Randomized, Double Blind Evaluation of Late Boost Strategies With IHV01 (FLSC in Aluminum Phosphate) and A244 With or Without ALFQ for HIV-uninfected Participants in the HIV Vaccine Trial RV306 / WRAIR 1920
Phase: PHASE1
Conditions: HIV
Sponsor: U.S. Army Medical Research and Development Command

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TITLE PAGE 
RANDOMIZED, DOUBLE BLIND EVALUATION OF 
LATE BOOST STRATEGIES WITH IHV01 (FLSC IN 
ALUMINUM PHOSPHATE) AND A244 WITH OR 
WITHOUT ALFQ FOR HIV-UNINFECTED 
PARTICIPANTS IN THE HIV VACCINE TRIAL RV306 / 
WRAIR 1920 
 
Sponsor 
The Surgeon General, Department of the Army 
 
Sponsor’s Representative 
Emily D. Badraslioglu, MS, PMP, RAC 
US Army Medical Research and Development Command 
(USAMRDC) 
1430 Veterans Drive 
Ft. Detrick, MD 21702-5009 
Telephone: +1 301 619 0317 
DSN: 343-0317 
Fax: +1 301 619 0197 
Email: usarmy.detrick.medcom-usamrmc.mbx.usamrmc-
regulatory-affairs@health.mil 
 
Funding Source 
Cooperative Agreement #W81XWH-18-2-0040 
 
Protocol Chair/Sponsor's 
Medical Expert for the Trial 
Sandhya Vasan, MD 
Director, HJF Component of MHRP & EIDB 
Henry M Jackson Foundation (HJF) 
6720A Rockledge Drive, Suite 400 
Bethesda, MD 20817 
Telephone: +1 301 500 3622 
Email: svasan@global-id.org 
 
US Army HIV Vaccine Product 
Manager 
Elizabeth (Betsy) Heger, M.Sc., PMP 
Product Manager, HIV 
Pharmaceutical Systems PMO 
USAMMDA 
Office: +1 301 619 7853 
Email: elizabeth.a.heger.civ@health.mil 
 

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US MHRP HIV Vaccine Product 
Manager 
Tanya Krubit, MS 
Product Quality Director, Vaccine Development and Quality 
Affairs; HJF 
In support of the US Military HIV Research Program (MHRP) 
6720A Rockledge Drive, Suite 400 
Bethesda, MD 20817 
Telephone: +1 301 500 3783 
Email: tkrubit@hivresearch.org 
 
Mahidol University Site-
Principal Investigator and 
Main Contact for Thailand 
Punnee Pitisuttithum, MD, DTM&H 
Chief, Clinical Trials Section 
Vaccine Trial Centre, Faculty of Tropical Medicine,  
Mahidol University 
420/6 Rajvithi Road, Bangkok, 10400 Thailand 
Telephone: +66 8 1829 4906, +66 2 354 9173, +66 2643 5598, 
+66 8 1700 1890 (24 hours) 
Fax: +66 2 354 9174 
Email: punnee.pit@mahidol.ac.th 
 
RTA-AFRIMS Site-Principal 
Investigator 
LTG Sorachai Nitayaphan, MD, PhD 
Senior Researcher 
Royal Thai Army (RTA), Clinical Research Center 
Armed Forces Research Institute of Medical Sciences 
315/6 Rajvithi Road 
Bangkok, Thailand 10400 
Telephone: +66 8 1625 1531, +66 2 354 7586, +66 9 0214 3119 
(24 hours) 
Fax: +66 2 354 7848 
Email: sorachain.rta@afrims.org 
 
Associate Investigators 
Nittaya Phanuphak, MD, PhD 
SEARCH Office/The Thai Red Cross AIDS Research Centre 
104 Rajdumri Road, Bangkok, Thailand 10330 
and 
Institute of HIV Research and Innovation (IHRI) Foundation  
319 Chamchuri Square Building, 11th Floor, Unit 1109-1116,  
Phayathai Road, Pathumwan  
Bangkok, Thailand 10330 
Telephone: +66 2 253 0966, +66 2 160 5372, +66 8 1825 3544, 
+66 8 3010 8544 (24 hours)  
Email: nittaya.p@ihri.org 
 
 
Benjaluck Phonrat, MS 
Department of Clinical Tropical Medicine 
Faculty of Tropical Medicine, Mahidol University 
420/6 Rajvithi Road 
Bangkok, Thailand 10400 
Telephone: +66 2 643 5599 
Email: benjaluck.pho@mahidol.ac.th 
 

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Jittima Dhitavat, MD 
Clinical Infectious Diseases Research Unit 
Department of Clinical Tropical Medicine 
Faculty of Tropical Medicine, Mahidol University 
420/6 Rajvithi Road 
Bangkok, Thailand 10400 
Telephone: +66 2 643 5599 
Email: jittima_pu@yahoo.com 
 
 
Arom Pitisuthitham, MD 
Vaccine Trial Centre, Faculty of Tropical Medicine,  
Mahidol University 
420/6 Rajvithi Road, Bangkok, 10400 Thailand 
Telephone: +66 2 643 5599 
E-mail: docarom@yahoo.com 
 
 
Sant Muangnoicharoen, MD 
Vaccine Trial Centre, Faculty of Tropical Medicine,  
Mahidol University 
420/6 Rajvithi Road, Bangkok, 10400 Thailand 
Telephone: +66 8 5199 8989 
E-mail: sant.mua@mahidol.ac.th 
 
 
Supitcha Kamolratanakul, MD 
Vaccine Trial Centre, Faculty of Tropical Medicine,  
Mahidol University 
420/6 Rajvithi Road, Bangkok, 10400 Thailand 
Telephone: +66 8 1699 6660 
E-mail: supitcha.kam@mahidol.edu 
 
 
MG Narongrid Sirisopana, MD 
Senior Researcher 
Armed Forces Research Institute of Medical Sciences 
315/6 Rajvithi Road 
Bangkok 10400, Thailand 
Telephone: +66 8 1908 6888 
Email: narongrid1956@yahoo.com 
 
 
COL Khunakorn Kana, MD 
Director of Research Division 
Armed Forces Research Institute of Medical Sciences 
315/6 Rajvithi Road 
Bangkok 10400, Thailand  
Telephone: +66 86 034 2527 
Email: k_khunakorn@hotmail.com 
 

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COL Wuttikon Rodkvamtook, PhD 
Director of Analysis Division 
Armed Forces Research Institute of Medical Sciences 
315/6 Rajvithi Road 
Bangkok 10400, Thailand 
Telephone: +66 8 1584 9546 
Email: Itwutti@hotmail.com 
 
 
MAJ Worapong Nasomsong, MD 
Infectious Disease Division, Department of Internal Medicine, 
Phramongkutklao Hospital,315 Ratchawithi Rd. Ratchathewi 
Bangkok 10400, Thailand 
and 
Clinical Research Center, Armed Forces Research Institute of 
Medical Sciences 
315/6 Rajvithi Road,Bangkok, Thailand 10400 
Telephone: +66 2 644 4420, +66 8 4211 1220 
Email: nasomsong.w@gmail.com 
 
 
COL Jessica Cowden, MD, MSPH 
Chief, Department of Retrovirology 
US Army Medical Directorate – AFRIMS 
Telephone: +66 2 696 7777 ext. 4901 
Email: jessica.cowden.mil@afrims.org 
 
Laboratory Investigators 
Siriwat Akapirat, MT, PhD 
Department of Retrovirology 
Armed Forces Research Institute of Medical Sciences (AFRIMS) 
Bangkok, Thailand 
Office: +66 2 696 2700 ext. 4923 / 4951 
Email: siriwata@hiv-th.org 
 
 
Alexandra Schuetz, PhD 
Department of Retrovirology 
US Army Medical Component, AFRIMS 
315/6 Rajvithi Road 
Bangkok, Thailand 10400 
Telephone: +66 2 696 2700 ext. 4912  
Fax: +66 2 644 4824 
Email: schuetza@hiv-th.org 
 
 
Shelly J. Krebs, Ph.D 
Chief, B cell Biology Core; HJF 
In support of the US Military HIV Research Program (MHRP) 
Walter Reed Army Institute of Research 
503 Robert Grant Ave; 1N29  
Silver Spring, MD 20910 
Phone: +1 301 319 3961 
Email: skrebs@hivresearch.org 
 

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Independent Safety Monitor 
LTC Aaron Farmer, DO FIDSA 
Director, Clinical Study Sites, Department of Virology  
US Army Medical Directorate 
Telephone: +66 81 914 7985 
Email: aaron.farmer.mil@afrims.org 
 
Protocol Consultants 
Merlin Robb, MD 
Vice President and Chief Medical Advisor 
Henry M Jackson Foundation (HJF) 
Senior Investigator, MHRP and EIDB, WRAIR 
6720-A Rockledge Drive 
Bethesda, MD 20817 
Telephone: +1 240 694 2150 
Email: mrobb@hjf.org 
 
Community Representative 
Community Advisory Board Chairperson 
 
Clinical Trial Sites 
Vaccine Trial Centre 
Faculty of Tropical Medicine, Mahidol University 
420/6 Rajvithi Road, Bangkok, 10400 Thailand 
Telephone: +66 8 1700 1890, +66 2 643 5599 
Fax: +66 2 248 6610, +66 2 643 5598 
 
 
Royal Thai Army Clinical Research Center 
Armed Forces Research Institute of Medical Sciences 
315/6 Rajvithi Road, Bangkok 10400, Thailand 
Telephone: +66 2 354 7586 and +66 9 214 3119 
 
Optional Procedures Sites 
The Thai Red Cross AIDS Research Centre 
104 Rajdumri Road, Pathumwan, Bangkok, 10330, Thailand 
Telephone: +66 2 254 2566, Fax: +66 2 254 7577 
 
 
Institute of HIV Research and Innovation (IHRI) Foundation  
319 Chamchuri Square Building, 11th Floor, Unit 1109-1116,  
Phayathai Road, Pathumwan, Bangkok, 10330 
Telephone: +66 2 253 0966, +66 2 160 5372, +66 8 1825 3544  
 
 
Chulalongkorn Memorial Hospital 
1873 Rama IV, Pathumwan, Bangkok 10330 
Telephone: +66 2 256 4000, Fax: +66 2 256 4455, Ext. 15 
 
Study Pharmacist 
Pornsuk Visudhiphan Grandin 
AFRIMS 
315/6 Rajvithi Road 
Bangkok, Thailand 10400 
Telephone: +66 8 3091 8415 
Email: pornsukg@hiv-th.org 
 

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Clinical Laboratory 
AFRIMS Retrovirology Clinical Laboratory (ARCL) 
315/6 Rajvithi Road 
Bangkok, Thailand 10400 
Telephone: +66 2 696 2700 ext. 4961 
 
Research Laboratories 
Department of Retrovirology 
USAMD-AFRIMS 
315/6 Rajvithi Road 
Bangkok, Thailand 10400 
Telephone: +66 2 696 2700 ext. 4960 / 4924 
 
 
US Military HIV Research Program 
Walter Reed Army Institute of Research (WRAIR) 
503 Robert Grant Avenue 
Silver Spring, MD 20910  
 
 
External Research Collaborators are listed in Appendix L  
 
 
Statistician 
Peter Dawson 
The EMMES Corporation 
401 North Washington Street, Suite 700 
Rockville, MD 20850 
Telephone: +1 301 251 1161 
Fax: +1 301 251 1355 
Email: pdawson@emmes.com 
 
Data Management 
The EMMES Corporation 
401 North Washington Street, Suite 700 
Rockville, MD 20850 
 
 
BIOPHICS - Center of Excellence for Biomedical and Public 
Health Informatics 
The 60 Anniversary of His Majesty the King's Accession to the 
Throne Building, 3rd - 4th floor 
Faculty of Tropical Medicine 
420/6 Rajvithi Road Bangkok 10400, Thailand 
Telephone: +66 2 354 9181- 86 
Fax: +66 2 354 9187 
 
 
Data Coordinating and Analysis Center (DCAC) 
6720A Rockledge Drive, Suite 400 
Bethesda, MD 20817 
 

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Institutional Review Boards 
Ministry of Public Health (MoPH) Ethical Committee (EC)  
Office of the Secretary, the Ethical Review Committee of 
Research in Human Subjects 
3rd Floor, Department of Medicine Services Building 
Tiwanon Road, Nonthaburi 11000, Thailand 
Telephone: +66 2 591 8251, +66 2 590 6171-2 
Fax: +66 2 591 8251 
Email: ecmoph@gmail.com 
FWA#: 00001953 
 
 
Royal Thai Army Medical Department IRB 
5th floor, Phramongkutklaovejvithaya Building  
Phramongkutklao Medical School  
315 Rajvithi Road,  
Bangkok 10400, Thailand  
Telephone: +66 2 354 7600, Ext 94270; +66 2 763 4297 
Fax: +66 2 354 9011  
Email: irbrta@yahoo.com 
FWA #00001813 
 
 
Ethics Committee of the Faculty of Tropical Medicine  
Mahidol University c/o Research and Academic Services  
4th Floor, The 60th Anniversary of His Majesty the King’s 
Accession of the Throne Building  
420/6 Rajvithi Road,  
Bangkok 10400, Thailand  
Telephone: +66 2 354 9100-4 extension 1349, 1525  
Fax: +66 2 306 9126  
Email: tmectropmed@mahidol.ac.th 
FWA #00002882 
 
 
The Institutional Review Board, 
Faculty of Medicine, Chulalongkorn University 
1873, 3rd Floor, Ananthamahidol building, 
Rama IV Rd., Patumwan, 
Bangkok, Thailand 
Telephone: +66 2 256 4493 
Fax: +66 2 255 4493 
Email: medchulairb@chula.ac.th 
FWA #00000943 
 
 
Walter Reed Army Institute of Research IRB 
503 Robert Grant Avenue 
Silver Spring, MD 20910, USA 
Telephone: +1 301 319 9940 
Email: usarmy.detrick.medcom-wrair.mbx.hspb@health.mil 
FWA #00000015 
 

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Local Drug and Vaccine 
Authorities 
Ministry of Public Health Technical Subcommittee on 
HIV/AIDS Vaccine Development (TSHAVD) 
Bureau of AIDS TB and STIs 
Vaccine Research Development Coordinating Unit, Department 
of Disease Control 
Tiwanont Rd, Nonthaburi 
11000 Thailand 
Telephone: +66 2 590 3209 
 
 
Thai Food and Drug Administration 
IND Section, Medicine Regulation Division (Bureau of Drug 
Control),  
Food and Drug Administration 
Ministry of Public Health 
88/24 Tiwanond Road, Nonthaburi 
11000, Thailand  
Telephone: +66 2 590 7061 
 
USAMRDC Office of Human 
and Animal Research Oversight 
Office of Human Research Oversight (OHRO) 
Office of Human and Animal Research Oversight (OHARO) 
US Army Medical Research and Development Command,  
ATTN: FCMR-RPH 
504 Scott Street 
Fort Detrick, MD 21702-5012 
Fax: +1 301 619 7803 
Telephone: +1 301 619 2165 
Email: usarmy.detrick.medcom-usamrmc.other.hrpo@health.mil 
 
 
FOR OFFICIAL USE ONLY 
Information and data included in this document contain privileged and/or proprietary information, which is the 
property of the United States Army, the Royal Thai Army, Mahidol University Faculty of Tropical Medicine, 
Chulalongkorn University, Institute of Human Virology, Duke University, the US Army Medical Materiel 
Development Activity, the US National Institutes of Public Health and the Henry M. Jackson Foundation, and may 
not be reproduced, published or disclosed to others without written authorization. No person is authorized to make it 
public without express written permission of the United States Army. These restrictions on disclosure will apply 
equally to all future information, which is indicated as privileged or proprietary. 

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PRINCIPAL INVESTIGATOR’S AGREEMENT 
1. I agree to follow this protocol version as approved by the IRBs/ERCs.  
2. I will conduct the study in accordance with applicable IRB/ERC requirements, Federal 
regulations, and state and local laws to maintain the protection of the rights and welfare of 
study participants.  
3. I certify that I, and the study staff, have received the requisite training to conduct this 
research protocol.  
4. I will not modify the protocol without first obtaining an IRB/ERC approved amendment and 
new protocol version unless it is necessary to protect the health and welfare of study 
participants.  
5. I have read and understand the information in the Investigators’ Brochure (or Manufacturer’s 
Brochure) regarding the risks and potential benefits. I agree to conduct the protocol in 
accordance with Good Clinical Practices (ICH-GCP), the applicable ethical principles, the 
Statement of Investigator (Form FDA 1572), and with local regulatory requirements. In 
accordance with the U.S. FDA Modernization Act and NIH policy, I will ensure the 
registration and reporting results of the trial on the www.clinicaltrials.gov website.  
6. In accordance with WRAIR Command Policy 2008-35, I will ensure that the Commanding 
General receives a pre-brief (or Executive Summary) and approves the study prior to 
execution.  
7. I will ensure that the data (and/or specimens) are maintained in accordance with the data 
(and/or specimen) disposition outlined in the protocol. Any modifications to this plan should 
first be reviewed and approved by the applicable IRBs/ERCs.  
8. I will promptly report changes to the research or unanticipated problems to the WRAIR IRB 
immediately via the WRAIR Human Subjects Protection Branch at (301) 319-9940 (during 
duty hours) or to usarmy.detrick.medcom-wrair.mbx.hspb@health.mil and submit a written 
report within 10 working days of knowledge of the event. 
9. I will prepare continuing review reports at an interval established by the IRB/ERC, and a 
study closure report when all research activities are completed.  
10. I will immediately report to the WRAIR Human Subjects Protection Branch knowledge of 
any pending compliance inspection by any outside governmental agency.  
11. I agree to maintain adequate and accurate records in accordance with IRB policies, Federal, 
state and local laws and regulations.  
 
 
 
 
 
Punnee Pitisuttithum, MD, DTM&H 
Date 
Mahidol University Site Principal Investigator 
 
 
 
 
 
LTG Sorachai Nitayaphan, MD, PhD 
Date 
RTA-AFRIMS Site Principal Investigator 

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EMERGENCY CONTACT INFORMATION 
Table 1: 
Emergency Contact Information 
Role in Study 
Name 
Address and Telephone Number 
Mahidol Principal Investigator 
and 
Main contact for Thailand 
Punnee Pitisuttithum, MD 
DTM&H 
Vaccine Trial Centre, 
Faculty of Tropical Medicine,  
Mahidol University 
Telephone: +66 8 1829 4906;  
+66 2 354 9173; +66 2643 5599; 
+66 8 1700 1890 (24 hours) 
Email: punnee.pit@mahidol.ac.th 
RTA-AFRIMS Principal 
Investigator 
LTG Sorachai Nitayaphan, MD, 
PhD 
Clinical Research Center,  
AFRIMS 
Telephone: +66 8 1625 1531;  
+66 2 354 7586;  
+66 9 0214 3119 (24 hours) 
Email: sorachain.rta@afrims.org 
Associate Investigators 
Nittaya Phanuphak, MD, PhD 
Telephone: +66 2 253 0966; +66 2 
160 5372, +66 8 1825 3544; +66 8 
3010 8544 (24 hours) 
Email: nittaya.p@ihri.org 
Benjaluck Phonrat, MS 
Telephone: +66 2 643 5599 
Email: benjaluck.pho@mahidol.ac.th 
Jittima Dhitavat, MD 
Telephone: +66 2 643 5599 
Email: jittima_pu@yahoo.com 
Arom Pitisuthitham, MD 
Telephone: +66 2 643 5599 
E-mail: docarom@yahoo.com 
Sant Muangnoicharoen, MD 
Telephone: +66 8 5199 8989 
E-mail: Sant.mua@mahidol.ac.th 
Supitcha Kamolratanakul, MD 
Telephone: +66 8 1699 6660 
E-mail: Supitcha.kam@mahidol.edu 
MG Narongrid Sirisopana, MD 
Telephone: +66 8 1908 6888 
Email: narongrid1956@yahoo.com 
COL Khunakorn Kana, MD 
Telephone: +66 86 034 2527 
Email: k_khunakorn@hotmail.com 
COL Wuttikon Rodkvamtook, 
PhD 
Telephone: +66 8 1584 9546 
Email: Itwutti@hotmail.com 
MAJ Worapong Nasomsong 
Telephone: +66 2 644 4420,  
+66 8 4211 1220 
Email: nasomsong.w@gmail.com 

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Role in Study 
Name 
Address and Telephone Number 
U.S. Army Medical Research 
and Development Command 
(MRDC), Office of Regulated 
Activities (ORA), Director  
MAJ Jonathan D. Stallings, 
PhD 
USAMRDC ORA 
1430 Veterans Drive 
Ft. Detrick, MD 21702-5009 
Telephone: +1 301 619 1087 
Fax: +1 301 619 7662 
Sponsor’s Representative 
Emily D. Badraslioglu, MS, 
PMP, RAC 
USAMRDC ORA 
1430 Veterans Drive 
Ft. Detrick, MD 21702-5009 
Telephone: +1 301 619 0317 
Fax: +1 301 619 0197 
Email: usarmy.detrick.medcom-
usamrmc.mbx.usamrmc-regulatory-
affairs@health.mil  
Institutional Review Boards 
MoPH  
FWA #00001953 
RTA 
 
FWA #00001813 
FTM 
 
FWA #00002882 
Chula  
FWA #00000943 
WRAIR 
FWA #00000015 
See page 7 
Local Drug and Vaccine 
Authorities 
MoPH TSHAVD 
Thai FDA 
See page 8 
 

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2. 
SYNOPSIS 
Name of Sponsor: 
The Surgeon General, Department of the Army 
Name of Investigational Products: 
IHV01 (FLSC), A244 (with aluminum hydroxide fluid gel adjuvant), ALFQ Adjuvant, and 
Placebo 
Name of Active Ingredient: 
IHV01 consists of the Full-Length Single Chain (FLSC) gp120-CD4 chimera subunit HIV-1 vaccine 
formulated in aluminum phosphate adjuvant. It is encoded by a synthetic gene, which contains a 
human codon-optimized HIV (BaL) gp120 sequence followed by human CD4D1D2, with a flexible 
20-amino acid linker. 
 
