baltimore Street, Suite 100 
Telephone: 410-706-5037 
Fax: 410-706-4189 
E-mail: HRPO@som.umaryland.edu 
 
 
 
All notification will be provided to the sponsor’s safety office. Further, the investigator should 
comply with relevant study site SOPs on reporting SAEs. The minimum information that the 
investigator will provide to the sponsor’s safety office is specified in Table 10. The sponsor’s 
representative may request additional information for purposes of the study.  
 
 
 
 
 
 

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Table 10:  SAE Information to be reported to the Sponsor’s Safety Office 
Notification Method 
Information to Be Provided 
E-mail or Telephone 
(within 24 hours of 
site awareness) 
IND number, sponsor study number, name of the investigational product, and 
investigator name and contact number 
 
Volunteer identification number 
 
SAE, onset date, date of investigational product administration, severity, relationship, 
and volunteer’s current status 
AND 
 
 
E-mail or Fax 
Cover sheet or letter 
 
Adverse event case report form 
 
Serious adverse event report form 
 
Concomitant medication case report form or a list of concomitant medications 
 
Medical record progress notes including pertinent laboratory/diagnostic test results 
NOTE: When submitting SAE reports via e-mail, the volunteer line of each email notification will read as 
follows: 
SAFETY REPORT – IND # _____, Sponsor Study #_____, Volunteer# _____, Event Term: _____ 
 
10.9.3. Reporting to the UMB IRB  
UPIRTSOs, SAEs related to participation in the study, and all volunteer deaths related to 
participation in the study should be promptly reported within 24 hours of site awareness by 
telephone, email, or fax to the UMB IRB (Table 9). A complete written report should follow the 
initial notification. 
Investigators are required to forward safety information provided by the sponsor’s representative 
to the IRB. 
10.9.4. 
Sponsor Reporting Requirements to FDA 
In order to comply with 21 CFR 312.32 (c), the sponsor must report any suspected adverse reaction 
that is both serious and unexpected to the FDA as an IND safety report within 15 calendar days. 
Any unexpected fatal or life-threatening suspected adverse reaction must be reported to the FDA 
within 7 calendar days. 
10.10. Reporting Additional Immediately Reportable Events to the Sponsors Safety Office 
and HRPO  
10.10.1. 
Pregnancy 
Each pregnancy must be described on the Pregnancy Report Form and reported immediately 
(within 24 hours of site awareness) by email, fax or phone to the sponsor’s safety office and the 
sponsor’s office. 
Volunteers who become pregnant after Day 0 will be followed until 30 days after delivery to 
determine outcome, including spontaneous or voluntary termination, details of the birth, and the 
presence or absence of any birth defects, congenital abnormalities, or maternal and/or newborn 
complications. The following information will be gathered for outcome, date of delivery, health 

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status of the mother and child including the child’s gender, 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, an elective termination for medical rationale, or the infant has a 
congenital anomaly/birth defect. 
Report the incident to UMB HRPO in accordance with HRPO policy. 
10.10.2. 
AE-related Withdrawal of Consent 
Any AE-related withdrawal of consent during the study must be reported immediately (within 
72 hours of identification) by email or fax to the sponsor’s safety office as per Table 10. Report 
the withdrawal to local IRB in accordance with IRB policy. 
10.10.3. 
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 immediately to the local IRB and the sponsor’s representative. 
10.10.4. 
 
 
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 the sponsor’s representative for 
review and approval. The sponsor’s representative will use this report to prepare the final clinical 
study report for submission to the FDA. 
The PI will report all AEs to the UMB IRB 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 at the UMB CVD. 
The final study report submitted to the IRB, including a copy of any acknowledgement 
documentation and any supporting documents, will be submitted to the HRPO as soon as all 
documents become available. 
11.0. 
STATISTICS 
Detailed statistical procedures, listings, table shells and figures will be provided in a separate 
statistical analysis plan (SAP) written shortly after protocol approval but before any volunteer 
enrollment. The SAP will be finalized before study close-out and database lock. The following 
key statistical components will be considered, and a detailed description will be documented in 
the SAP: 

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• 
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,  
• 
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. 
11.1. 
Description of Statistical Methods 
Descriptive analysis of safety and reactogenicity outcomes will include all volunteers who meet 
the eligibility criteria, receive inoculation, and for whom safety data are available. Summary tables 
will be created in which incidence, severity, and relationship to the use of investigational product 
of individual solicited local and systemic signs, symptoms, or trending unsolicited events are 
delineated by study group, severity, gender, and overall. Unsolicited AEs and SAEs will be 
analyzed in a similar fashion. 
Analysis of the data from this study will be descriptive in nature. The primary endpoints align with 
the safety and clinical objectives. The nature, frequency and severity of adverse events (AEs) 
associated with the attenuated DHIM-1 will be evaluated in the vaccinated ADVP004 and 
ADVP003 volunteers and unvaccinated dengue-naïve control volunteers with statistical 
comparisons both within and between groups. 
For hematology and serum chemistry tests, any clinically significant changes from baseline value, 
and between groups, will be identified. As an example, but not limited to the following, dengue 
viremia will be examined both as a binary endpoint (present or not present) and as a function of 
area under the curve (AUC).  Mean, standard deviation, minimum, maximum, and possibly median 
and quartiles will be used for continuous data and number and percentage will be used for 
categorical data, unless specified otherwise in the section below. Confidence intervals and p values 
will be calculated as appropriate.  
There will be a final statistical analysis conducted following the end of all study visits. 
11.1.1. 
Analysis Addressing the Primary Endpoints 
The primary endpoints align with the safety and clinical objectives. The nature, frequency and 
severity of adverse events (AEs) associated with the attenuated DHIM-1 will be evaluated in the 
vaccinated ADVP003/ADVP004 volunteers and unvaccinated dengue-naïve control volunteers 
individually and as a comparison between groups. This analysis will be primarily descriptive in 
nature and is not expected to provide statistically significant differentiation between the study 
groups. This portion of the analysis will be performed primarily to contribute further data to the 
safety and reactogenicity performance parameters of the DHIM-1 challenge material in both 
vaccinated and unvaccinated volunteers. 

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The safety analysis set (SafAS) will be used for the analysis of safety data in this study. The SafAS 
population consists of all volunteers who are inoculated. 
Individual solicited local AEs over the 7-day follow-up period and solicited systemic AEs over the 
28-day follow-up period or 7 days post hospitalization (whichever is later) will be analyzed. 
The incidence, intensity, and relationship of individual solicited AEs to the inoculation will be 
calculated overall and by group. Abnormal laboratory measurements that occur following each 
inoculation will be summarized overall, and by toxicity grade for each component of the trial. 
Presentations will include the number and percentage of volunteers with at least 1 solicited 
symptom (local or systemic), at least 1 local symptom, and at least 1 general (systemic) symptom, 
as well as the incidence of each symptom individually. 
The number of volunteers with at least 1 report of an unsolicited adverse event reported up to 
28 days after inoculation or 7 days post hospitalization (whichever is later) will also be 
summarized overall and by dose group. The intensity and temporal relationship of the unsolicited 
symptoms to inoculation will also be assessed. Presentations will also summarize unsolicited AEs 
by body system, grade, and relatedness to virus inoculation. For the tabulation of the AEs by body 
system, a volunteer will be counted only once in a given body system. For example, a volunteer 
reporting nausea and diarrhea will be reported as 1 volunteer, 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 volunteers within the body system reporting AEs. SAEs occurring at 
any point during the trial will be summarized and relatedness to virus inoculation will be assessed. 
Serious and/or unexpected AEs will also be discussed on a case-by-case basis. 
A Dengue Illness Index (Table 11) will be calculated for each volunteer.  A calculated score will 
be generated on each volunteer which will allow for a statistical analysis of the values tabulated 
between the vaccinated and unvaccinated controls.  The Dengue Illness Index will be compared 
between vaccinated and unvaccinated volunteers using statistical testing such as Fisher’s exact test 
or T-test to evaluate for statistically significant differences between these two groups. P-values 
less than 0.05 will be considered significant for analysis performed within this protocol. The 
number, percentage and severity score of volunteers in each group with each of the following 
clinical or laboratory symptoms as measured by the Dengue Illness Index will be determined 
based upon the following: 
-  Fever greater than or equal to 38°C (100.4°F) [measured at least 2 times at least four hours 
apart in 24 hours] 
-  Headache/retro-orbital pain  
- Rash  
-  Fatigue and/or malaise 
-  Myalgia 
-  Arthralgia and/or bone pain  
- GI symptoms (nausea, vomiting, abdominal pain) 

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- Liver function tests (ALT or AST graded to the higher value)  
- Leukopenia  
- Thrombocytopenia  
 
A scoring system will be used to evaluate symptoms (0 = none, 1 = mild, 2 = moderate, and 3 = 
severe) on a daily basis and a total will be calculated representing the mean of the summation of 
total number of days of symptoms (A), the number of symptoms per day (B) and the maximum 
severity score (grade 0-3) (C) (See Table 11).  Volunteers may also be categorized based upon 
the 2009 revised WHO guidelines (Dengue without warning signs, dengue with warning signs 
and severe dengue).44 Note:  Tourniquet tests will be performed at investigator discretion. 

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Table 11: 
Dengue Illness Index Card 
 
 
 

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11.1.2. 
Multiple Comparison Endpoint 
Hypotheses will not be tested as part of the analysis of study data, and so control of the effect of 
multiple comparisons is not relevant to this study. 
11.1.3. 
Analysis Addressing the Secondary Endpoints 
The performance of the challenge virus at a certain dose up to 28 days after inoculation will be 
assessed descriptively using the following parameters: 
Analysis of the secondary endpoints will be applied on per protocol population. Only those 
volunteers who receive inoculations will be included in the analysis.  The analysis of secondary 
endpoints will include analysis viremia magnitude and kinetics (area under the curve) which will 
be compared between the vaccinated and unvaccinated groups using statistical tests such as t-test 
and Kaplan Meier analysis to evaluate for statistically significant differences between these 
groups. 
11.1.4. Analysis Addressing the Exploratory Endpoints 
There are no hypotheses. All of the main analysis will be descriptive. Analyses involving the 
immune/immunogenetic response to the challenge virus at a certain dose may be characterized 
descriptively (but are not limited to): 
• 
GMT and geometric mean titer rates (GMTRs) of neutralizing antibody titer levels (measured 
by dengue neutralizing titer) at 0, 1, 3, and 6 months after inoculation 
• 
Number and percentage of volunteers with a titer ≥10 
• 
CMI data: descriptive statistical summaries will be provided to describe data by treatment 
group. 
• 
Genetic (RNA) data to include minor variant sequence analysis, whole genome RNA-seq, 
and/or transcriptomic analysis 
• 
ADE assay data 
Analysis of the exploratory endpoints will be applied on the full analysis set and per protocol 
population. Only those volunteers who receive inoculations will be included in the analysis. 
11.2. Planned Enrollment and Reason for Sample Size 
As this study has no statistical hypothesis test, there is no formal power calculation. The sample 
of 10 total vaccinated volunteers with 5 volunteers from each of the two most immunogenic dengue 
prime boost vaccination schedules has been determined primarily on the basis of available funding 
for a study of this nature along with the available pool of previously vaccinated volunteers who 
remain available and potentially willing to participate in this study. The control arm of previously 
unvaccinated and flavivirus naïve volunteers has been set at 5 based upon standards set using 
previous challenge models which will allow a reasonable comparison group for those symptoms 
and criteria that are expected to occur in >90% of subjects inoculated which would include viremia 
and at least one dengue like symptoms based on previous studies with this challenge virus strain. 

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Due to the sample size, only adverse events with high incidence rates will be detected. With 10 
volunteers, the probability of observing at least 1 AE is approximately 95% if the true incidence 
rate is 26%.  At least 7/10 volunteers must seroconvert to establish a true seroconversion rate of 
no less than 30% with 95% confidence. 
With 10 consecutive successes of meeting the desired performance parameters, it can be concluded 
with 95% confidence that the future success rate of the DENV-1-LVHC virus challenge is expected 
to be greater than 74%. 
11.3. 
Procedures for Reporting Deviations from the Original Statistical Plan 
Any deviation(s) from the original statistical plan as indicated in the protocol will be described in 
an amendment to the protocol and in the SAP.  
11.4. 
Selection of Volunteers to be Included in Analyses 
Three analysis sets will be used: The Per-Protocol Analysis Set (PPAS), the Full Analysis Set 
(FAS), and the Safety Analysis Set (SafAS). 
11.4.1. 
Safety Analysis Set 
The SafAS is defined as those volunteers, who meet the eligibility criteria, received the virus 
inoculation, and for whom safety data are available 
11.4.2. 
Full Analysis Set 
The FAS is defined as those volunteers, who meet the eligibility criteria, received the virus 
inoculation, and for whom performance data are available.  
11.4.3. 
Per-Protocol Analysis Set 
The PPAS will include all volunteers who meet the definition of the SafAS and had none of the 
following protocol deviations:  
1. Administration of inoculation was not done as per protocol (site and route of administration) 
2. Volunteer received a dose other than the one that he/she was expected to receive 
3. Volunteer received a protocol-restricted medication 
4. Volunteer did not complete the study due to being lost to follow up (during the 28 days after 
inoculation or through 7 days post hospital discharge, whichever is later), but not due to 
withdrawn consent 
11.4.4. 
Populations Used in Analyses 
The safety analyses will be performed on the SafAS. Volunteers will be analyzed according to the 
inoculation they actually received. 
The performance analyses will be performed on the FAS and PPAS. Volunteers will be analyzed 
according to the inoculation they actually received. 