A244 consists of the gp120 envelope glycoprotein HIV-1 subtype CRF_01AE A244 derived from the 
CM244 CRF_01AE. The A244 gp120 envelope has an 11 amino N-terminal deletion, similar to the 
A244 protein used in AIDSVAX B/E. The aluminum hydroxide fluid gel (AHFG) adjuvant that is 
mixed with A244 consists of Rehydragel HPA that is diluted with sterile water for injection to a 
concentration of 5 ± 1mg/mL. 
 
ALFQ (Army Liposomal Formulation) is a liposomal adjuvant containing a synthetic 
monophosphoryl lipid A (MPLA) with the addition of QS-21. 
 
Normal Saline (0.9% Sodium Chloride for Injection) will be used as a placebo. 
Title of Study:  
Randomized, Double Blind Evaluation of Late Boost Strategies with IHV01 (FLSC in aluminum 
phosphate) and A244 with or without ALFQ for HIV-uninfected Participants in the HIV Vaccine 
Trial RV306 / WRAIR 1920 
Study Center(s):  
Vaccine Trial Centre, Faculty of Tropical Medicine, Mahidol University; Royal Thai Army Clinical 
Research Center, Armed Forces Research Institute of Medical Sciences; Thai Red Cross AIDS 
Research Centre; Institute of HIV Research and Innovation Foundation; and the Chulalongkorn 
Memorial Hospital.  
Principal Investigators:  
Mahidol University: Punnee Pitisuttithum, MD, DTM&H 
RTA-AFRIMS: LTG Sorachai Nitayaphan, MD, PhD 
 
Associate Investigators: 
Nittaya Phanuphak, MD, PhD 
Benjaluck Phonrat, MS 
Jittima Dhitavat, MD 
Arom Pitisuthitham, MD 
Sant Muangnoicharoen, MD 
Supitcha Kamolratanakul, MD 
MG Narongrid Sirisopana, MD 

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COL Khunakorn Kana, MD 
COL Wuttikon Rodkvamtook, PhD 
MAJ Worapong Nasomsong, MD 
COL Jessica Cowden, MD, MSPH 
 
Laboratory Investigators:  
Siriwat Akapirat, MT, PhD 
Alexandra Schuetz, PhD 
Shelly Krebs, PhD 
Study Period (years):  
Estimated date first participant enrolled: June 2021 
Estimated date last participant completed: December 2023 
Estimated duration of the trial: 5 years (2-3 years screening 
and clinical activities: 2-3 years laboratory assays and data 
analysis) 
Phase of development: I 
 
Objectives:  
Primary: 
• To evaluate the safety of 3 novel investigational products in a late vaccine boost setting: the 
candidate IHV01 and A244/AHFG vaccines and the ALFQ adjuvant 
Secondary: 
• To evaluate the effect of a full dose of the IHV01 and A244/AHFG vaccines and the ALFQ 
adjuvant on cellular, humoral, and innate immune responses (peripheral, lymphoid and mucosal).  
• To evaluate the effect of fractional dosing of the IHV01 and A244/AHFG vaccines on cellular, 
humoral, and innate immune responses (peripheral, lymphoid and mucosal) 
Methodology:  
This study is exploratory in nature. 
 
The purpose of this study is to define the safety and immunogenicity of IHV01 and A244/AHFG with 
and without ALFQ at a full dose and at a fractional dose (one-fifth of a full dose) in a late boost 
setting for participants who had previously received a late boost of AIDSVAX®B/E with or without 
ALVAC in RV306. Safety will be assessed through the frequency of the overall and specific post-
vaccination reactions. Blood, lymph nodes, sigmoid tissue, and mucosal specimens/secretions will be 
collected to assess humoral, cell-mediated, innate, and mucosal immune responses. 
 
Healthy, HIV-uninfected participants, at a low risk for HIV infection, available for 12 months, who 
were randomized to receive active vaccine in RV306 and completed all vaccinations will be enrolled. 
A total of 80 participants will be enrolled across four vaccination groups with 20 participants per 
group. Within each group, participants will be randomized to receive either IHV01 and A244/AHFG 
at a full or fractional dose with or without ALFQ or placebo at a targeted ratio of 4:1 vaccine to 
placebo. Participants will receive 2 intramuscular (IM) injections into the quadriceps muscle at Day 0. 
The same quadriceps muscle will be used for both injections. Participants randomized to receive the 
vaccines will have one injection of IHV01 and one injection of A244/AHFG at a full or fractional 
dose with or without ALFQ, whereas participants randomized to receive placebo will get 2 separate 
injections of Normal Saline. All placebo injection volumes will match the vaccine injection volumes 
for the group in which a participant has been randomized. Participants will be followed-up for up to 
12 months after enrollment. Mucosal secretion collections and endocervical cytobrush/swab 

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procedures will be performed at Days 0, 14, 168, and 336 on consenting participants. Leukapheresis, 
sigmoid biopsy, and lymph node biopsy procedures will be performed only at Day 14 on consenting 
participants. 
Estimated Number of Participants Screened and Enrolled:  
Approximately 110 prior RV306 participants will be screened to enroll 80 participants across both 
sites (anticipated screening to enrollment ratio of 1.375:1) 
Note: More than 80 participants may be enrolled to accommodate for participant replacement (see 
Section 8.6.3). 
Main Criteria for Inclusion/Exclusion:  
Inclusion Criteria 
1. Healthy, HIV-uninfected male and female participants 
2. Prior RV306 recipients who were randomized to receive active vaccine with late boosting at 
month 12, 15, or 18 and who completed all vaccinations 
3. Have a Thai identity card 
4. Must be at low risk for HIV infection per investigator assessment 
5. Must be able to understand and complete the informed consent process 
6. Must be capable of reading Thai 
7. Must successfully complete a Test of Understanding prior to enrollment 
8. Must be in good general health without clinically significant medical history 
9. HIV-uninfected per diagnostic algorithm within 45 days of enrollment 
10. Laboratory screening analysis: 
a. Hemoglobin: Women ≥11.0 g/dL, Men ≥11.5 g/dL 
b. White cell count: 4,000 to 11,000 cells/mm3 
c. Platelets: 150,000 to 450,000/mm3 
d. ALT and AST ≤1.25 institutional upper limit of reference range 
e. Creatinine: ≤1.25 institutional upper limit of reference range 
f. Urinalysis: blood and protein no greater than 1+ and negative glucose 
Note: Each laboratory screening test that is out of acceptable range can be repeated during the 
screening window to confirm the result. A second screening visit may be conducted outside of 
the initial screening visit window for individuals who meet certain criteria (see Section 8.3.1) 
only if study enrollment is ongoing. 
11. Female-Specific Criteria: 
a. Not currently pregnant or breastfeeding and not planning to become pregnant during the 
first 3 months after study vaccine/placebo injections 
b. Negative pregnancy test for women at screening, prior to vaccination (same day), and prior 
to any of the invasive procedures 
c. Be using an adequate birth control method for 45 days prior to receipt of vaccine/placebo 
and for at least 3 months after receipt of the vaccine/placebo. Adequate birth control is 
defined as follows: Contraceptive medications delivered orally, intramuscularly, vaginally, 
or implanted underneath the skin, surgical methods (hysterectomy or bilateral tubal 
ligation), condoms, diaphragms, intrauterine device, or abstinence 
12. Male-Specific Criteria: 
a. Be using an adequate birth control method for at least 3 months after receipt of the 
vaccine/placebo. For non-vasectomized male participants with female partners of child-
bearing potential this includes the use of condoms or abstinence and/or their partner’s use 
of contraceptive medications delivered orally, intramuscularly, vaginally, or implanted 
underneath the skin, surgical methods (hysterectomy or bilateral tubal ligation), 
diaphragms, or intrauterine device. 
 

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Exclusion Criteria 
1. Asplenia: any condition resulting in the absence of a functional spleen 
2. Bleeding disorder diagnosed by a medical doctor (e.g., factor deficiency, coagulopathy, or 
platelet disorder requiring special precautions) 
3. History of allergic reaction, anaphylaxis, or other serious adverse reaction to vaccines or 
components of the vaccines 
4. Volunteer has received any of the following substances: 
a. Chronic use of therapies that may modify immune response, such as IV immune globulin 
and systemic corticosteroids (in doses of >20 mg/day prednisone equivalent for periods 
exceeding 10 days) 
Note: The following exceptions are permitted and will not exclude study participation: use 
of corticosteroid nasal spray for rhinitis, topical corticosteroids for an acute uncomplicated 
dermatitis; or a short course (duration of 10 days or less, or a single injection) of 
corticosteroid for a non-chronic condition (based on investigator clinical judgment) at least 
14 days prior to enrollment in this study 
b. Blood products within 120 days prior to HIV screening 
c. Immunoglobulins within 30 days prior to HIV screening 
d. Any licensed vaccine within 14 days prior to study vaccine administration in the present 
study 
e. Receipt of any investigational HIV vaccine other than RV306 products 
f. Investigational research agents or vaccine within 30 days prior to enrollment in the present 
study 
g. Receipt of a Coronavirus disease 2019 (COVID-19) vaccine that has been given Emergency 
Use Authorization (or those that become licensed) by the Thai FDA within 14 days prior to 
study vaccine administration in the present study 
Note: Volunteers receiving a COVID-19 vaccine that requires 2 doses will not be enrolled 
until 14 days after the second dose has been administered 
h. Anti-tuberculosis prophylaxis or therapy during the past 90 days prior to enrollment 
Note: Participants determined to be ineligible at screening due to receipt of the above listed 
substances may be re-screened once the applicable window of receipt has expired. 
5. Active sexually transmitted infection confirmed by clinical exam and diagnostic test 
6. Any medical, psychiatric, social condition, occupational reason, or other responsibility that, in 
the judgment of the investigator, is a contradiction to protocol compliance or impairs a 
volunteer’s ability to give informed consent 
7. Psychiatric condition that precludes compliance with the protocol; past or present psychoses; 
past or present bipolar disorder; disorder requiring lithium; or within 5 years prior to enrollment, 
a history of suicide ideation or attempt 
8. Study site employees who are involved in the protocol and/or may have direct access to study 
related area 
 
Final evaluation of eligibility will be based on the medical judgment of the investigator based on 
his/her medical and research experience. 
Investigational Product Dosage, Schedule, and Mode of Administration:  
Participants will be randomized to receive either a full dose of IHV01 (approximately 300µg) and 
A244 (approximately 300µg) or a fractional dose of IHV01 (approximately 60µg) and A244 
(approximately 60µg). Participants in two of the four randomization groups will also receive ALFQ 
(approximately 0.5ml) as part of the A244 vaccination. Participants will receive 2 IM injections, 
either vaccine or placebo, into the same quadriceps muscle at Day 0 only. Enrollment into Groups 1 
and 2 will begin with no more than two participants per site per week for the first two weeks. Once 
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(SMC) will review 7-days of safety data from these participants to determine whether enrollment can 
continue. Enrollment into Groups 1 and 2 will continue unrestricted after any safety concerns raised 
by the SMC are addressed and the Sponsor gives their approval for the continuation of enrollment. 
Enrollment into Groups 3 and 4 will commence after enrollment into Groups 1 and 2 has been 
completed, the SMC has reviewed 7-days of safety data from all Group 1 and Group 2 participants, 
and the Sponsor gives their approval. Enrollment into Groups 3 and 4 will follow a similar approach 
with no more than two participants per site per week for the first two weeks, followed by an SMC 
review of 7-days of safety data from the first eight participants enrolled across both Groups, and 
Sponsor approval before enrollment can continue unrestricted in Groups 3 and 4. 
 
For participants randomized to receive full dose IHV01 and A244 in Group 1, the A244 product will 
be prepared by mixing AHFG with A244 and Normal Saline. Using a sterile syringe, 1.2mL of the 
mixture will be withdrawn for IM injection. No mixing is necessary to prepare the IHV01 product. 
Using a sterile syringe, 1.0mL IHV01 will be withdrawn from the vial for IM injection. 
 
For participants randomized to receive fractional dose IHV01 and A244 in Group 2, the A244 product 
will be prepared by mixing Normal Saline, AHFG, and A244. Using a sterile syringe, 1.2mL of the 
resulting product will be withdrawn for IM injection. The IHV01 product will be prepared by mixing 
Normal Saline and IHV01. Using a sterile syringe, 1.0mL of the mixture will be withdrawn for IM 
injection. 
 
For participants randomized to receive full dose IHV01 and A244 + ALFQ in Group 3, the A244 + 
ALFQ product will be prepared by mixing AHFG and A244 with ALFQ. After mixing, 1.2mL will be 
withdrawn into a sterile syringe for IM injection. No mixing is necessary to prepare the IHV01 
product. Using a sterile syringe, 1.0mL IHV01 will be withdrawn from the vial for IM injection. 
 
For participants randomized to receive fractional dose IHV01 and A244 + ALFQ in Group 4, the 
A244 + ALFQ product will be prepared by mixing Normal Saline, AHFG, and A244 with ALFQ. 
After mixing, 1.2mL will be withdrawn into a sterile syringe for IM injection. The IHV01 product 
will be prepared by mixing Normal Saline and IHV01. Using a sterile syringe, 1.0mL of the mixture 
will be withdrawn for IM injection. 
 
Normal Saline (0.9% Sodium Chloride for Injection) will serve as a placebo for the trial. All placebo 
injection volumes will match the vaccine injection volumes for the group in which a participant has 
been randomized. 
Duration of Treatment:  
Participants will receive 2 IM injections of either the investigational products or placebo at Day 0, 
after which they will be followed for 12 months for safety and immunogenicity analyses. 
Criteria for Evaluation: 
Immunogenicity: 
Humoral responses will be assessed by HIV-specific binding antibody assays, HIV-specific 
neutralizing antibody assays, and non-neutralizing antibody function assays at Days 0, 14, 168, and 
336. Humoral mucosal immune responses in the rectal, penile, semen and cervico-vaginal 
compartments will be assessed using non-invasive sampling methods (sponge, menstrual disc, and 
masturbation) at the same time points. Genital secretion samples will be assayed for cytokines and 
inflammatory markers and be used to assess the proteome/microbiome. 
 

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HIV-specific cell-mediated responses will be assessed by intracellular cytokine staining, CD4 and 
CD8 T cell proliferation, and related assays. Innate immune responses and genetic responses will be 
quantified. In addition, invasive sigmoid and lymph node biopsies performed on a subset of willing 
participants at Day 14 will be utilized. Cells isolated from sigmoid biopsies and endocervical 
cytobrush/swab collection will be used to assess vaccine-specific immune responses and the dynamics 
of key mucosal cell populations and innate responses. 
 
Inguinal lymph node biopsies will be performed on a subset of willing participants at Day 14 to assess 
the effect of ALFQ on immune activation and HIV-specific immune responses in lymph nodes. 
Analysis will be done to determine whether ALFQ improves immune responses in the lymphoid 
follicles and stimulates the generation of T follicular helper cells, which may play a critical role in the 
generation of broadly neutralizing antibodies through interactions with germinal center B cells in the 
lymph nodes 
 
Leukapheresis will be performed on a subset of willing participants to allow mapping of innate and 
HIV-specific functional responses within a given antigen, where in excess of 30 million cells are 
usually required for each antigen.  
 
Safety: 
Safety will be assessed both by direct physical examination and by diary cards, which serve as 
memory tools for better identification of reactions. Participants will be assessed at pre-vaccination 
baseline and will remain in clinic under direct observation for at least 30 minutes post vaccination. 
Participants will return to clinic on Days 1 and 7 post-vaccination to be assessed for symptoms of 
local and systemic reactogenicity.  
 
Participants will be followed for a total of 12 months post vaccination. Adverse Events (AEs) and 
Serious Adverse Events (SAEs) will be recorded at all visits along with timing and possible 
attribution to Investigational Product. Because this clinical trial involves an adjuvant, Adverse Events 
of Special Interest (AESIs) will also be assessed. Safety laboratory analyses of complete blood count, 
liver function tests, urinalysis, and pregnancy test in females will also be performed. Safety data for 
the ALFQ product will be used to supplement safety data produced from other studies in support of 
its use with experimental COVID-19 vaccine candidates.  
Statistical Methods: 
This study intends to descriptively characterize safety and immune responses following delayed 
boosting of individuals previously receiving a late boost of AIDSVAX®B/E with or without ALVAC 
in the RV306 protocol. Comparisons of interest include full dose vs. fractional dose (Groups 1+3 vs. 
Groups 2+4) and effect of ALFQ (Groups 1+2 vs. Groups 3+4). For safety analyses, the occurrence of 
local and systemic reactogenicity symptoms, AEs and SAEs will be assessed as the proportions of 
participants experiencing such safety events. The primary immunogenicity endpoint is the magnitude 
of immune response to the endpoint titer of plasma IgG, the same variable assessed in RV306. 
Immune response over time (durability) will be assessed by the positive incremental area under the 
curve (AUC) based on a graph with log endpoint titer on the y-axis and visit day on the x-axis. Safety 
and immunogenicity analyses will also be sub-stratified by gender to explore gender-specific 
differences in immune responses. 
 