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NOTE: If the FAS and PPAS include the same volunteers, only 1 set of analyses will be 
produced. They will generally be identified as summarizing or listing data from the PPAS. 
If exploratory endpoint data become available, exploratory analysis set will be defined in separate 
documents defining the analyses planned for those populations and data. 
11.5. 
Handling of Missing Data and Outliers 
Missing data will be handled according to Table 12.  
Table 12: 
Methods for Handling Missing Data and Outliers 
Data 
Handling Method 
Safety  
Missing data will not be replaced with imputed values. 
Causality 
Non-serious unsolicited AEs and SAEs with missing causality will be considered as 
related to inoculation. 
Measurements  
Missing measurement (for temperature) will not be replaced. Nevertheless, the following 
rule will be applied: If temperature is partially missing after decimal point, the data will be 
analyzed replacing "MD" by zero (whatever the group). By example, a "39.MD" daily 
temperature (MD means missing data) will be considered as "39.0°C" at the time of 
analysis. 
Intensity 
Missing intensity will not be imputed. 
Start and Stop Dates 
Missing or partially missing stop dates after Day 28 for injection site or Day 28 for 
systemic reactions will not be recorded. 
Action Taken 
Missing action taken will not be imputed. 
Assessment of Outcome 
Assessment of outcome will not be imputed.  
Seriousness (for SAE) 
Missing seriousness will not be imputed. Missing seriousness will be indicated as such in 
the data listings. 
12.0.  
Direct Access to Source Data/Documents 
Volunteers will be identified on eCRFs by a unique volunteer 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 volunteer identification number will be 
used if it becomes necessary to identify data specific to a single volunteer. Representatives of the 
sponsor’s representative, the UMB IRB, and the FDA are eligible to review medical and research 
records related to this study as a part of their responsibility to protect human volunteers in clinical 
research. Personal identifiers will be removed from photocopied medical and research records. 
Personal identifying information and clinical/scientific data on volunteers previously participating 
in ADVP003 or -004 will be shared between USAMMDA and UMB-CVD under a CTA. Data 
will be maintained on secure servers or within locked file cabinets indefinitely or until directed to 
be destroyed by a governing authority. 

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13.0.  
QUALITY CONTROL AND QUALITY ASSURANCE 
13.1. 
Study Monitoring 
Study monitoring will be the responsibility of ICON. 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 volunteers and the investigators’ compliance with the 
protocol and GCPs. 
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 
volunteer has completed the study. A report of monitoring observations will be provided to the PI 
and the sponsor’s office, at a minimum.  
13.2. 
Audits and Inspections 
Auditing of the clinical trial may be conducted at any time during the study to ensure continued 
compliance with regulations, policies, and procedures. Authorized representatives of the sponsor, 
the FDA, the independent ethics committee or IRB may visit the site to perform audits or 
inspections, including source data verification. 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. 
Audit findings will be documented in a formal audit report that will detail the conduct of the audit 
and summarize the observations noted.  
The investigator should contact the sponsor’s office immediately if contacted by a regulatory 
agency about an inspection. 
14.0. 
 
ETHICS 
14.1. 
Ethical Conduct of the Study 
The study will be conducted in accordance with all applicable regulatory requirements including 
ICH Guideline for GCP, all applicable volunteer privacy requirements, and the study will be 
conducted in accordance with applicable regulations and policies including the Declaration of 
Helsinki, ICH Guidelines, US 32 CFR 219 (Protection of Human Volunteers), US 21 CFR Part 50 
[Protection of Human Volunteers (Informed Consent)] and Part 56 (IRBs) and Part 312 
(Investigational New Drug Application), AR 40-7, and AR 70-25, and the principles of respect for 
persons, beneficence, and justice described in the Belmont Report.  
The procedures set out in this study are designed to ensure that the sponsor’s representative and 
all study personnel abide by the principles of the ICH GCP Guideline and other applicable 
regulatory requirements. The investigator confirms this by signing approval for this study protocol 
and FDA Form 1572. 

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14.1.1. Confidentiality 
The Health Insurance Portability and Accountability Act (HIPAA) requires that researchers obtain 
the volunteer’s permission (HIPAA Authorization) to use and disclose health information about 
the volunteer that is either created by or used in connection with this research. The information 
includes the entire research record and supporting information from the volunteer’s medical 
records, results of laboratory tests, and both clinical and research observations made during the 
individual’s participation in the research. 
In this research, the volunteer’s health information will be collected and used to conduct the study; 
to monitor the volunteer’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 volunteer has the right to see and 
receive a copy of his/her information. 
The sponsor’s representative, the IRB, and the FDA are eligible 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 eligible to witness the applicable study procedures to assure 
the safety of volunteers. 
No personal identifier will be used in any publication or communication used to support this 
research study. The volunteer’s identification number will be used in the event it becomes 
necessary to identify data specific to a single volunteer. 
 
14.1.2. Compensation 
Volunteers will be compensated for time and inconvenience in accordance with the standards for 
compensation at the site. Compensation may vary depending upon the number of days that may 
be required for the daily evaluations in the period after a DHIM as those who become viremic 
early will require fewer days of clinic visits than those who have delayed or no viremia.  Details 
are provided in the ICF. 
 
14.1.3. Written Informed Consent 
The site will prepare a model ICF which will embody the ICH GCP as well as sponsor-required 
elements. Freely given and written informed consent must be obtained from each volunteer prior 
to participation in the study.  
The informed consent process and document will be reviewed and approved by the IRB and 
sponsor’s representative prior to initiation of the study. The consent document contains a full 
explanation of the possible risks, advantages, and any alternate vaccination/preventative options, 
and availability of treatment in the case of injury, in accordance with 21 CFR 50. The consent 
document indicates that by signature, the volunteer permits access to relevant study records by the 
sponsor’s representative and by representatives of the 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 IRB. 

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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 volunteer before any study-related procedures are 
initiated for that volunteer. This consent document must be retained by the investigator as part of 
the study records. The investigators or their designees will present the protocol in lay terms to 
individual volunteers. Questions on the purpose of the protocol, protocol procedures, and risks to 
the volunteers will then be solicited. Any question that cannot be answered will be referred to the 
principal investigator. No volunteer should grant consent until he or she has had ample time to 
read the informed consent document and questions have been answered to his/her satisfaction. The 
volunteer should understand that the study product is an investigational drug and is not licensed 
by the FDA for commercial use but is permitted to be used in this clinical research. Informed 
consent includes the principle that it is critical the volunteer be informed about the principal 
potential risks and benefits. This information will allow the volunteer to make a personal risk 
versus benefit decision and understand the following:  
• Participation is entirely voluntary 
• Withdrawal from participation can occur at any time 
• Refusal to participate involves no penalty 
• Questions to understand the nature of the protocol can be asked 
• A description of this clinical trial will be available on http://www.ClinicalTrials.gov, 
as required by US law.  
Should the protocol be modified, the volunteer ICF must be reviewed and revised, if applicable, 
to reflect the changes made to the protocol. If a previously enrolled volunteer is directly affected 
by the change, the volunteer will receive a copy of the revised informed consent document to 
read. If the volunteer agrees to continue participating in the study, the approved revision will be 
signed and dated by the volunteer. 
14.1.4. Medical Care for Research-Related Injury 
All non-exempt research involving human volunteers shall, at a minimum, meet the requirement 
of 32 CFR 219.116(a)(6). 
If a volunteer suffers an injury directly related to participation in this project, UMB and/or one of 
its affiliated institutions or health care groups will help obtain medical treatment for the specific 
injury and provide referrals to other health care facilities, as appropriate. UMB and/or its affiliated 
institutions or health care groups will not provide financial compensation or reimbursement for 
the cost of care provided to treat a research-related injury or for other expenses arising from a 
research-related injury. The institution or group providing medical treatment will charge the 
volunteer’s insurance carrier, the volunteer, or any other party responsible for treatment costs. If 
the volunteer incurs uninsured medical costs, they are the responsibility of the volunteer. 
14.2. 
Ethics Review 
The study is based on adequately performed laboratory and animal experimentation; the study will 
be conducted under a protocol reviewed by the UMB IRB; the study is to be conducted by 
scientifically and medically qualified persons; the benefits of the study are in proportion to the 
risks; the rights and welfare of the volunteers will be respected; the physicians conducting the 

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study will ensure that the risks do not outweigh the potential benefits; the results to be reported 
will be accurate; volunteers will give their informed consent and 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. 
14.2.1.  Review/Approval of Study Protocol 
Before the clinical study is initiated, the study protocol and other required documents will be 
submitted to the University of Maryland Human Research Protection Program IRB for review 
and/or approval, with the final review by the FDA. 
Enrollment and screening will not begin until IRB approvals have been obtained and the formal 
authorization letter is received by the PI. Initial IRB approval and all materials approved by the 
IRB for this protocol, including the patient consent form and recruitment materials, must be 
maintained by the protocol physician (PI) and made available for inspection.  
Volunteer informed consent will be obtained prior to the initiation of any study procedure. The PI 
will be responsible for preparing and submitting continuing review reports per institution and IRB 
requirements. 
14.2.2. Institutional Review Board 
The IRB of record (The University of Maryland HRPO) 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 patient consent form and recruitment materials, must be 
maintained by the protocol physician (PI) and made available for inspection. 
The WRAIR Human Subjects Protection Branch (HSPB) and Office of Regulated Activity (ORA) 
will be required to provide oversight given the role of WRAIR laboratory activity, data transfer 
and genetic analysis.  
The PI will be responsible for preparing and submitting continuing review reports per institution 
and IRB requirements. 
14.2.3. Protocol Modifications 
All modifications to the protocol and supporting documents (i.e., informed consent, study-specific 
procedures, SOPs, recruitment materials, etc.) must be reviewed and approved by the sponsor and 
by the IRB prior to implementation. Any protocol amendment will be agreed upon and approved 
by the sponsor’s representative prior to submission to the local IRB and prior to implementation 
of said change or modification. The ICF must be revised to concur with any amendment as 
appropriate and must be reviewed and approved with the amendment. Any volunteer already 
enrolled in the study will be informed about the revision and asked to sign the revised informed 
consent document if the modification directly affects the individual’s participation in the study. A 
copy of the revised, signed, and dated informed consent document will be given to the volunteer. 

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All original versions of the informed consent document will be retained in the protocol regulatory 
file, and a copy will be retained in the volunteer study chart. 
Any modification that could potentially increase risk to volunteers must be submitted to the IRBs 
for approval prior to implementation.  
15.0.  DATA HANDLING AND RECORD KEEPING  
The primary source document for this study will be the volunteer study case report form (CRF). 
If separate research records are maintained by the investigator(s), the medical record and the 
research records will be considered the source documents for the purposes of auditing the study. 
The source documents will be retained at the site. 
Any study information shared between the USAMMDA, WRAIR, and UMB-CVD will be coded 
and only transferred utilizing the technology transfer platform, SAFE, to ensure data 
confidentiality. Data in question includes clinical and immunologic data from ADVP003 and -
004 participants relevant to the conduct of ADVP005. 
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 collected 
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. 
All individuals who will be expected to use the EDC will be given the training necessary to perform 
their assigned tasks as described in (21 CRF 11.10(i)). Training will be conducted by qualified 
individuals initially and on a continuing, as needed basis. The training documentation will be 
maintained at the trial site. The sponsor will also keep a record of the training files.  
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.  All updates to the data management 
plan must be approved before study close-out and database lock. 
15.1. Inspection of Records 
The sponsor’s representative or designee will be allowed to conduct site visits at the study site for 
the purpose of monitoring any aspect of the study. The investigator agrees to allow the monitor to 
inspect the storage area, investigational product stocks, accountability records, volunteer charts, 
study source documents, and other records relative to study conduct. 
Volunteers’ study chart information will be 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 