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3. 
TABLE OF CONTENTS, LIST OF TABLES, AND LIST OF 
FIGURES  
TABLE OF CONTENTS 
1. TITLE PAGE ..........................................................................................................................1 
2. SYNOPSIS .............................................................................................................................12 
3. TABLE OF CONTENTS, LIST OF TABLES, AND LIST OF FIGURES .....................18 
4. LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS ....................................23 
5. INTRODUCTION .................................................................................................................25 
5.1. Military Relevance ...................................................................................................27 
5.2. Thailand Relevance ..................................................................................................28 
5.3. Rationale for Study ...................................................................................................29 
5.4. Name and Description of the Investigational Product .............................................29 
5.5. Summary of Nonclinical and Clinical Trials ............................................................29 
5.5.1. IHV01 (FLSC) Vaccine ..................................................................................29 
5.5.2. A244 Vaccine .................................................................................................30 
5.5.3. ALFQ Adjuvant ..............................................................................................31 
5.6. Known and Potential Risks and Benefits to Human Participants ............................33 
5.6.1. Risks/Discomfort to Participants and Precautions to Minimize Risk .............33 
5.6.2. Alternatives to this IND Product or Study ......................................................36 
5.6.3. Potential Benefit for Participants ....................................................................36 
5.6.4. Risks to the Study Personnel and the Environment ........................................37 
5.7. Route of Administration, Dosage Regimen, Treatment Period, and Justification ...37 
5.8. Compliance Statement ..............................................................................................37 
5.9. Study Population ......................................................................................................38 
5.10. Study Site .................................................................................................................38 
6. TRIAL OBJECTIVES AND PURPOSE ............................................................................40 
6.1. Primary Objective ....................................................................................................40 
6.2. Secondary Objectives ...............................................................................................40 
7. TRIAL DESIGN ....................................................................................................................41 
7.1. Study Endpoints .......................................................................................................41 
7.1.1. Safety Endpoints .............................................................................................41 
7.1.2. Immunogenicity Study Endpoints ..................................................................41 
7.2. Overall Study Design ...............................................................................................42 
7.3. Measures Taken to Minimize/Avoid Bias ................................................................47 
7.3.1. Randomization ................................................................................................47 
7.3.2. Blinding ..........................................................................................................47 
7.3.3. Unblinding ......................................................................................................48 
7.4. Investigational Product .............................................................................................48 
7.4.1. Investigational Product Packaging and Labeling ...........................................49 
7.4.2. Investigational Product Storage ......................................................................49 
7.4.3. Investigational Product Preparation ................................................................49 
7.4.4. Investigational Product Accountability ..........................................................50 

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7.5. Duration of Participation ..........................................................................................50 
7.6. Dose-adjustment Criteria ..........................................................................................50 
7.6.1. Safety Criteria for Stopping Doses .................................................................50 
7.6.2. Pharmacokinetic Criteria for Dose Adjustment or Stopping Doses ...............51 
7.6.3. Study Termination ..........................................................................................51 
7.7. Trial Treatment Randomization Codes ....................................................................51 
7.8. Identification of Data to be Recorded on the Case Report Forms (CRFs) ...............51 
8. SELECTION AND WITHDRAWAL OF PARTICIPANTS ............................................52 
8.1. Recruitment of Participants ......................................................................................52 
8.2. Informed Consent Process ........................................................................................52 
8.3. Eligibility Screening (Visit S1) ................................................................................52 
8.3.1. Participant Re-screening .................................................................................53 
8.4. Inclusion Criteria ......................................................................................................54 
8.5. Exclusion Criteria .....................................................................................................55 
8.6. Early Discontinuation or Withdrawal of Study Participants ....................................56 
8.6.1. Investigator Withdrawal of Participants .........................................................56 
8.6.2. Data Collected for Withdrawn Participants ....................................................56 
8.6.3. Replacement of Withdrawn Participants ........................................................56 
8.6.4. Follow-up for Withdrawn Participants ...........................................................57 
9. STUDY PROCEDURES .......................................................................................................58 
9.1. Vaccination and Follow-up Visits ............................................................................58 
9.1.1. Day 0 Through Day 336 .................................................................................58 
9.1.2. Post-Database Lock ........................................................................................59 
9.1.3. Mucosal Secretion Collection .........................................................................59 
9.1.4. Sigmoid Biopsy ..............................................................................................60 
9.1.5. Leukapheresis .................................................................................................61 
9.1.6. Inguinal Lymph Node Biopsy ........................................................................61 
9.1.7. Future/Genetic Use of Study Samples ............................................................62 
9.1.8. HIV Risk Assessment and Risk Counseling ...................................................62 
9.2. Concomitant Medications ........................................................................................63 
9.3. Management of Participants Who Become Pregnant ...............................................63 
9.4. Management of Participants Who Become Incarcerated .........................................63 
9.5. Management of Clinical Test Results and Participant Referrals ..............................63 
9.6. Procedures for Monitoring Participant Compliance .................................................64 
10. IMMUNOGENICITY ASSESSMENTS .............................................................................65 
10.1. Specification of Immunogenicity Endpoints ............................................................65 
10.2. Immunogenicity Endpoints ......................................................................................66 
10.2.1. Humoral Immune Responses ..........................................................................66 
10.2.2. Cellular Immune Responses ...........................................................................67 
10.2.3. Mucosal Immune Responses ..........................................................................67 
10.2.4. Innate Immunity ..............................................................................................67 
10.3. QA/QC for Immunogenicity Assays ........................................................................68 
10.4. Specimen Archiving and Transfer ............................................................................68 
11. PHARMACOVIGILANCE, SAFETY AND ADVERSE EVENT REPORTING ..........70 
11.1. Specification of Safety Endpoints ............................................................................70 

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11.1.1. Demographic/Medical History .......................................................................70 
11.1.2. Vital Signs ......................................................................................................70 
11.1.3. Physical Examination .....................................................................................70 
11.1.4. Laboratory Assessments .................................................................................70 
11.2. Adverse Event Reporting .........................................................................................73 
11.2.1. Adverse Event and Suspected Adverse Reaction ...........................................73 
11.2.2. Solicited Adverse Event .................................................................................74 
11.2.3. Serious Adverse Event or Serious Suspected Adverse Reaction ....................74 
11.2.4. Unexpected Adverse Event or Unexpected Suspected Adverse Reaction .....74 
11.2.5. Other Adverse Events .....................................................................................75 
11.3. Relationship to Investigational Product ...................................................................75 
11.3.1. Severity Assessment .......................................................................................75 
11.4. Independent Safety Monitor .....................................................................................76 
11.5. Protocol Safety Review Team ..................................................................................76 
11.6. Safety Monitoring Committee Reviews ...................................................................77 
11.7. Recording Adverse Events .......................................................................................77 
11.7.1. Methods/Timing for Assessing, Recording, and Analyzing Safety 
Endpoints ........................................................................................................77 
11.7.2. Duration of Follow-Up of Participants After Adverse Events .......................78 
11.8. Reporting Adverse Events ........................................................................................78 
11.8.1. Reporting Serious and Unexpected Adverse Events ......................................78 
11.8.2. Additional Immediately Reportable Events ....................................................81 
11.8.3. IND Annual Report to the U.S. FDA .............................................................83 
11.8.4. Final Report ....................................................................................................83 
12. STATISTICS .........................................................................................................................84 
12.1. Analysis Overview ...................................................................................................84 
12.2. Final Analysis Plan ...................................................................................................84 
12.2.1. Analysis Populations ......................................................................................84 
12.2.2. Demographics and Baseline Characteristics ...................................................84 
12.2.3. Safety Analysis ...............................................................................................85 
12.2.4. Immunogenicity Analysis ...............................................................................85 
12.2.5. Missing Values and Outliers ...........................................................................85 
12.2.6. Subgroup Analysis ..........................................................................................85 
12.2.7. Clinical Laboratory Data Analyses .................................................................86 
12.3. Planned Enrollment and Sample Size Consideration ...............................................86 
12.4. Level of Significance to be Used .............................................................................87 
12.5. Interim Analyses ......................................................................................................87 
12.6. Statistical Criteria for the Termination of the Trial ..................................................87 
12.7. Procedures for Reporting Deviations from the Statistical Plan ................................87 
12.8. Accounting for Missing, Unused, and Spurious Data ..............................................87 
13. DIRECT ACCESS TO SOURCE DATA/DOCUMENTS .................................................88 
13.1. Study Monitoring .....................................................................................................88 
13.2. Audits and Inspections .............................................................................................88 
13.3. Institutional Review Board .......................................................................................88 
14. QUALITY CONTROL AND QUALITY ASSURANCE ..................................................90 

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15. ETHICS ..................................................................................................................................91 
15.1. Ethics Review ...........................................................................................................91 
15.1.1. Review/Approval of Study Protocol ...............................................................91 
15.1.2. Protocol Modifications ...................................................................................91 
15.1.3. Protocol Deviation Procedures .......................................................................92 
15.1.4. Continuing Review and Closeout Reports ......................................................92 
15.2. Ethical Conduct of the Study ...................................................................................93 
15.2.1. Confidentiality ................................................................................................93 
15.2.2. Compensation for Participation ......................................................................94 
15.2.3. Medical Care for Research-Related Injury .....................................................94 
15.3. Written Informed Consent ........................................................................................94 
16. DATA HANDLING AND RECORDKEEPING ................................................................96 
16.1. Inspection of Records ...............................................................................................96 
16.1.1. Retention of Records ......................................................................................96 
17. PUBLICATION POLICY ....................................................................................................99 
18. LIST OF REFERENCES ...................................................................................................100 
19. APPENDICES .....................................................................................................................104 
Appendix A. Study Personnel Roles and Responsibilities ............................................104 
Appendix B. Division of AIDS (DAIDS) Table for Grading the Severity of Adult 
and Pediatric Adverse Events ..............................................................................106 
Appendix C. Main Informed Consent Form .................................................................107 
Appendix D. Optional Procedures Informed Consent Forms .......................................108 
Appendix E. Storage and Future Use Informed Consent Form ....................................109 
Appendix F. Withdrawal of Consent for Sample Storage Form ...................................110 
Appendix G. Test of Understanding and Answer Key ..................................................111 
Appendix H. Adverse Events of Special Interest (AESI) List ......................................112 
Appendix I. Gastrointestinal Questionnaires for Microbiome Analysis .......................113 
Appendix J. Female Genital Tract (FGT) Questionnaire (for female participants) ......114 
Appendix K. Dietary Questionnaires for Microbiome Analysis ...................................115 
Appendix L. External Collaborator List ........................................................................116 
Appendix M. Unblinding Letter ....................................................................................117 
Appendix N. Vaccine-Induced Seroreactivity Letter ....................................................118 
Appendix O. Diary Card ...............................................................................................119 
Appendix P. Volunteer Visit Schedule .........................................................................120 
Appendix Q. Appointment/Emergency Contact Card...................................................121 
Appendix R. Briefing Slides .........................................................................................122 
Appendix S. Volunteer Registry Data Sheet .................................................................123 
Appendix T. Menstrual Disc Self-Insertion Instructions ..............................................124 
Appendix U. Penile Swab Self-Collection Instructions ................................................125 
Appendix V. Informed Consent Form for Genetic Testing ..........................................126 
 

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LIST OF TABLES 
Table 1: 
Emergency Contact Information .................................................................................10 
Table 2: 
Abbreviations ..............................................................................................................23 
Table 3: 
Schedule of Evaluations .............................................................................................44 
Table 4: 
Immunology Assays ...................................................................................................65 
Table 5: 
Study Contacts for Reporting Serious and Unexpected Adverse Events ...................78 
Table 6: 
Sponsor’s AE Reporting Requirements ......................................................................80 
Table 7: 
Exact 95% Clopper-Pearson CI for the AE Rate in Each Study Group .....................86 
Table 8: 
Minimal Detectable Differences, in Standard Deviations, for a 2-Sample 
T-Test (2 sided, α=0.05) .............................................................................................87 
Table 9: 
Compensation for Participation ..................................................................................94 
 
LIST OF FIGURES 
Figure 1: 
Study Group Design ...................................................................................................43 
Figure 2: 
HIV Testing Algorithm ...............................................................................................72 
 

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4. 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
The following abbreviations are used in this study protocol. 
Table 2: 
Abbreviations  
Abbreviation 
Explanation 
ADCC 
Antibody-dependent Cellular Cytotoxicity 
ADCP 
Antibody-dependent Cellular Phagocytosis 
AE 
Adverse Event 
AESI 
Adverse Event of Special Interest 
AFRIMS 
Armed Forces Research Institute of Medical Sciences 
AHFG 
Aluminum Hydroxide Fluid Gel 
ALF 
Army Liposomal Formulation 
AR 
Army Regulation 
AUC 
Area Under the Curve 
C 
Celsius 
CFR 
Code of Federal Regulation 
cm 
Centimeter 
COVID-19 
Coronavirus Disease 2019 
CRF 
Case Report Form 
CRR 
Continuing Review Report 
DA 
Department of the Army 
DCAC 
Data Coordinating and Analysis Center 
DoD 
Department of Defense 
EC 
Ethical Committee 
F 
Fahrenheit 
FDA 
Food and Drug Administration 
FLSC 
Full Length Single Chain 
FGT 
Female Genital Tract 
FTM 
Faculty of Tropical Medicine 
GCP 
Good Clinical Practice 
GP 
Glycoprotein 
Hg 
Mercury 
HIPAA 
Health Insurance Portability Accountability Act 
HIV 
Human Immunodeficiency Virus 
HJF 
Henry M. Jackson Foundation 
IB 
Investigator’s Brochure 
ICH 
International Conference on Harmonization 
ICS 
Intra-cellular Cytokine Staining 
ID 
Infectious Disease 
IEC 
Independent Ethics Committee 
IHV 
Institute of Human Virology, University of Maryland School of Medicine 
IM 
Intramuscular 
IND 
Investigational New Drug 
IRB 
Institutional Review Board 
ISCOM 
Immune Stimulating Complex 
mg 
Milligram 
MHRP 
US Military HIV Research Program 

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Abbreviation 
Explanation 
mL 
Milliliter 
mm 
Millimeter 
MOP 
Manual of Procedures 
MoPH 
Ministry of Public Health 
MPLA 
Monophosphoryl Lipid A 
NAAT 
Nucleic Acid Testing 
OHRP 
Office for Human Research Protections, Department of Health and Human 
Services 
ORA 
Office of Regulated Activities, USAMRDC 
OHARO OHRO 
Office of Human and Animal Research Oversight, Office of Human Research 
Oversight 
PI 
Principal investigator 
The investigator who leads the study conduct at an individual study center. 
Every study center has a principal investigator. 
PSRT 
Protocol Safety Review Team 
PSSO 
Product Safety Surveillance Office, USAMRDC ORA 
PVG 
Pharmacovigilance 
RTA 
Royal Thai Army 
SAE 
Serious adverse event 
SAP 
Statistical analysis plan 
SMC 
Safety Monitoring Committee 
SOE 
Schedule of Evaluations 
SOP 
Standard operating procedure 
STI 
Sexually Transmitted Infection 
TOU 
Test of Understanding 
TSG-DA 
The Surgeon General, Department of the Army 
TSHAVD 
Technical Subcommittee on HIV/AIDS Vaccine Development, MoPH 
UPIRTSO 
Unanticipated Problem Involving Risk to Subjects or Others 
USAMC 
US Army Medical Component 
USAMMDA 
United States Army Medical Materiel Development Activity 
USAMRDC 
United States Army Medical Research and Development Command 
VTC 
Vaccine Trials Center 
WRAIR 
Walter Reed Army Institute of Research 
 

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5. 
INTRODUCTION 
In 2019, an estimated 38 million people [31.6–44.5] were living with HIV worldwide. This 
reflects the continued large number of new HIV infections and a significant expansion of access 
to antiretroviral therapy, which has helped reduce AIDS-related deaths, especially in more recent 
years (UNAIDS, 2020). Despite promising but still fragile successes in prevention and care and 
treatment, the development of a safe and efficacious preventive HIV vaccine as part of a 
comprehensive prevention program remains among the highest global health priorities and the 
best long-term tool for the control of the HIV-1 (Barouch, 2008; Johnston, 2008; Koff, 2010). 
In response to the epidemic, the Royal Thai Government developed and implemented a 
comprehensive plan for prevention and control of HIV; preventive HIV vaccines are an integral 
component of this plan (National AIDS Committee, 1997). Consequently, the National AIDS 
Commission of Thailand established a Subcommittee for HIV Vaccine Development and Trials 
with responsibility for coordinating and overseeing efforts in this area. In 1991, the World Health 
Organization (WHO) selected Thailand as a site for evaluation of candidate HIV vaccines 
(Esparza, 1993). Since then, WHO/UNAIDS has provided consultation to the Subcommittee for 
HIV Vaccine Development and Trials of the National Commission for the Prevention and 
Control of AIDS. Through multiple partnerships and collaborations, Thailand has actively 
carried out its National Plan and implemented several vaccine clinical trials including the first 
Phase III trial in the developing world (Pitisuttithum, 2010). The combination of planning, 
collaboration and commitment to HIV vaccine development has put Thailand in a position of 
international leadership concerning HIV vaccine development.  
To date, 5 efficacy trials of candidate HIV vaccines have been completed: VAX003 
(Pitisuttithum, 2006), VAX004 (Flynn, 2005), RV144 (Rerks-Ngarm, 2009), STEP (Buchbinder, 
2008), and HVTN505 (Hammer, 2013). Of these, only the RV144 phase III trial demonstrated 
any efficacy. Healthy participants who received the 6-month vaccination regimen containing 
ALVAC-HIV (vCP1521) (ALVAC) and AIDSVAX® B/E, formulated in aluminum hydroxide, 
were 31% less likely to become HIV infected over 3.5 years of follow-up than participants who 
received placebo. In a post-hoc analysis, efficacy was 60% at 12 months, indicating that 
protective immunity may have waned rapidly (Robb, 2012). A study of immunologic correlates 
found that the magnitude of plasma IgG antibodies to the scaffolded variable regions 1 and 2 
(V1V2) of HIV-1 envelope was inversely correlated with risk, while plasma IgA antibody titers 
to HIV-1 gp120 envelope were directly correlated with risk (Haynes, 2012a). 
To build upon the demonstrated efficacy in RV144, the RV305 clinical trial enrolled 
162 participants who received the active vaccine regimen in the RV144 trial 6 to 8 years earlier 
and boosted these individuals twice at months 0 and 6 with ALVAC and AIDSVAX® B/E, 
either alone or in combination, in a randomized, double-blinded placebo-controlled manner. 
Long-term memory responses to HIV-1 antigens persisted, allowing boosting of HIV-specific 
plasma antibody responses to levels higher than observed in the RV144 trial (Rerks-Ngarm, 
2017). Furthermore, late boosting expanded a subdominant pool of envelope CD4 binding site-
reactive memory B cells with increased somatic hypermutation, long third heavy chain 
complementarity determining regions (HCDR3), and Tier 2 neutralization capacity (Easterhoff, 
2017), all features of broadly neutralizing antibodies (Bonsignori, 2011; Walker, 2009; Walker, 
2011; Zhou, 2015). Mucosal sampling demonstrated that HIV-specific antibody responses were 
also present in cervicovaginal secretions, rectal secretions and seminal plasma (Akapirat, 2018), 

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the initial point of defense from sexually transmitted infections. Interestingly, immune responses 
to AIDSVAX® B/E were higher after the initial boost than after the second boost 6 months later, 
suggesting that a delayed interval between priming and boosting may generate stronger 
immunity to booster vaccinations. Thus, boosting with a protein-based candidate HIV vaccine 
after a delay of several years can boost memory and elicit high magnitude immune responses in 
multiple compartments. 
To further examine the impact of the interval between priming and boosting, the RV306 Phase II 
clinical trial was conducted in 360 healthy, unvaccinated participants to determine whether 
similar improvements in quality, magnitude or duration of humoral, cellular and mucosal 
responses could be afforded by boosting the RV144 regimen at either 12, 15, or 18 months post 
initial vaccination series, and to determine the optimal boosting interval for further clinical 
development. As with the primary RV144 vaccination series, boosting the RV144 vaccine 
regimen was safe overall, as there were no vaccine-related serious adverse events. Vaccination at 
month 12 with AIDSVAX® B/E with or without ALVAC, or at Month 15 or 18 with both 
vaccines, significantly improved humoral and cellular immunogenicity relative to participants 
who did not receive a late boost to the RV144 vaccine regimen. Overall, boosting at Month 15 or 
18 resulted in stronger humoral and cellular responses than boosting at Month 12. Specifically, 
peak plasma IgG responses were higher in participants boosted at Month 15 or 18 than at Month 
12 for all antigens. In addition, late boosting increased the durability of plasma IgG responses, as 
the median decrease of plasma IgG was 32-fold over 6 months without late boosting, but only 
8-fold over 6 months in participants receiving late boost vaccinations. Longer boosting intervals 
also preferentially improved neutralization breadth. While vaccine boosts at any time point 
improved neutralization of the Tier 1 pseudoviruses MW965.26 (Subtype C); SF162.LS 
(Subtype B) and TH023.6 (Subtype AE), only boosting at Month 15 or Month 18 improved 
neutralization of subtype B MN.3. 
The A244 protein, a candidate vaccine that consists of the gp120 envelope glycoprotein HIV-1 
subtype CRF_01AE A244 with an 11 amino N-terminal deletion, has demonstrated its ability to 
improve the antigenicity and immunogenicity in humans as a component of AIDSVAX® B/E 
and alone in non-human primate (NHP) studies (Alam, 2013). It has been re-manufactured for 
use as an additional boost to RV306 late boost volunteers in this clinical trial. An increasing 
body of literature now supports the concept of serial boosting with heterologous proteins to guide 
the evolution of broadly neutralizing antibodies from germline to mature B cells towards 
epitopes that confer protection. Dr. Haynes and others at Duke University have shown that 
evolution of broadly neutralizing antibodies occurs in a staged manner, and can be induced by 
serial protein immunogens (Bonsignori, 2017; Tian, 2016).  
The FLSC candidate vaccine, which consists of the FLSC gp120-CD4 chimera subunit HIV-1 
vaccine formulated in Aluminum phosphate adjuvant (Alum), has been manufactured for human 
use as IHV01 and has been safely administered to healthy participants in a Phase I trial. IHV01 
would provide a heterologous boost to prior RV306 participants, as it provides exposure to HIV 
envelope epitopes at the CD4 binding site (DeVico, 2007), distinct from AIDSVAX® B/E. 
Manufacture of protein-based vaccine antigens can be technically complex and expensive, both 
of which can limit vaccine supply, particularly in times of high demand during an epidemic 
outbreak. Optimization to reduce vaccine dosing can help to alleviate these issues. In a controlled 
human malaria parasite infection model, reducing the dose of the third boost of the candidate 