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signature, the volunteer permits access to relevant study chart information by the sponsor’s 
representative and by representatives of the FDA.  
15.2.   Retention of Records 
Following closure of the study, the investigator must maintain all site study records in a safe and 
secure location. The records must be maintained to allow easy and timely retrieval, when needed 
(e.g., audit or inspection), and, whenever feasible, to allow any subsequent review of data in 
conjunction with assessment of the facility, supporting systems, and staff. Where permitted by 
applicable laws/regulations or institutional policy, some or all of these records can be maintained 
in a validated format other than hard copy (e.g. microfiche, scanned, electronic for studies with an 
eCRF); however, caution needs to be exercised before such action is taken. The investigator must 
assure that all reproductions are legible and are a true and accurate copy of the original and meet 
accessibility and retrieval standards, including re-generating a hard copy, if required. Furthermore, 
the investigator must ensure there is an acceptable back-up of these reproductions and that an 
acceptable quality control process exists for making these reproductions. 
The investigator must maintain all documentation relating to the study. The CVD will retain 
records on site for the duration of the active IRB protocol.  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. An archival at an off-site facility, Iron Mountain, 
will hold records in perpetuity and the records are easily retrievable.  
The PI will be responsible for retaining sufficient information about each volunteer, i.e., name, 
address, telephone number, Social Security number, and volunteer identifier in the study, so that 
the sponsor’s representative, the local IRB, the FDA, employees of WRAIR or USAMRDC, or 
other regulatory authorities may have access to this information should the need arise. 
16.0. 
PUBLICATION POLICY 
16.1. IND Annual Reports and Final Clinical Study 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. 
 
16.1.1. IND Annual Report to the FDA 
The PI will be responsible for the preparation of a detailed annual synopsis of clinical activity, 
including adverse events, for submission to the sponsor’s representative.  Each annual report will 
summarize IND activity for one year. The sponsor’s representative will notify the PI of the due 
date with sufficient time for the PI to assemble the required information. 
 
16.1.2. Study Results and Clinical Trial Registries 
Following completion of the study, the investigator may publish the results of this research in a 
scientific journal. The International Committee of Medical Journal Editors (ICMJE) member 
journals have adopted a trials-registration policy as a condition for publication. This policy 

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requires that all clinical trials be registered in a public trials registry such as ClinicalTrials.gov, 
which is sponsored by the National Library of Medicine (NLM). Other biomedical journals are 
considering adopting similar policies. It is the responsibility of DMID to register this trial in an 
acceptable registry. In compliance with Public Law 110-85, the Food and Drug Administration 
Amendments Act of 2007 (FDAAA), DMID will also post the results of the trial in accordance to 
the legal requirements. 
The ICMJE defines a clinical trial as any research project that prospectively assigns human 
participants to intervention or comparison groups to study the cause-and-effect relationship 
between a medical intervention and a health outcome. Studies designed for other purposes, such 
as to study pharmacokinetics or major toxicity (e.g., Phase 1 trials), would be exempt from this 
policy. As a result, this study can but is not required to be registered in the NLM registry, 
ClinicalTrials.gov.  
17.0.  LIST OF REFERENCES 
 
1. World Health Organization G, Switzerland:  WHO. Dengue:  Guidelines for Diagnosis, 
Treatment, Prevention, and Control. 2009. 
2. Freedman DO, Weld LH, Kozarsky PE, et al. Spectrum of disease and relation to place of 
exposure among ill returned travelers. N Engl J Med 2006;354:119-30. 
3. Jensenius M, Han PV, Schlagenhauf P, et al. Acute and potentially life-threatening tropical 
diseases in western travelers--a GeoSentinel multicenter study, 1996-2011. Am J Trop Med Hyg 
2013;88:397-404. 
4. Schwartz E, Weld LH, Wilder-Smith A, et al. Seasonality, annual trends, and characteristics 
of dengue among ill returned travelers, 1997-2006. Emerg Infect Dis 2008;14:1081-8. 
5. Gibbons RV, Streitz M, Babina T, Fried JR. Dengue and US military operations from the 
Spanish-American War through today. Emerg Infect Dis 2012;18:623-30. 
6. Rigau-Perez JG, Clark GG, Gubler DJ, Reiter P, Sanders EJ, Vorndam AV. Dengue and 
dengue haemorrhagic fever. Lancet 1998;352:971-7. 
7. Burke DS, Nisalak A, Johnson DE, Scott RM. A prospective study of dengue infections in 
Bangkok. Am J Trop Med Hyg 1988;38:172-80. 
8. Vaughn DW, Green S, Kalayanarooj S, et al. Dengue in the early febrile phase: viremia and 
antibody responses. J Infect Dis 1997;176:322-30. 
9. Guzman MG, Alvarez M, Halstead SB. Secondary infection as a risk factor for dengue 
hemorrhagic fever/dengue shock syndrome: an historical perspective and role of antibody-
dependent enhancement of infection. Arch Virol 2013;158:1445-59. 
10. Halstead SB, Nimmannitya S, Cohen SN. Observations related to pathogenesis of dengue 
hemorrhagic fever. IV. Relation of disease severity to antibody response and virus recovered. 
Yale J Biol Med 1970;42:311-28. 
11. Anderson KB, Gibbons RV, Thomas SJ, et al. Preexisting Japanese encephalitis virus 
neutralizing antibodies and increased symptomatic dengue illness in a school-based cohort in 
Thailand. PLoS Negl Trop Dis 2011;5:e1311. 
12. Zulkipli MS, Dahlui M, Jamil N, et al. The association between obesity and dengue severity 
among pediatric patients: A systematic review and meta-analysis. PLoS Negl Trop Dis 
2018;12:e0006263. 

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13. USFDA. Dengvaxia. In: Administration USFD, ed. Vaccines, Blood and Biologics2020. 
14. Sridhar S, Luedtke A, Langevin E, et al. Effect of Dengue Serostatus on Dengue Vaccine 
Safety and Efficacy. N Engl J Med 2018;379:327-40. 
15. Statler J, Mammen M, Lyons A, Sun W. Sonographic findings of healthy volunteers infected 
with dengue virus. J Clin Ultrasound 2008;36:413-7. 
16. Mammen MP, Lyons A, Innis BL, et al. Evaluation of dengue virus strains for human 
challenge studies. Vaccine 2014;32:1488-94. 
17. Endy TP, Wang D, Polhemus ME, et al. A Phase 1, Open Label- Assessment of a Dengue 
Virus-1 Live Virus Human Challenge - (DENV-1-LVHC) Strain. J Infect Dis 2020. 
18. Sun W, Eckels KH, Putnak JR, et al. Experimental dengue virus challenge of human subjects 
previously vaccinated with live attenuated tetravalent dengue vaccines. J Infect Dis 
2013;207:700-8. 
19. Gunther VJ, Putnak R, Eckels KH, et al. A human challenge model for dengue infection 
reveals a possible protective role for sustained interferon gamma levels during the acute phase of 
illness. Vaccine 2011;29:3895-904. 
20. McKee KT, Jr., Bancroft WH, Eckels KH, Redfield RR, Summers PL, Russell PK. Lack of 
attenuation of a candidate dengue 1 vaccine (45AZ5) in human volunteers. Am J Trop Med Hyg 
1987;36:435-42. 
21. Kirkpatrick BD, Whitehead SS, Pierce KK, et al. The live attenuated dengue vaccine TV003 
elicits complete protection against dengue in a human challenge model. Sci Transl Med 
2016;8:330ra36. 
22. Graham H. The Dengue: a study of its pathology and mode of propagation. J Trop Med 1903. 
23. Ashburn PM, Craig CF. Experimental investigations regarding the etiology of dengue fever. 
J Infect Dis 1907;4:440-75. 
24. Cleland JB, Bradley B, MacDonald W. Further experiments in the etiology of dengue fever. 
The Journal of Hygiene 1919;18:217-54. 
25. Siler JF, Hall MW, Hitchens AP. Dengue - Its history, epidemiology, mechanism of 
transmission, etiology, clinical manifestations, immunity and prevention. The Philippine Journal 
of Science 1926;29:1-304. 
26. Simmons J. Dengue Fever. The American Journal of Tropcial Medicine and Hygiene 
1931;s1-11:77-102. 
27. Misao T. Clinical aspects of dengue fever. Chiryo (Therapy) 1944;26:199-218. 
28. Hotta S. Experimental studies in dengue 1 isolation, identification and modification of the 
virus. Journal of Infectious Diseases 1951;90:1-9. 
29. Sabin AB, Schlesinger RW. Production of immunity to dengue with virus modified by 
propagation in mice. Science 1945;101:640-2. 
30. Taniguchi T. Studies on the experimental inoculation of dengue fever. Medical Journal of 
Osaka University 1951;2:1-36. 
31. Schlesinger RW. Clinical and serologic response of man to immunization with attenuated 
dengue and yellow fever viruses. Journal of Immunology 1956;77:352-64. 
32. Wisseman CL, Jr., Kitaoka M, Tamiya T. Immunological studies with group B arthropod-
borne viruses. V. Evaluation of cross-immunity against type 1 dengue fever in human subjects 
convalescent from subclinical natural Japanese encephalitis virus infection and vaccinated with 
17D strain yellow fever vaccine. The American journal of tropical medicine and hygiene 
1966;15:588-600. 

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33. Eckels KH, Scott RM, Bancroft WH, et al. Selection of attenuated dengue 4 viruses by serial 
passage in primary kidney cells. V. Human response to immunization with a candidate vaccine 
prepared in fetal rhesus lung cells. Am J Trop Med Hyg 1984;33:684-9. 
34. Innis BL, Eckels KH, Kraiselburd E, et al. Virulence of a live dengue virus vaccine 
candidate: a possible new marker of dengue virus attenuation. J Infect Dis 1988;158:876-80. 
35. Edelman R, Tacket CO, Wasserman SS, et al. A live attenuated dengue-1 vaccine candidate 
(45AZ5) passaged in primary dog kidney cell culture is attenuated and immunogenic for humans. 
J Infect Dis 1994;170:1448-55. 
36. Dengue. 2019.  
37. Paixao ES, Teixeira MG, Costa M, Rodrigues LC. Dengue during pregnancy and adverse 
fetal outcomes: a systematic review and meta-analysis. Lancet Infect Dis 2016;16:857-65. 
38. Molton JS, Low I, Choy MMJ, et al. Dengue virus not detected in human semen. J Travel 
Med 2018;25. 
39. Live Dengue Vaccines Technical Consultation Reporting G, Bentsi-Enchill AD, Schmitz J, et 
al. Long-term safety assessment of live attenuated tetravalent dengue vaccines: deliberations 
from a WHO technical consultation. Vaccine 2013;31:2603-9. 
40. Hadinegoro SR, Arredondo-Garcia JL, Capeding MR, et al. Efficacy and Long-Term Safety 
of a Dengue Vaccine in Regions of Endemic Disease. N Engl J Med 2015;373:1195-206. 
41. Katzelnick LC, Montoya M, Gresh L, Balmaseda A, Harris E. Neutralizing antibody titers 
against dengue virus correlate with protection from symptomatic infection in a longitudinal 
cohort. Proc Natl Acad Sci U S A 2016;113:728-33. 
42. Wiwanitkit S, Wiwanitkit V. Excessive menstruation bleeding as a presentation of dengue 
hemorrhagic fever. Arch Gynecol Obstet 2013;287:1271. 
43. Langer J, Dufoe A, Brady J. U.S. Faces a Rise in Mosquito 'Disease Danger Days'.  Climate 
Central. https://www.climatecentral.org/news/us-faces-a-rise-in-mosquito-disease-danger-days-
21903: Cliimate Central; 2018:1-5. 
44. Severe falciparum malaria. World Health Organization, Communicable Diseases Cluster. 
Trans R Soc Trop Med Hyg 2000;94 Suppl 1:S1-90. 
 