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malaria vaccine RTS/S, AS-01 improved vaccine efficacy, accompanied by increased antibody 
somatic hypermutation and avidity, both characteristics of broadly neutralizing HIV antibodies 
(Regules, 2016). A separate study evaluating fractional dosing of inactivated polio administered 
intradermally demonstrated similar immunogenicity to full dose intramuscular vaccination 
(Resik, 2010). In a recent outbreak of yellow fever in Angola and the Democratic Republic of the 
Congo, vaccine shortage necessitated widespread deployment of fractional dosing of yellow 
fever vaccine at one fifth of the full dose to children and adults. Fractional dosing led to 
seroconversion in 98% of seronegative vaccine recipients, and immune responses in 66% of 
previously seroreactive vaccine recipients (Ahuka-Mundeke, 2018). Even a similar rather than 
superior immunogenicity of fractional dosing would result in significant cost savings, thereby 
facilitating scale-up and delivery to populations at greatest need for a preventive HIV vaccine. 
Relative to vaccine antigens from other pathogens, there is a particularly rapid decline of 
immune responses to HIV gp120 envelope proteins. Modulation of the response to vaccines with 
immunostimulatory adjuvants is one very promising approach to improve durability. The Army 
Liposome Formulation (ALF) family of adjuvants are ALFA (ALF adsorbed to aluminum salt), 
ALFQ (ALF mixed with the saponin QS21; US patent pending) (Beck, 2015), and ALFQA 
(ALFQ adsorbed to aluminum salt). Both ALFA and ALFQ are potent adjuvants, non-pyrogenic, 
and nontoxic in rabbit pyrogenicity and toxicology studies. ALFQ is an alternative to the 
successful but proprietary adjuvants, AS01B (liposomes containing both MPLA and the saponin 
QS21). ALFQ is potent in preclinical studies and contains 4 times the amount of MPLA and 
twice the amount of QS21 when compared to AS01B. An ongoing NHP study comparing HIV 
gp120 A244 formulated with either ALFQ or with aluminum hydroxide fluid gel (AHFG) has 
shown that animals that received ALFQ as the adjuvant compared to animals that received 
AHFG mounted significantly higher binding antibody responses to A244, gp70V1V2 A244, and 
V2 peptide (unpublished data). The responses were significantly higher after each immunization, 
as well as in ADCC responses, quantified using gp120 coated target cells. Given that antibody 
functions such as ADCC may have contributed to protection by non-neutralizing antibodies in 
RV144 vaccine recipients, ALFQ may contribute to increasing protective vaccine efficacy. In 
addition, data generated regarding the ALFQ adjuvant in this trial will help contribute to the 
safety database supporting its use with experimental antigens targeted towards other pathogens, 
including SARS-CoV-2. 
5.1. 
Military Relevance 
Historically, infectious diseases have had a major impact on US Armed Forces. With an 
estimated 34 million infected individuals worldwide at the end of 2011, HIV poses a significant 
and persistent threat in terms of force readiness and protection and may act as a war-starter by 
affecting the stability and security of nation-states. 
HIV’s relevance to the US Military has been recognized from the very beginning of the 
pandemic. In 1985, the US Military recognized the emerging HIV-1 epidemic as a new threat to 
US and allied forces worldwide. The United States Congress mandated the establishment of the 
US Military HIV Research Program to develop effective preventive measures to include 
prevention education, vaccine development and implementation of novel anti-viral therapies and 
clinical management tools for the US DoD and Allied Forces. In 2001, the Armed Forces 
Epidemiology Board identified HIV as a disease of military importance, and Army FOC 09-07, 

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Global Casualty Prevention, requires detection, identification, and vaccination in order to protect 
US personnel against potential infectious disease (ID) threats. The 2001 DoD Report on 
Biological Warfare Defense Vaccine Research and Development identified HIV as the 4th 
greatest infectious disease threat to DoD forces. Department of Army Headquarters designated 
HIV vaccine development as an Army Technology Objective (ATO), a status reserves for the 
highest priority science and technology efforts. Furthermore, The National Security Strategy of 
the United States (2002, 2006, and 2010) clearly identifies the threat of HIV/AIDS as a 
destabilizing force that threatens US National Security. Finally, in January 2011, The 
Department of the Army approved the HIV Vaccines Capability Development Document for 
HIV Vaccine, which addresses the need to provide service members with vaccine protection 
against HIV and AIDS.  
Medical care for HIV infection is extremely costly to the US Defense Health Program. The 
estimated lifetime cost of HIV infection is at least USD $400,000 and the estimated average 
yearly cost varies from $20,000 to $25,000, bringing the estimated lifetime healthcare cost of the 
5,000 HIV infected servicemen/women to $8B to $10B dollars. Total annual cost of treatment 
for HIV-infected service members ranges from $21 to $54 million per year.  
Infection rates could rise precipitously if forces are deployed to areas of high HIV prevalence. 
We developed a projection based on the US Army Surgeon General’s estimate of gonorrhea 
cases/year during the Vietnam War and determined the estimated number of visits to gonorrhea 
infected commercial sex workers necessary to generate that number. This equates to roughly one 
visit per service member per year during the height of the Vietnam War (this is widely viewed as 
an underestimate, since most cases of Sexually Transmitted Infections (STIs) were treated 
without being reported). Given the known infection rate of 1/300 and estimating commercial sex 
worker HIV prevalence at 30% (lower than currently estimated in Thailand, Vietnam or many 
African nations) this equates to 1,200 to 1,800 potential new HIV infections per year for a 
deployed force of 400,000. Developing a highly efficacious vaccine to protect the Warfighter 
thus remains of significant military relevance. 
5.2. 
Thailand Relevance 
Thailand has one of the highest rates of HIV in Asia and the Pacific, accounting for 9% of the 
region’s total population of people living with HIV. Although the epidemic is in decline, the 
prevalence remains high among key affected groups, with young people from key populations 
particularly at risk of acquiring HIV. In 2018, around half of new HIV infections in Thailand 
occurred among people aged 15-24. In 2019, 5,400 people in Thailand became HIV positive. 
Unprotected sex is estimated to account for 90% of all new HIV infections. The government has 
made important progress against HIV, but this epidemic will not end without a vaccine. The 
experimental vaccines that are in development present the greatest opportunity to save lives in 
human history as the work done in previous vaccine studies have helped to develop 
improvements in vaccine/adjuvant breadth and duration in order to protect more people for 
longer periods of time. Until the goal of a safe and effective vaccine is achieved, we must 
continue to use all available tools to help curb the HIV epidemic and accelerate progress towards 
that goal. Vaccines save lives today, and a new generation of vaccines can save even more lives 
tomorrow. There is still a lot of work to be done to achieve the goal of a safe and effective 
vaccine given the various HIV subtypes that are present across the world, however, there has 

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been a great deal of attention given to the HIV subtypes circulating in Thailand. In this study, 
A244 is subtype AE and is designed specifically to match circulating HIV subtypes in Thailand. 
If the vaccine is shown to improve immune responses in Thai people, the development of this 
vaccine will be monumentally beneficial to the Thai people. 
5.3. 
Rationale for Study 
A vaccine to prevent HIV infection remains of paramount importance that both to diminish the 
global epidemic (Medlock, 2017) and to reduce the burden to the DoD, given the cost of 
treatment for its HIV-infected service members ranges from $21 to $54 million per year. 
Similarly, improving the rapidity of our response to new emerging infectious diseases will be 
facilitated by development of a potent adjuvant that can be broadly coupled to various candidate 
vaccines, unencumbered by external intellectual property. RV546 addresses both of these goals 
by administering the IHV01 and A244/AHFG candidate vaccines with or without ALFQ in 
participants who have a “head start” on their vaccination series through participation in the prior 
RV306 trial, allowing for evaluation of safety and immunogenicity after a single vaccination to 
quickly inform product development efforts. 
5.4. 
Name and Description of the Investigational Product 
IHV01, an Institute of Human Virology (IHV) product, consists of the FLSC gp120-CD4 
chimera subunit HIV-1 vaccine formulated in Aluminum phosphate adjuvant (Alum). The 
formulation consists of 0.3 mg/mL of FLSC, 2.4 mg/mL of Alum, 5 mM NaOAc, 40 mg/mL 
mannitol, pH 6.2. 
A244, a Duke University product, consists of the gp120 envelope glycoprotein HIV-1 subtype 
CRF_01AE A244 derived from the CM244 CRF_01AE. The A244 gp120 envelop has an 
11-amino N-terminal deletion, similar to the bivalent AIDVAX® B/E protein used in RV144 
(Alam, 2013), which has been safely administered to over 9,000 participants. The AHFG 
adjuvant that is mixed with A244 consists of Rehydragel HPA that has been diluted with sterile 
water and has an aluminum concentration of 5 ± 1 mg/mL. 
ALFQ, a US Army product, is a liposomal adjuvant containing a synthetic monophosphoryl lipid 
A (3D-PHAD®) with the addition of QS-21.  
Normal Saline (0.9% Sodium Chloride for Injection) will serve as a placebo for the trial. 
Refer to Section 7.4 for additional information. 
5.5. 
Summary of Nonclinical and Clinical Trials 
5.5.1. 
IHV01 (FLSC) Vaccine 
Prior to clinical testing in humans, a rhesus macaque analogue, rhFLSC, was created containing 
rhesus macaque CD4D1D2 sequences matched to the immunized host to avoid xenogeneic 
responses against human CD4 domains. In separate experiments, rhesus macaques were 
immunized repeatedly with rhFLSC formulated with various adjuvants: ISCOMATRIX, a 
saponin-containing lipid immune stimulating complex (ISCOM), or RC529-SE, a stable 
emulsion of squalene, glycerol, phosphatidyl choline, and synthetic monophosphoryl lipid A (a 
TLR-4 agonist), or tat toxoid. Animals were assessed for protection from acquisition after 

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repeated low dose mucosal challenges with either SIVmac251 or SHIV162P3 to more accurately 
mimic exposure during sexual transmission in humans. Immunization with rhFLSC in various 
formulations afforded partial protection to challenges with either virus, allowing investigation of 
correlates of protection. Protection from acquisition was preferentially highest in those animals 
that developed antibody-dependent cellular cytotoxicity activity from antibodies directed to 
CD4i epitopes preferentially presented by the FLSC (Fouts, 2015). FLSC is vialed for human use 
with aluminum phosphate as IHV01. 
NCT02756208 is a recently completed Phase I dose escalation clinical trial to determine the 
optimal safe dose of IHV01 in healthy participants. This Phase I clinical trial was a randomized, 
placebo-controlled trial which enrolled 65 healthy HIV-1 negative adults who were at low risk of 
infection with HIV-1 in one of 3 different dose groups (75µg, 150µg, and 300µg). Of the 65 
enrolled participants, 49 received IHV01 and 16 received a Normal Saline placebo. Both groups 
received injections at four different time points. The primary endpoint of the trial was the safety 
and tolerability of the IHV01 vaccine product as measured by local and systemic vaccine 
reactogenicity, laboratory safety evaluation and medical history and physical examination. No 
vaccine-related SAEs had occurred and CD4+ T-cell counts remained stable throughout the trial 
(Chua, 2021). Of the 241 injections given, participants reported 26 instances of mild local 
reactogenicity (7 placebo, 19 vaccine), 3 instances of moderate local reactogenicity (0 placebo, 3 
vaccine), and 1 instance of severe local reactogenicity (0 placebo, 1 vaccine). The severe local 
reactogenicity was reported by an individual in the 300µg vaccine group. Despite this one 
instance of severe local reactogenicity, a majority of the injections were associated with no 
reactogenicity (196 of the 241 injections). The most common AEs related to the injections 
included injection site pain, pruritis, and headache. These AEs were not significantly different 
between the vaccinated and placebo groups. Additional safety information can be found in 
Section 5.3 of the IHV01 IB.  
In this study, IHV01 will be administered as a late boost to prior RV306 vaccine recipients. The 
unique conformational structure of FLSC provides exposure to HIV-1 envelope epitopes at the 
CD4 binding site that are not exposed in the monomeric gp120 expressed in the AIDSVAX® B/E 
vaccine used in RV144, RV305, and RV306. Exposure to these epitopes may likely improve the 
breadth of humoral responses, particularly to CD4i epitopes. 
5.5.2. 
A244 Vaccine 
Clinical trials involving Thai adults have demonstrated that gp120 vaccines, both monovalent 
and bivalent, are safe and immunogenic (Migasena, 2000; Pitisuttithum, 2003). This 
demonstrated proof of the safety and immunogenicity led to the VAX003 phase III randomized, 
double-blind, placebo-controlled vaccine efficacy trial of the AIDSVAX® B/E vaccine, which is 
a bivalent HIV gp120 envelope glycoprotein vaccine containing a subtype B envelope from the 
HIV-1 strain MN and a subtype E envelope from the HIV-1 strain A244. Although the trial was 
completed successfully, the vaccine did not show its ability to prevent HIV-1 infection or delay 
HIV-1 disease progression (Pitisuttithum, 2006). A subsequent RV144 efficacy study of 
AIDSVAX® B/E primed with ALVAC-HIV (vCP1521) demonstrated a 31% vaccine efficacy of 
this combination. 
The A244 construct to be used in RV546 has been remanufactured by the Duke Human Vaccine 
Institute (Durham, NC) and will utilize the A244 gp120 protein as a monovalent vaccine. This 

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protein retains the same 11 amino acid deletion (A244 Δ11 gp120) as in AIDSVAX® B/E, which 
was shown in nonhuman primates to improve both envelope antigenicity and immunogenicity 
(Alam, 2013). Though the A244 construct has been extensively tested in humans, this is the first 
time that the remanufactured version will be administered to humans. Given the extensive 
experience and positive safety profile of the A244 protein in prior studies, the U.S. FDA has 
agreed that a toxicology study is not necessary.  
The A244 construct will be mixed with an aluminum hydroxide gel fluid adjuvant. Aluminum 
hydroxide and aluminum phosphate have been extensively used as adjuvants for HIV-1 vaccines 
since the 1990s (Gamble, 2010; Ling Munier, 2011; Moody, 2014; Walker, 1995). A review of 
clinicaltrials.gov has 38 trials listed in which aluminum is or was being used as an adjuvant for 
HIV-1 Env antigens. The most well-known trials were the phase 3 trials with AIDSVAX®: 
VAX003 (Pitisuttithum, 2006), VAX004 (Flynn, 2005) and RV144 (Rerks-Ngarm, 2009). Both 
VAX003 and VAX004 were conducted in high-risk populations, IV injection drug users and men 
having sex with men, respectively. VAX003 assessed the efficacy of AIDSVAX® B/E and 
VAX004 assessed the efficacy of AIDSVAX® B/B immunization alone and failed to 
demonstrate vaccine efficacy against the acquisition of HIV-1. Both had high titer antibody 
responses. In contrast, the RV144 trial was conducted in a community-risk cohort that received 
both ALVAC and AIDSVAX® B/E. A modest 31.2% efficacy was observed in RV144. 
Antibodies to gp70V12 were identified as the inverse correlate for the acquisition of HIV-1 
(Haynes, 2012b). These antibodies also waned rapidly.  
5.5.3. 
ALFQ Adjuvant 
The WRAIR Laboratory of Antigen and Adjuvant Research (LAAR) developed the original 
multilamellar Walter Reed Liposomes formulation containing monophosphoryl lipid A (MPLA), 
saturated dimyristoyl fatty acids, and cholesterol as carriers of vaccines, adjuvants, and drugs 
have been used with several different antigens in 14 phase I and 2 phase II clinical vaccine trials 
with an excellent safety profile (Alving, 2012).  
Of these, the trial with the most direct relevance to this study was AVEG 015 (NCT00001042), a 
phase I randomized, double blind placebo controlled, clinical trial conducted in the US in healthy 
HIV-1 uninfected individuals. The purpose of this trial was to evaluate immune responses to SF-
2 gp120 protein formulated with 7 different adjuvants including AH (gp120 + AH) and AH-
adsorbed-L(MPLA) [L(gp120 + MPLA) + AH; Walter Reed Liposomes], wherein the antigen 
was encapsulated in multilamellar liposomes rather than mixing with unilamellar liposomes 
(McElrath, 1995). In a strikingly consistent pattern, 3 to 12-fold higher titers of IgG Abs were 
induced not only against the homologous clade B gp120 protein, but also against cross-clade A/E 
and C, gp120 proteins (Rao, 2018). The liposomal arm also induced 2.5 to 13-fold higher titers 
of V1V2-specific IgG Abs compared to the AH arm. Even 10 months after the last boost, the 
titers remained high with only a 5-fold or a 3-fold drop in the Ab titers. In addition, significantly 
higher levels of neutralizing antibodies and T-cell lymphoproliferation were observed in the 
liposomal arm compared to gp120 formulated with AH, the MF59 adjuvant, or monophosphoryl 
lipid A (MPLA) alone (Rao, 2018).  
The next generation formulations of liposomes, known as The Army Liposome Formulation 
(ALF) contain the same components as the Walter Reed Liposomes but are unilamellar and 
contain synthetic phospholipids, including MPLA (3D-PHAD®). The members of the ALF 

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family of adjuvants are ALFA (ALF adsorbed to aluminum salt; US patent pending), ALFQ 
(ALF mixed with the saponin QS21; US patent pending) (Beck, 2015), and ALFQA (ALFQ 
adsorbed to aluminum salt). Both ALFA and ALFQ are potent adjuvants, non-pyrogenic, and 
nontoxic in rabbit pyrogenicity and toxicology studies. ALFA and ALFQ are alternatives to the 
successful but proprietary adjuvants, AS04 (MPLA adsorbed to aluminum salt) and AS01B 
(liposomes containing both MPLA and the saponin QS21). ALFQ is potent in preclinical studies 
and contains 4 times the amount of MPLA and twice the amount of QS21 when compared to 
AS01B. This improved formulation has been demonstrated to increase the immunogenicity and 
protective efficacy of the candidate malaria vaccine P. falciparum circumsporozite protein (CSP) 
(FMP013) relative to the adjuvants Montanide, ALF, and ALFA (Genito, 2017). Similarly, 
ALFQ increased the immunogenicity and protective efficacy of a conjugate Campylobacter 
jejuni capsular polysaccharide antigen conjugated to CRM197 (CPS) in separate challenge 
experiments in mice and Aotus nancymaae monkeys relative to AH and ALF adjuvants 
(unpublished).  
The most direct evidence to support use of ALFQ with HIV envelope antigens arises from an 
ongoing study in rhesus macaques led by Dr. Bart Haynes at Duke University, which compares 
HIV gp120 A244 formulated with either ALFQ or with AHFG. Animals that received ALFQ as 
the adjuvant compared to animals that received AHFG mounted significantly higher binding 
antibody responses to A244, gp70V1V2 A244, and V2 peptide. The responses were significantly 
higher after each immunization, as well as in ADCC responses, quantified using gp120 coated 
target cells. Given that antibody functions such as ADCC may have contributed to protection by 
non-neutralizing antibodies in RV144 vaccine recipients, ALFQ may contribute to increasing 
protective vaccine efficacy. Challenge results in this experiment are pending.  
In light of the highly favorable data from non-clinical studies, ALFQ moved into human clinical 
trials in separate studies involving candidate malaria and COVID-19 vaccines. In the malaria 
vaccine trials, the ALFQ adjuvant was administered with the FMP013 (NCT04268420) and 
FMP014 (NCT04296279) formulations in low (20 µg protein antigen, 100 µg 3D-PHAD®, 50 
µg QS-21) and high doses (40 µg protein antigen, 200 µg 3D-PHAD®, 100 µg QS-21). In the 
COVID-19 vaccine trial (NCT04784767), the ALFQ adjuvant was administered with a spike-
ferritin nanoparticle (SpFN) antigen at consistent dosages in both the low SpFN dose (25 µg 
SpFN antigen, 200 µg 3D-PHAD®, 100 µg QS-21) and high SpFN dose (50 µg SpFN antigen, 
200 µg 3D-PHAD®, 100 µg QS-21) groups. Except for the vaccinations in Part B of FMP014, 
vaccinations in these studies have been completed and post-vaccination safety data is available. 
In summary, ALFQ local reactogenicity in these studies appears to be comprised primarily of 
local pain and tenderness that is omnipresent, mild to moderate, and short-lived across both 
products and doses. Systemic reactogenicity was mostly mild to moderate with the most 
commonly reported symptoms consisting of headache, fever, chills, fatigue, and myalgia. In 
addition, there was one case of severe fatigue in FMP013 that occurred 24 hours post-injection 
and lasted less than 6 hours and one case of severe flu-like symptoms (severe fever chills, 
myalgias, and moderate temperature) in a FMP014 high dose participant that lasted less than 24 
hours. No SAEs were reported in the FMP014 and EID030 trials; however, there were two SAEs 
reported in FMP013, Grade 3 right ankle hemarthrosis and Grade 4 acute appendicitis, both of 
which were assessed by the investigator as not related to the investigational products. No deaths 
or related SAEs were reported in these studies.  