 
 

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Toxicity Grading Scales for Laboratory (Chemistry and Hematology), Local Reactogenicity, 
Vital Signs and Systemic Adverse Events 
18.0 APPENDIX A: 
Laboratory Toxicity Grading Scale for Chemistries 
Table 13: 
Toxicity Grading Scale for Laboratory Abnormalities  
Seruma,d 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially Life 
Threatening 
(Grade 4)b 
Sodium – Hyponatremia (mEq/L) 
132 – 134 
130 – 131 
125 – 129 
< 125 
Sodium – Hypernatremia (mEq/L) 
144 – 145 
146 – 147 
148 – 150 
> 150 
Potassium – Hyperkalemia (mEq/L) 
5.1 – 5.2 
5.3 – 5.4 
5.5 – 5.6 
> 5.6 
Potassium – Hypokalemia (mEq/L) 
3.5 – 3.6 
3.3 – 3.4 
3.1 – 3.2 
< 3.1 
Glucose – Hypoglycemia (mg/dL) 
65 – 69 
55 – 64 
45 – 54 
< 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) 
23-26 
27 – 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 – 1.5 
1.1 – 1.2 
0.9 – 1.0 
< 0.9 
Phosphorous – hypophosphatemia 
(mg/dL) 
2.3 – 2.5 
2.0 – 2.2 
1.6 – 1.9 
< 1.6 
CPK (mg/dL) 
1.25 – 1.5 x 
ULNc 
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 LFT 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 LFT 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 
a The laboratory values provided in the tables serve as guidelines and are dependent upon institutional normal parameters. Institutional normal reference ranges should be provided to 
demonstrate that they are appropriate. 
b The clinical signs or symptoms associated with laboratory abnormalities might result in characterization of the laboratory abnormalities as Potentially Life Threatening (Grade 4). For 
example, a low sodium value that falls within a Grade 3 parameter (125-129 mEq/L) should be recorded as a Grade 4 hyponatremia event if the volunteer had a new seizure associated with the 
low sodium value. 
c   ULN is the upper limit of the normal range. 
d    Laboratory values that fall in the institutional normal reference range do not receive a toxicity grade 

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Table 14: 
Safety Laboratory Toxicity Grading Scale for Hematology Adverse Events  
Hematology Parametera,d 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially Life 
Threatening 
(Grade 4) 
Hemoglobin for Females (gm/dL)b 
11.0 – 12.0 
9.5 – 10.9 
8.0 – 9.4 
< 8.0 
Hemoglobin for females change from 
baseline value (gm/dL) 
Any decrease 
– 1.5 
1.6 – 2.0 
2.1 – 5.0 
> 5.0 
Hemoglobin for Males (gm/dL) 
12.5 – 13.5 
10.5 – 12.4 
8.5 – 10.4 
< 8.5 
Hemoglobin for Males change from 
baseline value (gm/dL) 
Any decrease 
– 1.5 
1.6 – 2.0 
2.1 – 5.0 
> 5.0 
WBC Increase (cell/mm3) 
10,800 – 
15,000 
15,001 – 
20,000 
20,001 – 25, 
000 
> 25,000 
WBC Decrease (cell/mm3) 
2,500 – 3,500 
1,500 – 2,499 
1,000 – 1,499 
< 1,000 
Lymphocytes Decrease (cell/mm3) 
750 – 1,000 
500 – 749 
250 – 499 
< 250 
Neutrophils Decrease (cell/mm3) 
1,500 – 2,000 
1,000 – 1,499 
500 – 999 
< 500 
Eosinophils (cell/mm3) 
650 – 1500 
1501 - 5000 
> 5000 
Hypereosinophilic 
Platelets Decreased (cell/mm3) 
125,000 – 
140,000 
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) 
400 – 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) 
a The laboratory values provided in the tables serve as guidelines and are dependent upon institutional normal parameters. Institutional normal reference ranges 
should be provided to demonstrate that they are appropriate. 
b In cases where ABNORMAL laboratory values fall below grade 1 the lab abnormality will be graded as GRADE 1. 
c In cases where ABNORMAL laboratory values fall between two FDA grades, the ABNORMAL value will be graded as the highest grade of the two. 
d Laboratory values that fall in the institutional normal reference range do not receive a toxicity grade 
 
 
 
 

Clinical Protocol ADVP005 
The University of Maryland, Baltimore  
IND 016332; CVD Dengue 12000 
Center for Vaccine Development and Global Health 
 
V04.0, 04 Jan 2021 
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Table 15: 
Toxicity Grading Scale for Local Reactions 
a  In addition to grading the measured local reaction at the greatest single diameter, the measurement should be 
recorded as a continuous variable. 
b  Induration/Swelling should be evaluated and graded using the functional scale as well as the actual measurement. 
Local Reaction 
Normal 
(Grade 0) 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially 
Life 
Threatening 
(Grade 4) 
Redness/Erythema 
<25 mm 
25-50 mm 
51-100 mm 
>100 mm 
Necrosis or 
exfoliative 
dermatitis 
Swelling/ 
Indurationa, c 
<25 mm 
25-50 mm 
and does not 
interfere with 
activity 
51-100 mm or 
interferes with 
activity 
>100 mm or 
prevents daily 
activity 
Necrosis 
Pain  
None 
Does not 
interfere with 
activity 
Interferes with 
activity or 
repeated use of 
non-narcotic pain 
reliever 
Prevents daily 
activity or 
repeated use of 
narcotic pain 
reliever 
ER visit or 
hospitalization 

Clinical Protocol ADVP005 
The University of Maryland, Baltimore  
IND 016332; CVD Dengue 12000 
Center for Vaccine Development and Global Health 
 
V04.0, 04 Jan 2021 
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Table 16: 
Toxicity Grading Scale for Vital Signs 
Vital Signsa 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially Life Threatening 
(Grade 4) 
Fever (°C)  
 (°F)b 
38.0 – 38.4 
100.4 – 101.1 
38.5 – 38.9 
101.2 – 102.0 
39.0 – 40 
102.1 – 104 
> 40 
> 104 
Tachycardia - beats 
per minute 
101 – 115 
116 – 130 
> 130 
ER visit or hospitalization for 
arrhythmia 
Bradycardia - beats 
per minutec 
50 – 54 
45 – 49 
< 45 
ER visit or hospitalization for 
arrhythmia 
Hypertension 
(systolic) - mm Hg 
141 – 150 
151 – 155 
> 155 
ER visit or hospitalization for 
malignant hypertension 
Hypertension 
(diastolic) - mm Hg 
91 – 95 
96 – 100 
> 100 
ER visit or hospitalization for 
malignant hypertension 
Hypotension 
(systolic) – mm Hg 
85 – 89 
80 – 84 
< 80 
ER visit or hospitalization for 
hypotensive shock 
a Volunteer should be at rest for all vital sign measurements. 
b Oral temperature, no recent hot or cold beverages or smoking. 
c When resting heart rate is between 60 to 100 beats per minute. Physician investigators will use clinical judgment.  
when characterizing bradycardia among some healthy volunteer populations, for example, conditioned athletes 
when it comes to inclusion into the study during screening. 