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Safety data from this study, the malaria vaccine studies, and the SpFN + ALFQ vaccine (Joyce, 
2021) study will contribute to the ALFQ safety profile, which will support its use with future 
experimental vaccine candidates.  
5.6. 
Known and Potential Risks and Benefits to Human Participants 
5.6.1. 
Risks/Discomfort to Participants and Precautions to Minimize Risk 
Outlined below are anticipated and unexpected adverse reactions, and a brief description of 
procedures to ameliorate risks and symptoms. All known risks and precautions described here 
are explained in detail in the main study informed consent (Appendix C) and optional procedures 
informed consent (Appendix D), as applicable. All possible safety precautions will be employed, 
however if a participant is injured or become ill as a direct result of their participation in the 
study procedures listed below, they will receive care and treatment for the injury with treatment 
costs covered by research-related injury insurance (see Section 15.2.3). 
5.6.1.1. 
Local Reactions 
Participants may exhibit post-vaccination reactions including local reactions at the injection sites 
such as erythema, induration, pain/tenderness, swelling and limitation of leg movement. These 
reactions are generally of short duration and rarely require treatment. Should such reaction 
persist and require treatment, the participant will be referred to appropriate medical care services. 
5.6.1.2. 
Systemic Reactions 
Participants may exhibit general signs and symptoms associated with administration of a vaccine 
injection, including fever, tiredness, chills, rash, myalgia, arthralgia, nausea, and headache. 
These side effects will be monitored but are generally of short duration and rarely require 
treatment. Should such reaction persist and require treatment, the participant will be referred to 
appropriate medical care services. As with all vaccines, an allergic reaction is possible and will 
be managed as per site SOP for anaphylaxis. To mitigate this risk, participants will be observed 
in the clinic for at least 30 minutes post-injections. 
5.6.1.3. 
Pregnancy 
The effect of the candidate HIV vaccines on pregnancy and fetus are unknown as they have not 
been specifically studied in pregnant women. Female participants who are pregnant or plan to 
become pregnant during the first 3 months post-vaccination will be excluded from enrollment. 
Female participants must use an adequate form of birth control from 45 days prior to the study 
injections until at least 3 months after receipt of the study injections. Adequate birth control is 
defined as follows: Contraceptive medications delivered orally, intramuscularly, vaginally, or 
implanted underneath the skin, surgical methods (hysterectomy or bilateral tubal ligation), 
condoms, diaphragms, intrauterine device, or abstinence. A pregnancy test will be performed on 
all female participants at screening; prior to the study injections (same day); at Visits 4, 5, and 6; 
and prior to any of the invasive procedures as indicated in the SOE. Should a female participant 
become pregnant within the 3 months post-study injections, she will be followed for safety for 
the remainder of the study period per the SOE. Every effort will be made to remain in contact 
with the pregnant participant until birth or termination of the pregnancy in order to record the 
outcome. 

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The possible effects of these vaccines on the offspring of a male participant is unknown. Male 
participants who have not had a vasectomy must use an adequate form of birth control for at least 
3 months after receipt of the study injections. Adequate forms of birth control will depend on the 
child-bearing potential of the participant’s female partner but will generally be the same as for 
female participants. For male participants who have undergone a vasectomy, no additional 
contraception will be required. 
Male and female participants with reproductive potential will be advised to use multiple methods 
of contraception to ensure their effectiveness, including but not limited to using barrier methods 
of contraception in combination (e.g., condom with diaphragm) or a male’s use of condoms and 
his female partner’s use of contraceptive medication or an intrauterine device. 
5.6.1.4. 
Lactation 
These candidate HIV vaccines have not been specifically studied in pregnant and lactating 
women. No data on lactating women are available from previous vaccine trials. There is no 
information about harm to an unborn child or a child who is breastfeeding. Breastfeeding women 
will not be enrolled. 
5.6.1.5. 
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 each study clinic. This is available to 
handle emergencies, such as anaphylaxis and cardiac arrest.  
5.6.1.6. 
Vaccine Induced Seroreactivity 
As with every candidate HIV vaccine trial, there is a risk that participants will mount an antibody 
response to the vaccine, which may cause them to test positive on a standard clinical diagnostic 
assay for HIV. In order to protect participants from any issues that may arise due to a false-
positive HIV tests that can be attributed to receiving the vaccinations, the PI will provide an 
explanatory letter (Appendix N) and the study will provide re-testing. In order to maintain 
blinding, the letter will be given to all participants at their vaccination visit regardless of whether 
they received the vaccines or placebo. The clinical study team will support and counsel any 
participant who remains seroreactive after study closure and will perform repeat diagnostic 
testing every 6 months until antibodies are negative.  
5.6.1.7. 
Venipuncture 
Blood sampling carries the risk of minor discomfort, bruising, fainting and, rarely, infection at 
the needle puncture site.  
5.6.1.8. 
Mucosal Secretion Collections 
Mucosal secretion samples will be collected in the clinic (semen, penile swab, rectal sponges, 
rectal swab, cervico-vaginal menstrual discs, vaginal swab, endocervical cytobrush/swab) by 
trained study personnel. Alternately, for the collection of cervical-vaginal secretions, women 
may elect to insert and remove cervical menstrual discs themselves. For the collection of penile 
swabs, men may elect to collect the swabs themselves during the study visit. Inserting an 

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instrument (speculum, anoscope, sponge, swab, menstrual disc, cytobrush) into the anus or the 
vagina may cause discomfort and slight irritation. There is no evidence of rectal sponge/swab, 
penile swab, cervical menstrual disc, or vaginal swab sampling contributing to risk of HIV or 
other sexually transmitted infection. Semen will be collected by masturbation.  
Cytobrush/swab collection: A cytobrush/swab is a small flexible plastic instrument with bristles 
at the tip used in gynecologic procedures to collect cervical cells for subsequent laboratory 
analysis. 
For these mucosal collections, men and women will be asked to refrain from receptive vaginal or 
anal intercourse, douching, or inserting any product into the rectum or vagina for 3 days prior to 
the mucosal collection. Men will be asked not to masturbate nor ejaculate for 3 days prior to 
semen collection. 
5.6.1.9. 
Sigmoid Biopsy 
Sigmoid biopsies will be performed with a flexible sigmoidoscope. Brief cramping and gas pains 
may be felt as air is inserted or as the scope advances. The passing of gas is necessary and should 
be expected after the procedures are terminated. Participants may choose to receive sedation 
during the procedure in order to ameliorate the discomfort and anxiety they may feel. There may 
be slight bleeding from the biopsy site, which generally stops spontaneously. In rare cases, an 
interventional endoscopic technique is needed to stop bleeding. There is a remote possibility that 
a biopsy may result in significant bleeding or even perforation requiring emergency medical 
care. A gastroenterologist will perform this procedure in order to minimize these risks. As a 
precautionary measure, however, all participants will be counseled to avoid receptive anal 
intercourse for 3 days prior to and 7 days following sigmoid biopsies. 
5.6.1.10. Inguinal Lymph Node Biopsy 
The excisional inguinal lymph node biopsy will be performed under local anesthesia by a 
qualified surgeon. As with all surgical procedures, there is a risk of scarring, bruising, bleeding 
or infection at the surgical site. These risks are minimized by the small size of the incision 
(approximately 1-2 centimeters), clinical evaluation to determine any additional bleeding risks 
(such as anticoagulant use or a congenital clotting disorder), and the adherence by the surgeon to 
aseptic technique. Participants may experience some discomfort following the procedure despite 
use of local anesthesia; for participants who complain of any subsequent discomfort, additional 
analgesia will be made available. There is a possibility of seroma formation at the biopsy site, 
which may require subsequent drainage and further management, or sensory nerve injury during 
the procedure, which could result in temporary or permanent local reduction in feeling. There is 
a small risk of chronic swelling of the leg on the side that the lymph node is taken from. For this 
reason, it is recommended that one participant have no more than 4 inguinal lymph node 
biopsies, or 2 biopsies per side, done in the subject’s entire life, which would include this study, 
and any other studies, and any medical procedures performed elsewhere for non-research 
purposes. Finally, there is the unlikely possibility that the procedure will be unsuccessful and no 
lymph node will be recovered. This procedure will be performed by trained personnel at 
Chulalongkorn Hospital in Bangkok, which has a long history of safely performing lymph node 
biopsies on both HIV infected and healthy individuals (Chintanaphol, 2018). Only a single 
lymph node biopsy will be performed per participant in this study.  

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5.6.1.11. Leukapheresis 
Adverse reactions to leukapheresis procedure are rare and include vaso-vagal episodes related to 
needle insertions and transient volume shifts, peri-oral paresthesias, chills, nausea, and heartburn 
caused by the citrate anticoagulant used during the procedure. Vaso-vagal reactions are handled 
by postural manipulation and fluid administration. Participants will be observed closely by an 
experienced blood bank technician or trained medical staff during the procedure. Citrate 
reactions are usually relieved by slowing the rate of the anticoagulant infusion and by 
administering oral calcium carbonate tablets. Allergic reactions can occur with the use of 
chemical during this procedure. There is limited data on allergic reactions with individuals 
undergoing leukapheresis but the possibility of having anaphylactic reactions cannot be 
completely eliminated or prevented during the procedure. An emergency cart will be on standby 
to respond to an allergic reaction if it occurs.  
5.6.1.12. Social Harm and Discrimination 
In the unlikely event of HIV infection during this study, the primary concern is related to 
ascertaining and providing HIV diagnostic information and, in particular, involuntary disclosure 
of HIV status to others. These disclosures may result in depression and rarely suicide among 
individuals learning that they are infected with the HIV virus. Furthermore, involuntary 
disclosure to others may result in prejudice by the community, family, employers, and 
psychosocial factors including stigma and discrimination. This risk will be minimized by the fact 
that all counselors will be trained in pre- and post-test counseling for HIV and will aim at fully 
informing participants of all activities in the study and attendant risks and benefits. The 
candidate vaccines may also induce false positivity to standard HIV antibody tests and may 
result in problems when applying for life or health insurance, international travel, employment or 
hospitalization. If there are any problems related to the above situations, the investigators will 
provide HIV testing to confirm HIV status. They will provide this information to the persons 
involved. Currently, people who have received experimental HIV vaccines are deferred from 
blood donation even though they do not have HIV infection.  
The study staff will take appropriate action to assist participants with any discrimination they 
may experience by participating in this study. Such measures will be accompanied with 
appropriate counseling.  
5.6.1.13. Unknown Risks 
As with all research there is the possibility of risks that are unknown or that cannot be foreseen 
based on current information. It is currently unknown whether the vaccination will be effective at 
attenuating or preventing HIV or if it will affect any future HIV vaccinations. 
5.6.2. 
Alternatives to this IND Product or Study 
At this time, there are no known alternatives to these vaccines that afford the same potential 
protection from acquiring HIV. 
5.6.3. 
Potential Benefit for Participants 
Study participants will receive no direct benefit from study participation. Female participants 
who choose to undergo cervical mucosal samplings may benefit from early detection of cervical 

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cancer by Pap smear. Participants who choose to undergo sigmoid biopsies may benefit from 
early detection of lower bowel cancer through the evaluation of suspicious polyps that may be 
found during these biopsies. Findings of medical concern will be referred for appropriate care 
and treatment. Others may benefit from knowledge gained in this study that may contribute to 
the development of an HIV vaccine. 
5.6.4. 
Risks to the Study Personnel and the Environment 
The principal risk in the clinical setting is in the handling of needles that may be contaminated 
and the attendant risks including hepatitis, HIV, and other human pathogens. Adherence to 
standard operating procedures (SOP) for working with infectious agents and universal 
precautions will reduce the risk of exposure.  
There are no known risks to the environment other than those associated with the generation of 
biohazardous waste attendant to [vaccination] of humans. All biohazardous waste will be 
disposed of as stipulated by local, state, and Federal regulations and in accordance with study 
site SOPs. 
5.7. 
Route of Administration, Dosage Regimen, Treatment Period, and 
Justification 
Participants will be randomized to receive either a full dose of IHV01 (approximately 300µg) 
and A244 (approximately 300µg), a fractional dose of IHV01 (approximately 60µg) and A244 
(approximately 60µg), or placebo. Participants in Groups 3 and 4 will receive ALFQ 
(approximately 0.5ml) as part of the A244 vaccination. Participants will receive 2 IM injections 
into the quadriceps1 muscle at Day 0. The same quadriceps muscle will be used for both 
injections. Participants randomized to receive the vaccines will have one injection of IHV01 and 
one injection of A244/AHFG at a full or fractional dose with or without ALFQ, whereas 
participants randomized to receive placebo will get 2 separate injections of Normal Saline. All 
placebo injection volumes will match the vaccine injection volumes for the group in which a 
participant has been randomized. 
Refer to Section 7.4 and Table 3 for more information. 
5.8. 
Compliance Statement 
The study will be conducted according to the protocol and in compliance with ICH GCP, 
Belmont Principles, and other applicable ethical, regulatory, Federal and DoD requirements. All 
identified study personnel will be trained to perform their roles and will carry out their 
responsibilities in accordance with the roles and responsibilities descriptions listed in 
Appendix A. 
 
 
1 Although the IHV01 investigator’s brochure notes that injection into the non-dominant deltoid will be used in all 
cases, the product owner has agreed to allow injections into the quadriceps for RV546. We do not anticipate any 
safety issues with injection into the quadriceps muscle, which was chosen to help participants tolerate the total 
injection volume. 

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5.9. 
Study Population 
A total of 80 healthy, HIV-uninfected participants at a low risk for HIV infection, available for 
12 months, who were randomized to receive active vaccine in RV306 and completed all 
vaccinations will be enrolled. A total of 274/300 (91.3%) active recipients from Groups 2, 3, 4a, 
and 4b received all vaccinations and completed all study visits in RV306. 
Each site will recruit participants who participated in RV306 at their respective sites, however 
recruitment may also include participants who participated in RV306 at the site in Chiang Mai 
University if the participant is willing to be enrolled and followed at one of the two Bangkok 
sites. It is anticipated that up to 110 prior RV306 participants will be screened in order to enroll 
80 participants into RV546. As much as possible, an equivalent proportion of male and female 
participants will be enrolled into each arm of the study. 
Study staff and participants will be blinded to vaccine/placebo and full dose/fractional dose 
assignment. As a byproduct of the sequential enrollment of the vaccine/placebo groups, study 
staff will not be blinded to a participant’s receipt of ALFQ. Groups 1 and 2 (without ALFQ 
adjuvant) will be enrolled first in a randomized, double-blinded manner. Enrollment into 
Groups 1 and 2 will begin with no more than two participants per site per week for the first two 
weeks. Once at least 7-days of safety data is available from the first eight participants enrolled 
across Groups 1 and 2, the Safety Monitoring Committee (SMC) will review 7-days of safety 
data from these participants to determine whether enrollment can continue. Enrollment into 
Groups 1 and 2 will be unrestricted after any safety concerns raised by the SMC are addressed 
and the Sponsor has given their approval for continuation of enrollment. Enrollment into 
Groups 3 and 4 will commence after enrollment into Group 1 and Group 2 has been completed, 
the SMC has reviewed 7-days of safety data from all Group 1 and 2 participants, and the Sponsor 
gives their approval. Enrollment into Groups 3 and 4 will follow a similar approach with no 
more than two participants per site per week for the first two weeks, followed by an SMC review 
of 7-days of safety data from the first eight participants enrolled across both Groups, and 
Sponsor approval before enrollment can continue unrestricted in Groups 3 and 4. The PI, 
investigators and study participants will remain blinded to vaccine and placebo allocation. 
The legal age at which individuals can provide their own consent to participate in research in 
Thailand is 18 years. Children will not be recruited for RV546 not only because it does not meet 
the guidelines for inclusion of children in research, but also because participants must have 
participated in RV306 to be eligible for RV546. Since the minimum age for eligibility in RV306 
was 20 years old at the time of enrollment, all potential participants will legally be considered 
adults. There are no limitations to race, ethnicity or sex. Refer to Section 12.3 for a statistical 
justification of the sample size. 
5.10. 
Study Site 
All clinical study activities, including mucosal secretion collections, will take place at the 
Vaccine Trial Center, Faculty of Tropical Medicine at Mahidol University and at the Royal Thai 
Army Clinical Research Center at the Armed Forces Research Institute of Medical Sciences, 
both in Bangkok. 
Leukapheresis and lymph node biopsy procedures will be completed at the Thai Red Cross AIDS 
Research Center, Institute of HIV Research and Innovation (IHRI) Foundation and the 

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Chulalongkorn Memorial Hospital, all in Bangkok. Sigmoid biopsy procedures will only be 
completed at the Chulalongkorn Memorial Hospital.  

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6. 
TRIAL OBJECTIVES AND PURPOSE 
6.1. 
Primary Objective 
• To evaluate the safety of 3 novel investigational products in a late vaccine boost 
setting: the candidate IHV01 and A244/AHFG vaccines and the ALFQ adjuvant 
6.2. 
Secondary Objectives 
• To evaluate the effect of a full dose of the IHV01 and A244/AHFG vaccines and the 
ALFQ adjuvant on cellular, humoral, and innate immune responses (peripheral, 
lymphoid and mucosal). 
• To evaluate the effect of fractional dosing of the IHV01 and A244/AHFG vaccines on 
cellular, humoral, and innate immune responses (peripheral, lymphoid and mucosal). 

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7. 
TRIAL DESIGN 
7.1. 
Study Endpoints 
7.1.1. 
Safety Endpoints 
Safety will be assessed both by direct physical examination and by diary cards (Appendix O), 
which serve as memory tools for better identification of reactions. Participants will be assessed at 
a pre-vaccination baseline and will remain in clinic under direct observation for at least 
30 minutes post-vaccination. Participants will return to clinic on Days 1 and 7 post-vaccination 
as per the Schedule of Evaluations (SOE; Table 3). During these visits, participants will be 
assessed for symptoms of local (at the injection sites) reactogenicity such as erythema, 
induration, pain/tenderness, swelling and limitation of leg movement as well as for symptoms of 
systemic reactogenicity such as fever, tiredness, chills, myalgia, arthralgia, headache, nausea, 
and rash. After review of the diary card and discussion with the participant, the study staff will 
document any post-vaccination reaction(s) and all related information (severity, frequency, etc.) 
concerning such a reaction in the participant’s clinic source documents. Results will be expressed 
as percent of participants with the overall and specific post-vaccination reactions. 
AEs and SAEs will be recorded at all visits along with timing and possible attribution to 
Investigational Product. Because this clinical trial involves an adjuvant, AESIs will also be 
assessed. Results will be expressed as percent of participants with the AEs and SAEs. Safety 
laboratory analyses of complete blood count, liver function tests, urinalysis, and pregnancy test 
in females will also be performed according to the SOE. 
7.1.2. 
Immunogenicity Study Endpoints 
Vaccine-induced immune responses will be assessed in study participants as detailed in the SOE 
(Table 3). 
Humoral responses will be assessed by HIV-specific binding antibody assays, HIV-specific 
neutralizing antibody assays, and non-neutralizing antibody function assays at Days 0, 14, 168, 
and 336. Humoral mucosal immune responses in the rectal, semen and cervico-vaginal 
compartments will be assessed using non-invasive sampling methods (sponge, menstrual disc, 
and masturbation) at the same time points. Cell-mediated immune responses will be assessed 
utilizing invasive sigmoid and lymph node biopsies that will be performed at Day 14. Results of 
immunology assessments through invasive collection methods in the mucosal compartment are 
exploratory and will be mostly descriptive as performed in a limited number of participants. 
Measurement of CD4+ and CD8+ T-cell effector function will be evaluated through response by 
cytokines such as IFN-γ and IL2. The CD4+ function will be assessed by the lymphoproliferation 
responses as measured by the functional CFSE assay. Innate immunity (e.g., NK cells) will be 
assessed by quantifying ADCC and ADCP, determining NK cell phenotype using various flow 
cytometric panels, cytokine array assays to characterize the type of cytokines elicited by this 
vaccine regimen with or without adjuvants, by the assessment of gene expression by DNA 
microarray and related techniques, and other related assays. B-cell responses will be 
characterized in blood and in diverse anatomical compartments (provided by sigmoid biopsies 
and lymph nodes biopsies) in a subset of participants. Host gene transcription profile and 
signature to vaccine antigens will be assessed by RNA sequencing and related techniques.  

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Leukapheresis will be performed at Day 14 in a subset of willing participants for in-depth 
investigations of the T-cell responses.  
In the context of unknown immune correlates of protection, additional assessments may be 
performed as new scientific technologies and assessment tools are made available. 
For further details, see Section 10.1. 
7.2. 
Overall Study Design 
The purpose of this Phase I randomized, double-blind, placebo-controlled clinical trial is to 
define the safety and immunogenicity of IHV01 and A244/AHFG with and without ALFQ at a 
full dose and at a fractional dose (one-fifth of a full dose) in a late boost setting for participants 
who had previous received the AIDSVAX®B/E with or without ALVAC in RV306. Blood, 
lymph nodes, and mucosal specimens will be collected to assess innate, cell-mediated, and 
humoral immune responses. 
A total of 80 healthy, HIV-uninfected previous RV306 participants who were randomized to 
receive active vaccine with late boosting will be enrolled into this study. Only the RV306 
participants who were randomized to receive active investigational product with a late boost in 
Groups 2, 3, 4a or 4b, and completed all vaccinations, will be eligible for enrollment. 
Within each study Group, participants will be randomized to receive 2 IM injections, either 
IHV01 and A244/AHFG with or without ALFQ or Normal Saline placebo on Day 0 (see 
Figure 1) at a targeted ratio of 4:1 vaccine to placebo. Half of the IHV01 and A244/AHFG 
recipients will receive the full dose (Groups 1 and 3) of each vaccine and the other half will 
receive a fractional dose (Groups 2 and 4) of each vaccine, which is one fifth of the full dose. 
Groups 3 and 4 will also receive ALFQ. Once the first eight participants are enrolled across 
Groups 1 and 2 at a pace of no more than two participants per site per week for the first two 
weeks, the SMC will review 7-days of safety data from these participants to determine whether 
enrollment can continue. Any safety concerns raised by the SMC will be addressed prior to the 
continuation of enrollment. Enrollment into Groups 1 and 2 will continue unrestricted after any 
safety concerns raised by the SMC are addressed and the Sponsor gives their approval for the 
continuation of enrollment. Enrollment into Groups 3 and 4 will commence after enrollment into 
Groups 1 and 2 has been completed, the SMC has reviewed 7-days of safety data from all Group 
1 and Group 2 participants, and the Sponsor gives their approval. Enrollment into Groups 3 and 4 
will follow a similar approach with no more than two participants per site per week for the first 
two weeks, followed by an SMC review of 7-days of safety data from the first eight participants 
enrolled across both Groups, and Sponsor approval before enrollment can continue unrestricted 
in Groups 3 and 4. 
At the screening visit, the participant’s general health will be evaluated via medical history, 
physical exam, pregnancy testing, urinalysis, and testing for HIV, syphilis, and Hepatitis B. 
Participants will return for follow-up at 1, 7, 14, 168, and 336 days after vaccination.  
Safety and tolerability will be assessed with both clinical and laboratory monitoring. Vaccine-
related reactions will be observed and solicited for 30 minutes post-vaccination and with the aid 
of a diary card and interview of participants during the 7 days post-vaccination. The information 
gained from the review of the diary card and the interview with the participants will be 

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documented in the clinical study chart. In addition, AEs will be documented at each clinical 
encounter. AEs will be graded for seriousness, severity and relationship to the investigational 
product.  
A subset of willing participants will undergo longitudinal mucosal collections for semen, rectal 
secretions, and/or cervico-vaginal secretions at Days 0, 14, 168, and 336 as well as 
leukapheresis, sigmoid biopsy, and/or lymph node biopsy at Day 14 as described in the 
SOE (Table 3). Participants will be followed until 12 months after vaccination for safety and 
immunogenicity analyses. 
Figure 1: 
Study Group Design 

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Table 3: 
Schedule of Evaluations 
Visit 
S1 
111 
2 
3 
4 
5 
6 (Exit) 
7 
Visit Day 
-45 to -16 
0 
1 
7 
14 
168 
336 
post database 
lock 
Visit Week 
 
0 
1 
1 
2 
24 
48 
Visit Month 
 
0 
 
 
0.5 
6 
12 
Visit Window 
 
 
+2d 
-/+2d 
-2/+3d 
-/+7d 
-/+7d 
 
Clinical 
Final Results 
and 
Unblinding5 
 
Informed Consent (Main, Storage/Future 
Use, Genetic, and Optional Procedures) 
X 
 
 
 
 
 
 
Test of Understanding 
X 
 
 
 
 
 
 
Enrollment and Randomization 
 
X 
 
 
 
 
 
Vaccination 
 
X 
 
 
 
 
 
Vital Signs8 and Physical exam1 
X 
X 
 
X 
X 
X 
X 
Medical History & Concomitant Medications 
X 
X 
 
X 
X 
X 
X 
Adverse Event Documentation (AE, SAE, 
AESI) 
 
X 
X 
X 
X 
X 
X 
Diary Card 
 
X 
X 
X 
 
 
 
HIV Risk Assessment/Risk Counseling 
X 
X 
 
 
X 
X 
X 
Pregnancy Test & Pre/Post Counseling 
X 
X 
 
 
X 
X 
X 
Urinalysis (blood, protein and glucose) 
X 
X 
X 
X 
X 
X 
X 
CBC w/ diff & CD4 
2 
2 
2 
2 
2 
2 
2 
Creatinine, ALT/AST 
3.5 
 
 
3.5 
3.5 
3.5 
3.5 
Syphilis and Hepatitis B Serology 
3.5 
 
 
 
 
 
 
HIV Testing & Pre/Post Counseling9 
6 
 
 
 
6 
6 
6 
Optional Procedures 
Mucosal Secretions2,7; Plasma LH and FSH 
(cervical secretions only) 
 
X 
 
 
X 
X 
X 
STI Testing7 
X 
 
 
 
 
 
 
Vaginal2,7 and/or rectal3,7 and/or penile7 swab 
for microbiome and proteome 
 
X 
 
 
X 
X 
X 
Endocervical Cytobrush/swab2,7 
 
X 
 
 
X 
X 
X 
Pap Smear, if none available in last two 
years7 
X 
 
 
 
 
 
 
Sigmoid Biopsy3,7 
 
 
 
 
X 
 
 

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Visit 
S1 
111 
2 
3 
4 
5 
6 (Exit) 
7 
Visit Day 
-45 to -16 
0 
1 
7 
14 
168 
336 
post database 
lock 
Visit Week 
 
0 
1 
1 
2 
24 
48 
Visit Month 
 
0 
 
 
0.5 
6 
12 
Visit Window 
 
 
+2d 
-/+2d 
-2/+3d 
-/+7d 
-/+7d 
 
Lymph Node Biopsy 
 
 
 
 
X 
 
 
Leukapheresis4 
 
 
 
 
X (10ml) 
 
 
Safety Tests for Biopsy or Leukapheresis  
(up to max possible volume)10 
 
 
 
 
20 
 
 
Research 
HIV Binding Antibody 
 
SP 
 
 
SP 
SP 
SP 
HIV Neutralizing Antibody Assays 
 
6 
 
 
6 
6 
6 
Functional Antibody Assays 
 
SP 
 
 
SP 
SP 
SP 
B-Cell Analysis6 
 
30 
 
30 
30 
30 
30 
Multiparameter Flow Cytometry 
 
20 
 
 
20 
20 
20 
Innate Cell Analysis 
 
20 
20 
20 
20 
20 
20 
Transcriptomics 
 
20 
20 
20 
20 
20 
20 
Additional Immunogenicity Testing6 
 
30 
 
30 
30 
30 
30 
Daily Volume (mL) 
15 
128 
42 
105.5 
157.5 
137.5 
137.5 
Daily volume (mL) for participant 
weighing below 50 kgs 
15 
93 
42 
70.5 
122.5 
137.5 
137.5 
Cumulative Volume (mL) 
15 
143 
185 
290.5 
448 
585.5 
723 
Cumulative Volume (mL) for participant 
weighing below 50 kgs 
15 
108 
150 
220.5 
343 
480.5 
618 
12-Week Cumulative Volume (mL)6 
 
 
 
 
448 
137.5 
137.5 
12-Week Cumulative Volume (mL) for 
participant weighing below 50 kgs6 
 
 
 
 
343 
137.5 
137.5 
SP = Assay performed from stored plasma, no additional blood volume required 
1 Full physical examination at screening (Visit S1) and targeted physical examination at all other visits. 
2 FGT questionnaire (Appendix J) will be administered to all participants who choose to undergo mucosal secretion collections and/or endocervical 
cytobrush/swab for microbiome/proteome assessment and cell collection prior to procedure at each collection visit 
3 Gastrointestinal and dietary questionnaires (Appendix I & Appendix K) will be administered to all participants who choose to undergo a sigmoid biopsy and/or 
rectal swab for microbiome/proteome assessment prior to the procedure at each collection visit 
4 Participants undergoing leukapheresis at Visit 4 will not have peripheral blood drawn for B-cell analysis, multiparameter flow cytometry, innate cell analysis, 
transcriptomics, or additional immunogenicity testing. In addition to the blood drawn for the remaining tests on the SOE, 8.5ml of blood will be collected by 
phlebotomy for PBMC and plasma storage. Red blood cell loss from leukapheresis is estimated to be 10mL. 

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5 Participants will be contacted for the unblinding visit after the clinical data is locked.  
6 The blood volumes drawn are in accordance with the Thai National Blood Bank policy: donation every 3 months but no more than 4 times a year; the amount of 
blood for each donation is according to weight that applies to both male and female: >50 Kg, 450 mL; ≤50 Kg, 350 mL. For participants who weigh below 50 
Kg, blood for “Additional Immunogenicity Testing” will not be collected at Visits 1, 3 and 4 and only 25 mL of blood will be collected for “B-Cell Analysis” at 
Visits 1, 3, and 4. 
7 Participants who consent for optional mucosal collections (semen, penile, rectal, and cervico-vaginal secretions, and cytobrush) and sigmoid biopsies will 
undergo additional testing for gonorrhea/chlamydia genital infections. Female participants who consent for optional mucosal collections will also undergo a 
pelvic examination that includes a Pap smear (if not done in 2 years prior to the mucosal collection) and visual inspection to evaluate for abnormalities. Female 
participants who choose to self-insert the menstrual disc collection device will be given instructions for self-insertion (Appendix T). Male participants who 
consent for optional mucosal collections will undergo a brief visual examination at the screening visit to assess the presence of urethral inflammation and will be 
given instructions for self-collection (Appendix U). Abnormalities that affect the participant’s safety or welfare or those that would increase the risk to a 
participant will preclude their participation in optional procedures. 
8 Vitals to be collected both pre- and at about 30 minutes post-vaccination at Day 0. 
9 Due to the time required to receive results of HIV testing, post-test counseling will occur at the next study visit after the results are available. HIV testing will 
be completed as described in Section 11.1.4.4 and management of positive results will be as described in Section 9.5. 
10 Up to 10mL of blood will be drawn for safety tests within 2 days before the biopsy procedures for safety purposes. For the leukapheresis procedure, up to 
10mL of blood will be drawn for safety tests within 2 days before and after the procedure for a total of 20mL of blood collected across both blood draws. 
11 Repeat testing that is necessary to establish a participant’s eligibility will not affect the post-screening enrollment window unless the repeat testing is necessary 
to establish a baseline CD4 count, in which case enrollment will occur no sooner than 16 days after the date that blood was drawn for the baseline CD4 count. 
 

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7.3. 
Measures Taken to Minimize/Avoid Bias 
7.3.1. 
Randomization 
A randomization schedule will be centrally generated by EMMES. The study will recruit 
approximately equal numbers of males and females, but formal targets will not be established. 
Randomization will be performed creating a random ordinal listing. Sets of contiguous 
recruitment slots will be assigned to each clinic. 
The individual site lists will be kept under lock and key by the pharmacy staff at the respective 
clinical sites. At the end of the study after unblinding of the participants, the lists will be returned 
to the study sponsor. 
7.3.2. 
Blinding 
The PI, study staff, and participants will be blinded as to receipt of active vaccines or placebo but 
the PI and study staff will not be blinded to group allocation as it pertains to the inclusion of 
ALFQ. Since the vaccines and placebos are not identical in appearance, to preserve blinding the 
material inside the syringe will be masked by the pharmacist. In addition, the pharmacy staff 
preparing the vaccine syringes will not be involved in the clinical assessment of participants and 
will be instructed not to comment on the appearance of experimental agent to study staff. For all 
participants, the volume of injections will be consistent.  
Pharmacy staff will be trained in GCP and instructed not to discuss the vaccine randomization 
lists, codes, or participant assignments with study personnel. Pharmacy staff will be required to 
sign a confidentiality agreement and will be the only person(s) on site who will know the 
randomization assignment. The randomization assignment will not appear on any label or source 
document leaving the pharmacy. Samples will be labeled on site using labels containing 
participant study numbers, protocol number and visit number. The samples will be accompanied 
by a specimen tracking form that records the study number, date and time of sample collection.  
Since HIV-1 binding antibody and Western Blot/immunochromatography results 
(Section 5.6.1.6) may reflect vaccine or placebo assignments, access to such data will be limited 
to the laboratory personnel who are performing the tests and managing the data, and the 
independent statistician. If diagnostic testing of original and verification samples reveals true 
HIV infection, the participant will be notified by the clinic staff / physician. The PI and Sponsor 
will be informed of HIV infection by the laboratory. HIV-infected participants will be informed 
about their HIV infection, but the participant, study site investigators, Sponsor, and manufacturer 
staff will remain blinded as to their treatment assignment until the study is closed and the 
database is locked. Diagnostic HIV testing will utilize a sequence of validated tests that will 
differentiate between vaccine-induced seroreactivity and true HIV infection. Information to the 
study staff of each vaccine trial site will not include the results of specific tests but will state only 
HIV "infected" or "not infected", or that repeat testing is needed (as in the case of need for a 
verification specimen). Report of results will be standardized so that the timing of HIV test 
reporting does not compromise the double-blind nature of the trial, as a result returned 
immediately after EIA testing could signal the clinical team that this specimen did not require the 
additional testing which would be needed if vaccine had induced an antibody response. Some 
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HIV testing. Participants will be actively discouraged from having HIV testing outside of the 
trial protocol. If specific needs arise, the research team will provide HIV testing and assist 
participants who need HIV status determination. Further, participants will be counseled that if 
they have engaged in behavior that may have increased risk of exposure to HIV, they should 
have HIV testing done and this should be done through the vaccine trial system. 
7.3.3. 
Unblinding 
In case of vaccine-related death or life-threatening SAEs, knowledge of whether a participant 
received vaccine or placebo can be critical for the interpretation of the significance of clinical 
findings and thus impact decisions regarding continuation of study participation. In such cases, 
the PI will consult with a pharmacovigilance (PVG) physician from the USAMRDC ORA 
Product Safety Surveillance Office (PSSO), the Protocol Chair/Sponsor’s Medical Expert for the 
Trial and/or the HIV Vaccine Product Manager before making the decision to unblind. If the 
group determines that unblinding is necessary, the PI will request the unblinding of the 
participant’s assignment from the Study Pharmacist. 
Any other request for unblinding, with its rationale, must be forwarded through the PI. The PI 
will evaluate the request and will notify the Protocol Chair/Sponsor's Medical Expert for the 
Trial and HIV Vaccine Product Manager. They will evaluate the request and will advise the 
USAMRDC ORA PSSO regarding a course of action. A PVG physician from the USAMRDC 
ORA PSSO will decide whether to approve the request for unblinding.  
In the case of a decision to unblind, the Sponsor will authorize the independent statistician at 
EMMES to provide this information to the PI, who must notify the IRBs (see Table 5) and 
provide the study assignment (vaccine vs. placebo) to the site physician. It should be noted that 
there are very few circumstances in which unblinding will be essential to the medical 
management of a vaccine (or placebo) recipient. The site investigator will report episodes of 
accidental unblinding, with an explanation, to Protocol Chair/Sponsor's Medical Expert for the 
Trial, the IRBs and the USAMRDC ORA PSSO. Follow-up of unblinded participants will 
continue through the duration of the trial. 
After completion of the study and database lock, the Sponsor will authorize the independent 
statistician to prepare a list containing each participant’s unblinded treatment assignment. 
Participants will be contacted to return to clinic or will be notified via phone or letter 
(Appendix M) of their unblinded treatment assignment. 
7.4. 
Investigational Product 
The investigational products to be utilized in this trial include IHV01, A244/AHFG with and 
without ALFQ, and a Placebo. 
IHV01, an IHV product, consists of the FLSC gp120-CD4 chimera subunit HIV-1 vaccine 
formulated in Aluminum phosphate adjuvant (Alum). The formulation consists of 0.3 mg/ml of 
FLSC, 2.4 mg/ml of Alum, 5 mM NaOAc, 40 mg/mL mannitol, pH 6.2.  
A244, a Duke University product, consists of the gp120 envelope glycoprotein HIV-1 subtype 
CRF_01AE A244 derived from the CM244 CRF_01AE. The A244 gp120 envelop has an 11 
amino N-terminal deletion, similar to the protein used in RV144 (Alam, 2013). The formulation 
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AHFG adjuvant that is mixed with A244 consists of Rehydragel HPA that has been diluted with 
sterile water and has an aluminum concentration of 5 ± 1 mg/mL.  
ALFQ, a U.S. Army product, is a liposomal adjuvant containing a synthetic monophosphoryl 
lipid A (3D-PHAD®) with the addition of QS-21. The liposomal lipid bilayer of ALFQ contains 
synthetic phospholipids DMPC and DMPG, and synthetic cholesterol (55 mol%). The 
concentrations of synthetic phospholipid (DMPC + DMPG) and cholesterol in an ALFQ vial are 
22.9µM and 27.9µM, respectively. A full dose of ALFQ vaccine formulation contains 200µg of 
MPLA and 100µg of QS-21.  
Normal Saline (0.9% Sodium Chloride for Injection), purchased from a commercial vendor, will 
serve as a placebo for the trial. All placebo injection volumes will match the study injection 
volumes for the group to which a participant has been randomized.  
7.4.1. 
Investigational Product Packaging and Labeling 
The investigational products IHV01 (FLSC), A244 and ALFQ are covered under an IND 
application. Each vial label will include a variation of the following statement: “Caution: New 
Drug − Limited by Federal (or United States) law to investigational use”. Product labels will 
comply with both US and Thai labeling requirements. The Pharmacy Manual of Procedures 
(MOP) details specific product labels. 
7.4.2. 
Investigational Product Storage 
The investigational products will be stored in a secure pharmacy refrigerator or freezer at the 
clinical sites. Doses of IHV01 (FLSC), ALFQ, and AHFG must be kept at +2-8°C; A244 at 
≤-65°C; and placebo (normal saline) at ≤30°C until preparation. The vaccine/placebo should be 
given within 2 hours after preparation. 
If a deviation in storage temperature occurs outside of the allowable excursions defined in the 
Pharmacy MOP, the pharmacy staff must report the storage temperature excursion promptly to 
the Principal Investigator (PI) and to USAMRDC ORA. The excursion must be investigated and 
action must be taken to restore and maintain the required temperature limits. Vaccine products, 
adjuvants, and placebo products that have experienced a temperature excursion must be 
quarantined until USAMRDC ORA approves it for further use or authorizes its destruction (see 
Section 7.4.4). 
7.4.3. 
Investigational Product Preparation 
All investigational product preparation will be performed by the Site Pharmacy personnel on the 
day of vaccine administration, after confirmation by the clinical staff that the participant is 
present, is eligible for enrollment into the study and has received their randomization 
assignment. The pharmacy personnel will perform all manipulations of investigational products 
in a biological safety cabinet in accordance with GCP guidelines and the RV546 Pharmacy 
MOP. The pharmacy personnel will wear a lab coat and gloves for all manipulations. The center 
seals of the vials will be removed and the vial stoppers wiped with an alcohol wipe prior to 
insertion of a sterile needle attached to a sterile syringe. The RV546 Pharmacy MOP outlines 
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7.4.4. 
Investigational Product Accountability 
The site pharmacy personnel will be responsible for and will maintain logs of investigational 
product receipt, storage, reconstitution, accountability by participant, and investigational product 
remaining before final disposition. These logs will be securely maintained in the pharmacy at 
each site.  
The empty vials and the unused portion of the vial should be discarded in a biohazard 
containment bag and incinerated after verification from the monitor. Any unopened vials (past 
the re-test date or otherwise) that remain will be destroyed at the discretion of the sponsor in 
accordance with policies that apply to investigational agents in Thailand. Partially used vials or 
expired prepared doses will not be administered to other participants or used for in vitro 
experimental studies. They will be disposed of in accordance with institutional pharmacy policy. 
Retest dates shall be updated as per analytical analyses of the IP. No IP shall be administered to a 
participant beyond the current retest/expiry date until stability testing has been completed and it 
has been determined that the retest/expiry date can be extended without increasing the risk to 
participants.  
7.5. 
Duration of Participation 
The total duration of study participation is about 12 months from enrollment. 
7.6. 
Dose-adjustment Criteria 
No modification of dosage for any of the vaccine products will be allowed for this study. 
7.6.1. 
Safety Criteria for Stopping Doses 
Site investigators will notify the PI who will notify the PSRT by email of any safety events that 
potentially meet the study pause criteria listed below. The PSRT will expedite a review of the 
pertinent safety data to determine if the following pause criteria has been met. The PSRT may 
pause vaccination at any time but will use as a guide, based upon an abundance of data 
supporting safety of these vaccinations, the occurrence of: 
• One Grade 3 event judged to be probably or definitely related to vaccination; or  
• One Grade 4 or Grade 5 event judged to be possibly, probably or definitely related to 
vaccination; or 
• One SAE judged to be possibly, probably, or definitely related to vaccination; or 
• Unexplained decline in CD4 cell count (>30% from baseline) in more than 5 
participants in a single Group, as confirmed by a similar result (>30% decline from 
baseline) from a repeat assay performed at least 4-weeks after the first assay from 
which a >30% decline in CD4 cell count resulted. 
Note: An unexplained drop in CD4 cell count is defined as a decline that cannot be 
explained by an intercurrent illness or other event known to cause decreased CD4 
cell counts 
If immediate action is needed to protect the rights, welfare and safety of a study participant, this 
must be promptly reported to the IRBs. This information should include participant ID, 

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diagnosis, severity, relation to product, clinical description of event to the extent possible and 
actions taken, if any.  
7.6.2. 
Pharmacokinetic Criteria for Dose Adjustment or Stopping Doses 
Not applicable. 
7.6.3. 
Study Termination 
The PI, Sponsor’s Medical Expert/Protocol Chair, sponsor, MoPH EC, RTA IRB, FTM EC, 
Chulalongkorn IRB, WRAIR IRB, Thai FDA, USAMRDC OHARO, U.S. FDA, or U.S. DoD 
may stop or suspend the study at any time. The PI will notify the IRBs in writing of any stoppage 
or cancellation of the study in accordance with Section 11.8.2.2. 
7.7. 
Trial Treatment Randomization Codes 
See Section 7.3.1 and Section 7.3.2. 
7.8. 
Identification of Data to be Recorded on the Case Report Forms 
(CRFs) 
Source data will be collected at the study site. For more information on data handling, refer to 
Section 16. 