Clinical Protocol ADVP005 
The University of Maryland, Baltimore  
IND 016332; CVD Dengue 12000 
Center for Vaccine Development and Global Health 
 
V04.0, 04 Jan 2021 
88 
 
Table 17: 
Toxicity Grading Scale for Systemic Adverse Events  
Systemic 
(General) 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially Life 
Threatening  
(Grade 4) 
Headache/retro-orbital 
pain 
No interference 
with activity 
Repeated use of non-
narcotic pain reliever 
> 24 hours or some 
interference with 
activity 
Significant; any use 
of narcotic pain 
reliever or prevents 
daily activity 
ER visit or 
hospitalization 
Fatigue/malaise 
No interference 
with activity 
Some interference with 
activity 
Significant; 
prevents daily 
activity 
ER visit or 
hospitalization 
Myalgia 
No interference 
with activity 
Some interference with 
activity 
Significant; 
prevents daily 
activity 
ER visit or 
hospitalization 
Arthralgia/bone pain 
No interference 
with activity 
Some interference with 
activity 
Significant; 
prevents daily 
activity 
ER visit or 
hospitalization 
Nausea/vomiting/ 
abdominal pain 
No interference 
with activity or 1-2 
episodes/24 hours 
Some interference with 
activity or > 2 
episodes/24 hours 
Prevents daily 
activity, requires 
outpatient IV 
hydration 
ER visit or 
hospitalization 
for hypotensive 
shock 
Rash 
Localized skin 
eruption 
Diffuse skin eruption 
up to 50% of the body 
surface area (BSA) 
Generalized skin 
eruption involving 
> 50% BSA, or – 
Rash with bullae, 
vesicles, mucous, 
membrane 
ulceration, target 
lesions, purpura, or 
with epidermal 
detachment 
Stevens Johnson 
Syndrome / 
Toxic epidermal 
syndrome  
Other Systemic 
Symptoms: Illness or 
clinical AE (as defined 
according to applicable 
regulations) 
No interference 
with activity 
Some interference with 
activity not requiring 
medical intervention 
Prevents daily 
activity and 
requires medical 
intervention 
ER visit or 
hospitalization 
WRAIR required reporting addendum 
In accordance with the Department of Defense (DoD) Institutional Agreement for 
Institutional Review Board (IRB) Review (IAIR), the WRAIR will rely upon the 
University of Maryland for the ethical review and oversight of this protocol. The WRAIR 
Human Subjects Protection Branch (HSPB) will still perform an administrative review of 
the protocol to ensure that the WRAIR reporting requirements are being met.  In 
accordance with the IAIR, WRAIR will provide initial approval authorization for 
WRAIR’s participation on this study, as well as WRAIR Commander approval 
authorizations for all subsequent amendments.  Additionally, a shadow file will be 
maintained within the WRAIR HSPB and acknowledgements of all continuing reviews and 
reported lifecycle actions will be provided by the WRAIR HSPB to the WRAIR study point 
of contact (POC). 

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Headquarters Level Review 
 
Documentation of review and approval by the U.S. Army Medical Research and 
Development Command (USAMRDC), Office of Research Protections (ORP), Human 
Research Protection Office (HRPO) must be provided to the WRAIR HSPB, as 
appropriate. 
 
Unanticipated Problems Involving Risks to Subjects or Others 
 
Unanticipated problems involving risks to subjects or others encompass a broader category 
of events than serious adverse events (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 (e.g., program personnel) must also 
be reported. 
 
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. 
 
Unanticipated problems involving risk to subjects or others, should be promptly reported 
(48 hours) by telephone, email, or fax to the WRAIR HSPB. A complete written report 
should follow the initial notification within 10 working days. All unanticipated problems 
occurring within the reporting period should also be summarized in the continuing review 
reports submitted to the WRAIR HSPB.  The contact information for the WRAIR HSPB 
is as follows: 
 
Director, Human Subjects Protection Branch 
503 Robert Grant Avenue 
Silver Spring, MD 20910 
Telephone: 301-319-9940 
Fax: 301-319-9961 
E-mail: usarmy.detrick.medcom-wrair.mbx.hspb@mail.mil  
  
Serious Adverse Events 
 
All related SAEs and deaths should be reported to the WRAIR HSPB within 48 hours by 
telephone, email, or fax.  A complete written report should follow the initial notification 
within 10 working days.  All SAEs occurring within the reporting period should also be 

Clinical Protocol ADVP005 
The University of Maryland, Baltimore  
IND 016332; CVD Dengue 12000 
Center for Vaccine Development and Global Health 
 
V04.0, 04 Jan 2021 
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summarized in the continuing review reports submitted to the WRAIR HSPB.  The contact 
information for the WRAIR HSPB is as follows: 
 
Director, Human Subjects Protection Branch 
503 Robert Grant Avenue 
Silver Spring, MD 20910 
Telephone: 301-319-9940 
Fax: 301-319-9961 
E-mail: usarmy.detrick.medcom-wrair.mbx.hspb@mail.mil  
 
Protocol Modifications/Amendments 
 
All amendments/modifications to the protocol and supporting documents (informed 
consent, site specific procedures [SSPs], standard operating procedures [SOPs], 
recruitment materials, etc.) must be reviewed by the WRAIR HSPB and a WRAIR 
Commander Authorization Approval issued prior to WRAIR participation on the 
amended/modified protocol.  
 
Protocol Deviations 
 
All major protocol deviations that adversely affect the safety or rights of a subject or 
scientific integrity of the study, will be reported to the WRAIR HSPB within 48 hours and 
a written report should be submitted within 10 working days.  The contact information for 
the WRAIR HSPB is as follows: 
 
Director, Human Subjects Protection Branch 
503 Robert Grant Avenue 
Silver Spring, MD 20910 
Telephone: 301-319-9940 
Fax: 301-319-9961 
E-mail: usarmy.detrick.medcom-wrair.mbx.hspb@mail.mil  
   
All protocol deviations occurring within the reporting period should be summarized in the 
continuing review reports that are submitted to the WRAIR HSPB. 
 
Continuing Reviews and Closeout Report 
 
The WRAIR POC is responsible for preparing and/or submitting continuing review reports 
and a closeout report as per WRAIR Standard Operating Procedure UWZ-C-618. The 
WRAIR HSPB will review and acknowledge the reports in order for WRAIR personnel to 
continue their participation on the study.  Once all study activities have been completed, to 
include data analysis, a closeout report will need to be submitted to the WRAIR HSPB to 
close the study.