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8. 
SELECTION AND WITHDRAWAL OF PARTICIPANTS 
8.1. 
Recruitment of Participants 
As the study population is a fixed subset of healthy individuals who participated in RV306, a 
targeted recruitment strategy will be employed. Prior RV306 participants who received all 
RV306 vaccinations, completed all RV306 study visits, and consented to be contacted for future 
research will be contacted by phone or through the mail to be invited to attend an optional 
information session at their convenience. Information sessions will be conducted at the clinical 
sites by the PI or designee and will contain information about the study, vaccines, and 
participation requirements. The site staff will make all necessary provisions to assure the privacy 
of these discussions with all potential participants participating in these information sessions. 
The briefing session will be followed by an opportunity for questions from the participants. All 
participants interested in this study will subsequently, individually be administered informed 
consent. 
Refer to Section 5.9 for a detailed description of the participant population. 
8.2. 
Informed Consent Process 
At the beginning of the screening visit, written informed consent will be obtained from the 
participant by the PI or their designee. Separate informed consent forms will be administered for 
the main study and for the optional procedures. Written informed consent will be obtained from 
each participant before any study procedures are performed. All informed consent forms will be 
administered individually, in a private setting, with strict respect of confidentiality. Participants 
will be given ample time and opportunity to inquire about details of the study, discuss with close 
family members or friends and ask any questions before dating and signing the consent forms. A 
copy of the informed consent form, signed by both the participant and the investigator 
conducting the consent process, will be given to the participant along with a copy of the 
volunteer event schedule (Appendix P). The consenting process will be documented in the CRFs. 
After providing signed informed consent the participant will complete a Test of Understanding 
(TOU) (Appendix G). Participants are allowed to take the TOU 3 times but must have a passing 
score (80% or greater) including two compulsory questions to be answered correctly by the third 
attempt to participate in the study. If after 3 attempts to pass the TOU the participant is unable to 
do so, the participant will become ineligible for study participation. Participants who 
successfully pass the TOU will then be screened as per the study protocol. 
8.3. 
Eligibility Screening (Visit S1) 
Screening for eligible participants will be performed within 45 days prior to administration of the 
investigational vaccine. Participants who have passed the TOU and have given written informed 
consent will undergo a complete medical history, physical examination, and screening laboratory 
assessments to determine eligibility for study participation. The following screening assessments 
will be completed after the informed consent process has been completed: 
• Medical history, including any medications taken 
• Physical exam 
• Vital signs 

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• HIV risk assessment and risk counseling 
• HIV testing (including pre/post-test counseling) 
• Urine pregnancy testing and counseling 
• Urinalysis 
• Creatinine 
• CBC w/diff 
• CD4 
• ALT/AST 
• Syphilis testing 
• Hepatitis B testing 
• Optional Procedures: 
− STI testing 
− Pap smear (if none available in the 2 years prior to screening) 
Screening evaluations for specific eligibility criteria (see Section 8.3.1 and Section 8.5) must be 
completed within the screening visit window specified (see Table 3) prior to enrollment but may 
be repeated within the screening visit window to confirm the results. A second screening visit 
may be conducted outside of the initial screening visit window for individuals who meet certain 
criteria (see Section 8.3.1) only if study enrollment is ongoing. 
Determination of a participant’s risk for HIV infection will be completed at screening as 
described in Section 9.1.8. Counseling for HIV risk reduction and the potential risks of becoming 
pregnant during participation will be provided by trained counselors at screening and throughout 
the study (see Table 3).  
Participants agreeing to optional mucosal collections (semen, penile, rectal, cervico-vaginal 
secretions, and cytobrush) and sigmoid biopsies will undergo additional testing to evaluate for 
sexually transmitted infections. This includes testing for gonorrhea/chlamydia by urine NAAT 
and by rectal swabs for individuals practicing receptive anal intercourse in the 6 months prior to 
testing for those undergoing rectal secretion collection or sigmoid biopsy. Female participants 
will undergo a pelvic examination that includes Pap smear testing with visual inspection to 
evaluate for further abnormalities. Individuals with abnormalities noted on examination and 
laboratory testing not explained by these conditions may undergo additional testing (e.g., wet 
prep for Trichomonas vaginalis in female participants) or be referred for further care and 
treatment. Abnormalities that affect the participant’s safety or welfare or those that would 
increase the risk to a participant will preclude their participation in optional procedures. 
The PI or designee will make the final decision regarding eligibility. Clinical data and specimens 
collected during screening from participants subsequently found to be ineligible for participation 
in the study will become part of the study records and specimens will be evaluated in accordance 
with protocol procedures. Only eligible participants will be given the investigational product at 
the enrollment visit. 
8.3.1. 
Participant Re-screening 
Participants who are determined to be ineligible at screening due to out-of-range laboratory 
values can be re-screened if, in the medical judgement of the investigator, the out-of-range value 
is due to a temporary condition. Participants who are ineligible due to a condition that meets 

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study exclusion criteria can be re-screened if the condition is considered to be temporary. 
Exclusionary conditions that are considered temporary include but are not limited to syphilis, 
gonorrhea, and chlamydia. Re-screening will be completed only after resolution of the temporary 
condition. 
Participants who are determined to be ineligible at screening due to their receipt of substances 
listed in exclusion criterion 4 (see Section 8.5) within the specified windows can be re-screened 
once the applicable windows have expired.  
Participants who are not enrolled prior to expiration of their screening visit window, whether 
initially determined as eligible or temporarily ineligible, and who remain interested in study 
participation must repeat the full screening visit in order to ensure their eligibility for enrollment. 
Re-screening will only be completed if study enrollment is ongoing. Individuals who are 
re-screened will retain the same participant ID number as was assigned at their first screening 
visit. 
8.4. 
Inclusion Criteria 
Participants must meet all of the following criteria to be included in the study: 
1. Healthy, HIV-uninfected male and female participants  
2. Prior RV306 recipients who were randomized to receive active vaccine with late boosting 
at month 12, 15, or 18 and who completed all vaccinations 
3. Have a Thai identity card 
4. Must be at low risk for HIV infection per investigator assessment 
5. Must be able to understand and complete the informed consent process 
6. Must be capable of reading Thai 
7. Must successfully complete a Test of Understanding prior to enrollment  
8. Must be in good general health without clinically significant medical history 
9. HIV-uninfected per diagnostic algorithm within 45 days of enrollment 
10. Laboratory screening analysis: 
a. Hemoglobin: Women ≥11.0 g/dL, Men ≥11.5 g/dL 
b. White cell count: 4,000 to 11,000 cells/mm3 
c. Platelets: 150,000 to 450,000/mm3 
d. ALT and AST ≤1.25 institutional upper limit of reference range 
e. Creatinine: ≤1.25 institutional upper limit of reference range 
f. Urinalysis: blood and protein no greater than 1+ and negative glucose 
Note: Each laboratory screening test that is out of acceptable range can be repeated 
during the screening window to confirm the result. A second screening visit may be 
conducted outside of the initial screening visit window for individuals who meet certain 
criteria (see Section 8.3.1) only if study enrollment is ongoing. 
11. Female-Specific Criteria: 
a. Not currently pregnant or breastfeeding and not planning to become pregnant during 
the first 3 months after study vaccine/placebo injections 
b. Negative pregnancy test for women at screening, prior to vaccination (same day), and 
prior to any of the invasive procedures 
c. Be using an adequate birth control method for 45 days prior to receipt of 
vaccine/placebo and for at least 3 months after receipt of the vaccine/placebo. 

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Adequate birth control is defined as follows: Contraceptive medications delivered 
orally, intramuscularly, vaginally, or implanted underneath the skin, surgical methods 
(hysterectomy or bilateral tubal ligation), condoms, diaphragms, intrauterine device, 
or abstinence. 
12. Male-Specific Criteria 
a. Be using an adequate birth control method for at least 3 months after receipt of the 
vaccine/placebo. For non-vasectomized male participants with female partners of 
child-bearing potential this includes the use of condoms or abstinence and/or their 
partner’s use of contraceptive medications delivered orally, intramuscularly, 
vaginally, or implanted underneath the skin, surgical methods (hysterectomy or 
bilateral tubal ligation), diaphragms, or intrauterine device. 
8.5. 
Exclusion Criteria 
Participants meeting any of the following criteria will be excluded from the study: 
1. Asplenia: any condition resulting in the absence of a functional spleen 
2. Bleeding disorder diagnosed by a medical doctor (e.g., factor deficiency, coagulopathy, 
or platelet disorder requiring special precautions) 
3. History of allergic reaction, anaphylaxis, or other serious adverse reaction to vaccines or 
components of the vaccines 
4. Volunteer has received any of the following substances: 
a. Chronic use of therapies that may modify immune response, such as IV immune 
globulin and systemic corticosteroids (in doses of >20 mg/day prednisone equivalent 
for periods exceeding 10 days) 
Note: The following exceptions are permitted and will not exclude study 
participation: use of corticosteroid nasal spray for rhinitis, topical corticosteroids for 
an acute uncomplicated dermatitis; or a short course (duration of 10 days or less, or a 
single injection) of corticosteroid for a non-chronic condition (based on investigator 
clinical judgment) at least 14 days prior to enrollment in this study. 
b. Blood products within 120 days prior to HIV screening 
c. Immunoglobulins within 30 days prior to HIV screening 
d. Any licensed vaccine within 14 days prior to study vaccine administration in the 
present study 
e. Receipt of any investigational HIV vaccine other than RV306 products 
f. Investigational research agents or vaccine within 30 days prior to enrollment in the 
present study 
g. Receipt of a Coronavirus disease 2019 (COVID-19) vaccine that has been given 
Emergency Use Authorization (or those that become licensed) by the Thai FDA 
within 14 days prior to study vaccine administration in the present study 
Note: Volunteers receiving a COVID-19 vaccine that requires 2 doses will not be 
enrolled until 14 days after the second dose has been administered 
h. Anti-tuberculosis prophylaxis or therapy during the past 90 days prior to enrollment 
Note: Participants determined to be ineligible at screening due to receipt of the above 
listed substances may be re-screened once the applicable window of receipt has expired. 
5. Active sexually transmitted infection confirmed by clinical exam and diagnostic test  

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6. Any medical, psychiatric, social condition, occupational reason, or other responsibility 
that, in the judgment of the investigator, is a contradiction to protocol compliance or 
impairs a volunteer’s ability to give informed consent 
7. Psychiatric condition that precludes compliance with the protocol; past or present 
psychoses; past or present bipolar disorder; disorder requiring lithium; or within 5 years 
prior to enrollment, a history of suicide ideation or attempt 
8. Study site employees who are involved in the protocol and/or may have direct access to 
study related area 
8.6. 
Early Discontinuation or Withdrawal of Study Participants 
Each participant has the right to withdraw from the study at any time for any reason without 
penalty. In those cases where a “withdrawal of consent” is requested by the participant, 
documentation of the withdrawal of consent and the reason(s) for the request will be captured in 
the participant’s clinical research records and/or on the status change CRF. Although the 
participant is not obligated to give reason(s) for withdrawing, the investigator should make a 
reasonable effort to ascertain the reason(s) while fully respecting the participant’s rights. The 
participant will also be asked if she/he will agree to complete the clinical assessments listed for 
the study termination/exit visit (Visit 6).  
8.6.1. 
Investigator Withdrawal of Participants 
The PI may withdraw a participant without their consent for the following reasons: 
• Failure to comply with study procedures or requirements 
• A participant’s health or safety may be compromised by further participation 
• The PI assesses that it’s not in the best interest of the participant to continue 
participation in the study 
If a participant is withdrawn by an investigator, the reason should clearly be stated in the source 
documents and on the status change CRF. 
When a participant withdraws due to an AE or is withdrawn by the PI due to an AE, the 
sponsor’s safety office, the USAMRDC ORA PSSO, must be notified within 5 business days 
(usarmy.detrick.medcom-usamrmc.mbx.sae-reporting@health.mil). Investigators must follow 
specific policies regarding the timely reporting of AEs and SAEs to the IRBs (Section 11.8.1.2). 
8.6.2. 
Data Collected for Withdrawn Participants 
Only data and samples already collected will be analyzed according to the protocol. The study 
team will not utilize samples or data from this participant for any future use if he/she has not 
consented to future use and will discard residual samples when the study is completed as is done 
for all participants who refuse storage and future use of their samples.  
8.6.3. 
Replacement of Withdrawn Participants 
Participants who withdraw or are withdrawn at any time prior to receiving the study injections 
until 30 days after receiving the injections may be replaced. A participant who has been assessed 
as eligible for the study through screening will take the place of the withdrawn participant. 

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Participants who withdraw or are withdrawn within this window will not count toward the total 
number of participants enrolled in the study. 
8.6.4. 
Follow-up for Withdrawn Participants 
After withdrawal, the study team will engage in no further communication with the participant 
except as directed by an IRB and/or USAMRDC regarding information concerning participant 
safety. 

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9. 
STUDY PROCEDURES 
9.1. 
Vaccination and Follow-up Visits 
9.1.1. 
Day 0 Through Day 336 
Day 0 is defined as the day of protocol enrollment and vaccine/placebo injections. At the 
enrollment visit, screening results will be reviewed and general eligibility will be assessed. All 
screening results, including results from any repeat testing, must be received prior to enrollment. 
Repeat testing will not affect the post-screening enrollment window (see Table 3) unless the 
repeat testing is necessary to establish a baseline CD4 count, in which case enrollment will occur 
no sooner than 16 days after the date that blood was drawn for the baseline CD4 count. 
If clinical assessment on Day 0 suggests significant changes may have occurred since the 
screening visit, then the physical examination, complete blood count, prothrombin / international 
normalized ratio of partial thromboplastin time, blood chemistries and urinalysis done at 
screening must be repeated, as appropriate, and results received before the participant receives 
the study injections. Pregnancy test results for female participants must be obtained prior to the 
study injections.  
After Day 0, deviations from the visit windows in completing study visits are discouraged and 
will be recorded as protocol deviations, but are permitted, at the discretion of the PI (or 
designee), in consultation with the sponsor, in the interest of obtaining participant safety and 
immunogenicity evaluations following study injections. 
Study visit procedures and tests through Day 336 include the following as indicated in the SOE 
(Table 3): 
• Clinical evaluations: targeted physical examination and vital signs (both pre- and at 
about 30 minutes post-vaccination on Day 0) 
• Interim medical history, including any new medications taken 
• Adverse event documentation 
• Study vaccine/placebo injections 
• Diary Card: Baseline in the evening on the day of the injections; 7-day diary card for 
self-assessment by participant 
• HIV risk assessment and risk counseling 
• HIV testing (including pre/post-test counseling) 
• Urine pregnancy testing and counseling 
• Urinalysis 
• CBC w/ diff 
• CD4 
• Optional Procedures: 
− Leukapheresis (with safety tests before and after procedure) (Day 14) 
− Sigmoid Biopsy (with GI and dietary questionnaires and safety tests prior to 
biopsy) (Day 14) 
− Lymph node biopsy (with safety tests prior to biopsy) (Day 14) 

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− Mucosal secretion collection, plasma LH and FSH (cervical secretions only) and 
Female Genital Tract (FGT) Questionnaire (Days 0, 14, 168 and 336) 
− Endocervical cytobrush/swab and FGT Questionnaire (Days 0, 14, 168 and 336) 
At intervals specified in the SOE, blood will be drawn for safety and immunologic assays. For 
participants who weigh >50 kg, the total blood volume drawn from each participant will not 
exceed 450 mL in any 12-week period. For participants who weigh ≤50 kg, the total blood 
volume drawn from each participant will not exceed 350 mL in any 12-week period. 
9.1.2. 
Post-Database Lock 
Upon study completion and locking of the database, participants will return for a final visit to 
receive their final clinical laboratory tests and unblinding assignment. Participants who received 
active investigational product will be counseled that these remain experimental candidate 
vaccines that have no proven ability to protect against subsequent HIV infection, so continued 
appropriate precautions for HIV behavioral prevention must be exercised. In the event that an 
individual participant has undergone vaccine-induced seroreactivity, they will be informed that 
they may face discrimination if undergoing a HIV testing outside the clinic facility, as disclosed 
in the informed consent. These participants will be offered the opportunity to return to the clinic 
for routine monitoring of seroreactive status every 6 months as a clinical service even after study 
closure until it disappears. 
9.1.3. 
Mucosal Secretion Collection 
Mucosal secretion collection (cervico-vaginal, semen, rectal secretions, vaginal swab, 
endocervical cytobrush/swab, penile swab and rectal swab) will be performed at Visits 1, 4, 5, 
and 6 on participants who provide separate informed consent for these procedures. 
Consenting women will have a pelvic examination done by an experienced doctor, who will 
inspect the tissue of the vagina and cervix and will collect endocervical cells using an 
endocervical cytobrush/swab as well as collecting a vaginal swab to assess the 
microbiome/proteome. Participants will also complete the FGT questionnaire. After the 
examination, the participant will be instructed on how to use a menstrual disc device to collect 
cervical and vaginal secretions by either self-insertion the night before the visit or the morning of 
the visit, or with assistance by trained clinic staff during the visit. Participants who choose self-
insertion will be given a menstrual disc at a prior visit and instructions (Appendix T) on how to 
insert the menstrual disc on the night before or the morning of the collection visit. The menstrual 
disc will remain in place for 4 to 12 hours. Female participants will be asked to avoid receptive 
vaginal intercourse or insertion of objects intravaginally (tampons, douching, etc.) for the 3 days 
prior to specimen collection. Pregnant women and those with a history of toxic shock syndrome 
will be excluded. Collection will not take place if the woman is menstruating or has symptoms of 
active inflammation or infection of the vagina or cervix. For participants who choose to self-
insert the menstrual disc, study staff will call the participant the day before their study visit to 
remind them about inserting the menstrual disc and to ask about pregnancy, menstruation, and 
any abnormalities that may preclude them from using the menstrual disc. Participants will be 
advised to not self-insert the menstrual disc as necessary. For menstruating females, mucosal 
collection window can be extended to -3/+14 days. The date of last menstrual period will be 
recorded at each collection visit and blood samples for hormone levels will be evaluated. If, 

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during the course of the study, a woman becomes pregnant, she will be excluded from further 
genital secretion collections. 
Consenting men will have a brief visual examination at the screening visit which will be 
completed by an experienced doctor who will inspect the participant’s penis for signs or 
symptoms of urethral inflammation. The participant will be given instructions (Appendix U) on 
how to self-collect the swabs and will be asked about any signs or symptoms of urethral 
inflammation prior to the collection of penile swabs and/or a semen sample at follow-up visits. 
Both penile swabs and semen collections will be deferred if there are signs or symptoms of 
urethral inflammation. Penile swab will be collected by full circumference of the penis at the 
coronal sulcus, the junction between the glans and the shaft of the penis. This procedure can be 
completed by the participant after receiving instruction from study staff or if the participant 
prefers, the study staff can complete the collection. For the semen collection, the participant will 
be asked to ejaculate into a sterile container. A private room in clinic will be provided for both 
procedures. Male participants providing semen will be asked to avoid ejaculation for the 3 days 
prior to specimen collection.  
Both male and female participants may elect to provide rectal secretions and rectal swabs for 
microbiome/proteome assessment. Participants providing rectal secretions will be asked to avoid 
receptive anal intercourse or insertion of objects into the anus (enema, etc.) for the 3 days before 
specimen collection. The anal rectal sponge will be inserted and removed by trained medical 
staff and will be placed in the rectum for a maximum of 5 minutes. Examination and specimen 
collections take up to 30 minutes to complete. Rectal sponge and swab collections will be 
deferred if there are signs or symptoms of perianal inflammation. Gastrointestinal and dietary 
questionnaires will be administered to all participants who choose to collect rectal secretion/swab 
collection.  
9.1.4. 
Sigmoid Biopsy 
Sigmoid biopsies will be performed only at Visit 4 on participants who provide separate 
informed consent for the procedure. The procedure will be performed by trained personnel at the 
King Chulalongkorn Memorial Hospital in Bangkok, Thailand.  
Prior to undergoing a sigmoid biopsy, participants will be tested for COVID-19 (if required by 
institutional procedures) and a pregnancy test will be performed on female participants. All 
participants tested for COVID-19 must have a negative test result to proceed with the biopsy. 
Female participants must also have a negative pregnancy test to proceed. Participants who test 
positive for COVID-19 will be referred for care and treatment if required by institutional 
procedures. 
Gastrointestinal and dietary questionnaires will be administered to all participants who choose to 
undergo a sigmoid biopsy prior to the procedure.  
Using a GI endoscope, a gastroenterologist will collect about 16 to 30 tissue samples from the 
participant’s gut. This procedure carries the possibility of pain, so prior to and during the 
sigmoid biopsy a licensed physician will offer participant intravenous sedative and pain 
medications. Medication will be provided to the participant free of charge by the study team. The 
use of aspirin and non-steroid anti-inflammatory drugs by participants will be prohibited 7 days 
before and 1 day after the procedure. Participants who undergo sigmoid biopsies will also be 

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asked to avoid receptive anal intercourse 3 days before and 7 days after the biopsy. The sigmoid 
biopsy procedure will take up to 1.5 hours to complete and participants will be observed for 
about 3 hours post-procedure. 
9.1.5. 
Leukapheresis 
Leukapheresis will be performed only at Visit 4 on participants who provide separate informed 
consent for the procedure. The procedure will be performed by trained personnel at the King 
Chulalongkorn Memorial Hospital, the Thai Red Cross AIDS Research Centre, or the Institute of 
HIV Research and Innovation (IHRI) Foundation. 
For the leukapheresis procedure, participants will be connected to a cell separator machine via 
IV, which will separate white blood cells for laboratory testing while returning red blood cells 
either through the same needle or through a second needle placed in the opposite arm. When the 
procedure is completed, the maximum total red blood cell loss is about 10 mL (less than one 
tablespoon). Participants who undergo the leukapheresis procedure will not have blood drawn for 
immunogenicity assays at Visit 4. In addition to the white blood cells that are collected, 
approximately 150 mL of plasma will be removed at the end of each leukapheresis procedure for 
storage. Participants who undergo leukapheresis will have blood collected by phlebotomy in 
accordance with the SOE (Table 3), however they be exempted from the immunogenicity blood 
draw scheduled at the same visit. An additional 8.5ml of blood will be collected from these 
participants for PBMC and plasma storage in order to have comparator cells from each visit. 
Leukapheresis will be performed on participants whose platelet counts or hemoglobin counts 
meet the protocol inclusion criteria. 
The entire procedure will take up to 2-3 hours, during which time the participant will be under 
constant observation by trained leukapheresis staff. If tingling in the face, lips or hands is 
experienced, study staff may offer calcium carbonate tablets to correct the calcium loss. 
Leukapheresis can occasionally cause nausea and may temporarily lower platelet count. More 
rarely, vomiting, fainting, seizures, dizziness, muscle cramps, itching, flushing, or burning 
sensation, rash, swelling of hands and feet, headache, nasal congestion, sneezing, wheezing, or 
chest tightness can occur. After the procedure is complete, participants will be observed for 
30 minutes. 
9.1.6. 
Inguinal Lymph Node Biopsy 
Inguinal lymph node biopsies will be performed only at Visit 4 on participants who provide 
separate informed consent for the procedure. The procedure will be performed by trained 
personnel at the King Chulalongkorn Memorial Hospital, the Thai Red Cross AIDS Research 
Centre, or the Institute of HIV Research and Innovation (IHRI) Foundation. 
Inguinal lymph node biopsy will be performed by a qualified surgeon. Participants will be placed 
under local anesthesia and an inguinal lymph node will be removed via a surgical incision. The 
procedure will last approximately 30 to 40 minutes, however, participants will also be asked to 
rest under observation for up to 4 hours post-procedure. The use of aspirin and non-steroid anti-
inflammatory drugs by participants will be prohibited 7 days before the procedure so as to 
decrease the risk of bleeding. 
Participants agreeing to inguinal lymph node biopsy may be asked to undergo photography of the 
biopsy wound immediately after the procedure and again 6 months later in order to provide an 

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objective reference for assessment of wound healing and for possible use as participants’ 
educational material. Participants are free to refuse photography of their wound at any point 
should they not be amenable for any reason. 
Transportation will be organized by the study staff and provided at no cost to the participant. 
Accommodations will be offered for the participant’s convenience and not for medical need as 
all procedures are outpatient. The sigmoid biopsies, leukapheresis and Inguinal Lymph Node 
biopsies will be performed at the Thai Red Cross AIDS Research Centre, King Chulalongkorn 
Memorial Hospital, Bangkok, and/or Institute of HIV Research and Innovation (IHRI) 
Foundation.  
9.1.7. 
Future/Genetic Use of Study Samples 
Samples collected under this protocol will be used to conduct protocol-related safety and 
immunogenicity evaluations as noted in Table 3. Research samples will be processed and stored 
at the AFRIMS Department of Retrovirology Specimen Processing Laboratory (SPL).  
Stored samples with consent for genetic testing (Appendix V) may be used to elucidate genetic 
factors associated with immune response to a vaccine and to further evaluate responses to the 
vaccine. Results from genetic testing, whether for this study or from future genetic testing, will 
not be provided to participants as the results are not validated for clinical use. Any remnant 
tissue, cells, serum, or plasma will be stored for future exploratory virological and 
immunological assays as per individual participant’s consent for future use (Appendix E). Any 
future use of stored samples will require IRB review and approval prior to use. At the time of 
protocol termination, samples will remain at the AFRIMS Department of Retrovirology SPL or, 
after IRB approval, transferred to another repository. At this time, a list of participants who 
refused future use of their samples will be generated from the study database and the 
corresponding samples will be destroyed.  
9.1.8. 
HIV Risk Assessment and Risk Counseling 
Participants must be considered at low risk for HIV infection in order to meet eligibility criteria. 
This determination was also required for participation in the RV306 trial. Given that all 
participants in this study will be recruited from the pool of RV306 participants who completed 
all vaccinations, these participant’s risk level for HIV infection have already been determined as 
low. Despite their prior risk level assessment determining that they were at low risk, each 
participants risk level will be re-assessed in this study. 
Investigators will determine a participant’s level of risk for HIV infection based on various 
self-reported metrics, including but not limited to medical history as it pertains to STIs, use of 
condoms, use of drugs and alcohol, sexual preference, number of sexual partners, and other self-
reported measures with consideration of the low risk for HIV infection assessment given prior to 
participation in RV306. Information about a participant’s level of risk for HIV infection that is 
not already collected via questionnaire or CRF will be recorded in the visit notes, which are kept 
in the participants study file. Counseling for HIV risk reduction will be provided by trained 
counselors throughout the study (see Table 3). 

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9.2. 
Concomitant Medications 
Information pertaining to receipt of non-study vaccines, research agents, immunoglobulin 
preparations, immunosuppressive medication, antiretroviral drugs, any blood products, and any 
other medications that a participant uses will be elicited at study visits and recorded in source 
documents. 
To ensure appropriate medical follow-up for study participants, information regarding 
concomitant medications used in association with an AE will be collected and recorded in the 
source documents.  
9.3. 
Management of Participants Who Become Pregnant 
Pregnant women and women who plan to become pregnant during the first 3 months after study 
vaccine administration are excluded from enrollment. If a participant becomes pregnant during 
the course of the study, they will continue to be followed for the remainder of the study period 
per the SOE, however the participant will be excluded from optional invasive procedures and 
blood draws will be conducted for safety purposes only. Every effort will be made to remain in 
contact with the pregnant participant until birth or termination of the pregnancy in order to 
record the outcome of the pregnancy. 
Pregnancy outcomes will be recorded via a standardized pregnancy CRF. Information 
documented on this form will include date of last menstrual period, date pregnancy confirmed, 
history of complications during prior pregnancies (such as congenital abnormalities or 
spontaneous abortions) and pregnancy outcome information including date of termination or 
delivery, any complications of pregnancy, the gender, weight, and presence of any congenital 
abnormalities. 
9.4. 
Management of Participants Who Become Incarcerated 
Participation of prisoners is not planned, and any participant will be suspended from study visits 
while incarcerated, except visits or telephone contact for the purpose of ensuring participant 
safety. No study product will be administered to a participant who is incarcerated. The IRBs (see 
Table 5) will be notified of the period of incarceration. Data and samples that were collected 
before the period of incarceration may still be stored and used for analyses. After release from 
incarceration, the participant may either return to participation in study visits according to the 
SOE or study participation may be terminated at the discretion of the PI. 
Participants who have returned to the clinic after a period of incarceration will be counseled 
again about the potential risks of being a participant in the study. A note to the effect that the 
counseling was done will be written in the progress notes in the participant's binder. 
Any participant who is incarcerated for more than 6 months will be re-consented to include 
taking and passing the TOU. 
9.5. 
Management of Clinical Test Results and Participant Referrals 
Only results from approved clinical tests will be shared with participants, specifically: HIV test 
results, complete blood count, liver function tests and creatinine, urinalysis, pregnancy tests, 
gonorrhea/chlamydia test results, syphilis and hepatitis B serology. Results will be shared with 

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participants at a subsequent study visit unless it is of medical concern. For any results that are of 
medical concern, the participant will be contacted and notified as soon as possible and will be 
referred for care and treatment. At no point during the study will individual research results be 
shared with participants. 
Participants found to be infected with HIV or syphilis at screening will be counseled 
appropriately by a study physician/associate investigator or designee, and then referred to a local 
health care facility for care and treatment. 
Participants who are found to have signs or symptoms of reportable communicable diseases (see 
Section 15.2.1) will be referred to a local health care facility for immediate care. 
9.6. 
Procedures for Monitoring Participant Compliance 
All vaccinations and study procedures will be conducted under the direct supervision of the 
investigational staff. Study staff will be trained to perform study procedures in accordance with 
the protocol and Clinical MOP. Source documents, such as visit checklists will be kept to ensure 
that the appropriate procedures are completed at each visit. Participant charts will undergo 
regular quality control checks by study staff in order to confirm that all study procedures are 
being completed accurately and thoroughly. In addition to the internal QC processes, the study 
sponsor will perform periodic monitoring and quality assurance checks (see Section 14). 

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10. 
IMMUNOGENICITY ASSESSMENTS 
10.1. 
Specification of Immunogenicity Endpoints 
Table 4: 
Immunology Assays 
Humoral Assays 
Sample Type 
Function Measurement 
ADCC, ADCP, and other non-
neutralizing antibody functions 
Plasma/ Serum 
Measures lysis of HIV expressing targets 
mediated by HIV specific antibodies 
HIV-specific binding  
Plasma/ Serum 
Binding antibody to vaccine antigens 
HIV-specific neutralizing 
antibodies  
Serum  
Neutralizing activity against luciferase 
reporter gene expression 
Cellular and Innate Assays 
Sample Type 
Function Measurement 
Cellular response by cytokines 
such as IFN-γ and IL2 after 
stimulation with HIV-specific 
antigens 
PBMC 
1. CD4+ and CD8+ antigen-specific 
response 
2. Cellular functionality (CD4+; CD8+ 
only if sufficient CD8+ univariate 
responses) 
3. Cellular polyfunctionality (CD4+; 
CD8+ only if sufficient CD8+ 
univariate responses) 
Lymphocyte proliferation  
PBMC, sigmoid 
biopsies, or lymph 
node biopsies 
Characterize the function of proliferating 
cells in response to HIV antigens 
B-cell ELISPOT 
PBMC, sigmoid 
biopsies, or lymph 
node biopsies 
Measures cytokine secretion from B cells 
in response to HIV antigens 
Flow cytometry for innate 
immune cell phenotyping and a 
cytokine array assay 
Plasma/ Serum, 
sigmoid biopsies, or 
lymph node biopsies 
Phenotype NK and other innate cells and 
characterize the cytokines elicited by the 
different vaccine regimens 
DNA Microarray: gene 
expression to vaccine antigens 
PBMC, sigmoid 
biopsies, or lymph 
node biopsies 
Host gene expression profile and signature 
to vaccine antigens 
RNA sequencing: gene 
transcription to vaccine antigens 
PBMC, sigmoid 
biopsies, or lymph 
node biopsies 
Host gene transcription profile and 
signature to vaccine antigens 
Mucosal Assays 
Mucosal Secretions 
Function Measurement 
HIV-specific binding antibodies  
Semen, rectal 
secretions, cervico-
vaginal secretions 
Binding antibody to vaccine antigens 
 
CD4+, CD8+, and other T cell 
characterization 
Cells from lymph 
nodes biopsy, 
sigmoid biopsy, and 
cytobrush 
Defines phenotypic characteristics of T 
cells recognizing the antigen by multicolor 
flow cytometry 
Microbiome/Proteome 
Vaginal swab or 
cytobrush, penile 
swabs, rectal 
swabs/sponge, 
sigmoid biopsies 
Assessing the microbiome/proteome 

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10.2. 
Immunogenicity Endpoints 
Immunogenicity will be assessed using the following assays at time points defined in 
Section 7.1.2. 
10.2.1. 
Humoral Immune Responses  
HIV-specific Binding Antibody ELISA Assays will be performed to detect serum or plasma 
IgG and IgA binding antibodies to HIV-1 gp120, in particular to the V1V2 loops. Capture 
antigens will include V1V2 sequences from both subtype AE and Subtype B HIV-1 Env 
(gp70V1V2 92TH023 and gp70V1V2 Case A2), and HIV-1 Env gp120 proteins matched to 
sequences in AIDSVAX® B/E without the gD tag and with an 11 amino acid N-terminal 
deletion, represented as gp120 A244gD- D11 and gp120 MNgD- D11. Durability of IgG 
response will be assessed for each participant by estimating the decline in log10 IgG from peak to 
6- and 12-months post vaccination.  
Neutralizing Antibody Assays will be measured as a function of reductions in luciferase (Luc) 
reporter gene expression after a single round of infection in TZM-bl cells using high throughput 
analysis. Stocks of molecularly cloned envelope-pseudotyped viruses have been prepared by 
transfection in 293T/17 cells (American Type Culture Collection) and titrated in TZM-bl cells. 
This assay has been formally optimized and validated and was performed in compliance with 
Good Clinical Laboratory Practices, including participation in a formal proficiency testing 
program. Tier 2 neutralization was assessed using a panel of 11 pseudoviruses, and a global 
panel. 
Antibody Profiling will be conducted to assess antibody breadth and correlation of Fc receptor 
usage with antibody function. The current capacity for Antibody Profiling is the assessment of 
over 300 samples in one experiment. The beads presenting the HIV antigens will be distributed 
robotically in a high-throughput manner into 384 well plates. Similarly, the samples will be 
diluted robotically and incubated with the beads. Detection reagents will be added to the wells 
after washing. These detection reagents are inclusive of subclass, isotype and Fc receptor usage. 
Therefore, in one well, we will be able to capture 900 to 1000 data points from a single sample, 
and over 250,000 data points per experiment that will allow evaluation of the humoral immune 
response to these vaccine strategies. 
Antibody-dependent cell-mediated cytotoxicity and antibody-dependent cellular 
phagocytosis will evaluated using an amine reactive staining (Aqua) to identify killed target 
cells in addition to the loss of CFSE in the traditional RFADCC. This Aqua-based assay is high 
throughput, has a better signal to noise ratio and a lower coefficient of variation than the 
conventional CFSE-based RFADCC assay. The ADCP phagocytic score will also be calculated 
and compared with Antibody profiling data to evaluate the impacts on IgG subclass and Fc 
receptor usage on non-neutralizing function between groups. 
FLSC-Specific Antibody Assays. Sera collected at the various times pre- and post- vaccination 
are subjected to anti-FLSC, anti-gp120, and CD4i competitive ELISAs. FLSC and gp120 (BaL) 
assays are performed as a solid-phase ELISA using FLSC or gp120 as the antigens. The CD4i 
assay is a competitive solid-phase ELISA that analyzes the ability of the test samples to compete 
for biotinylated human monoclonal antibodies (e.g., A32). 

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10.2.2. 
Cellular Immune Responses 
Intra-cellular cytokine staining (ICS) assay. PBMCs will be stimulated with HIV-specific 
antigens and gated on cell surface markers such as CD4 and CD8 to determine antigen-specific 
cellular responses by individual cytokines, such as IFNγ and IL2, as well as a composite, 
calculated functional and polyfunctional response using the COMPASS analysis method. 
Cellular Proliferation. The proliferative responses of participant’s PBMC and/or tissue to HIV-
specific antigens and mitogens will be measured by the CFSE assay to characterize the function 
of proliferating cells.  
B-cell Responses. A highly sensitive B-cell ELISPOT system was developed, which allows the 
enumeration of antibod-secreting cells from diverse anatomical compartments directed against 
different structural determinants of HIV envelope. Both plasmablast and memory B cell 
responses will be quantified to detect gp120 A244-specific IgG producing plasmablasts and 
long-lived memory B cells using PBMCs and/or tissue. 
10.2.3. 
Mucosal Immune Responses 
Genital secretion samples will be assayed for cytokines and inflammatory markers using a 
Luminex® multiplex assay. IgG and IgA antibodies from anogenital secretions directed against 
HIV antigens such as gp120 and V1V2 will also be quantified by Luminex®. 
Vaginal swab, penile swab and rectal swab samples, and if available CVM and rectal sponge, 
will be assayed for microbiome and/or host proteome. Next-generation sequencing (NGS) has 
enabled the profiling of the microbiota within a single sample allowing for a high-throughput and 
cost saving approach using microbial 16S ribosomal RNA gene analysis. In addition, to 
understand how the microbiome and host immune functions are interacting in the context of 
vaccine responses the potential use of a novel proteomics approach will be employed. The 
proteome could be analyzed by mass spectrometry (MS), and protein identification and 
expression levels will be performed. This will provide information not only on microbial 
composition, but also on functional aspects of the microbiome and host immunological 
pathways. 
Sigmoid biopsy samples processing will include but is not limited to isolation of mucosal 
mononuclear cells for immediate assessment (due to poor recovery of cryopreserved sigmoid 
samples) of HIV-specific immune responses and cell phenotype including the percentage of 
CD4+ CCR5+ activated target cells. In addition, samples will be stored for evaluating the gut-
associated microbiome, immunohistochemical staining, and transcriptome analysis, and related 
studies. 
Lymph node biopsy samples will be processed by 2 methods. Part of the lymph node will be 
formalin fixed and embedded in paraffin for immunohistochemical staining for cellular 
phenotype and markers of immune activation. The other part will be processed for quantification 
of the frequency of antigen specific CD4 and CD8 T cells in responses to HIV 
immunostimulatory peptides similar to the ICS assay above, quantified by flow cytometry. 
10.2.4. 
Innate Immunity 
Characterization of Innate Immunity. Flow cytometric panels will be used to phenotype the 
different types of NK cells, while functional ICS will be used to characterize their function. 

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Luminex® multiplex assays will be performed to compare soluble cytokines and other factors in 
plasma/serum/tissue across vaccine regimens, to further characterize innate biologic pathways 
engaged. 
Innate and Early Adaptive Responses by Gene Activation Assessment. DNA microarrays 
will explore the innate and early adaptive immune responses to vaccination by screening 
genomic expression profile in PBMCs and/or tissue. 
Transcriptomics. Transcriptome data will be generated using RNASeq to investigate gene 
expression in correlation with increased immunogenicity to a particular vaccine combination. 
10.3. 
QA/QC for Immunogenicity Assays 
For all assays, standardized SOPs are used and technicians are trained theoretically and 
practically prior to conducting the assays, with all training documented. Cell viability and 
percent recovery of cryopreserved PBMC are monitored at USAMD-AFRIMS by a quarterly 
Immunology Quality Assessment (IQA) program (4 vials per interval prepared by 
2 technologists) conducted by Duke University School of Medicine. The ICS assays used by the 
laboratories in the US and Thailand are qualified and both laboratories are participating in the 
External Quality Assurance Program Oversight Laboratory (EQAPOL) from the Duke Human 
Vaccine Institute to allow external quality control of those assays.  
10.4. 
Specimen Archiving and Transfer 
Biological samples such as serum, plasma, PBMCs, whole blood, mucosal secretion (cervico-
vaginal secretion, penile swabs, semen, rectal secretion), sigmoid biopsies and lymph node 
biopsies remaining after all assays described in this protocol have been completed will be 
barcoded and archived using electronic specimen storage and tracking system. PBMCs will be 
stored at -125oC or lower and plasma/sera will be stored at -70oC or lower. Samples from 
participants who have provided consent for storage and future use will be archived in the 
AFRIMS Department of Retrovirology, SPL, Thailand for 5 years from protocol closure with 
possible extension after request to IRBs and Ethics Committees. If no archiving renewal is 
requested or approved, these specimens will be destroyed per applicable SOP’s of the archiving 
institutions. Samples from participants who have not provided consent for storage and future use 
will be destroyed at the end of the study in accordance with the archiving institutions SOP. The 
PI, the Sponsor, IRBs/ECs, SMCs, and authorized regulatory bodies may have access to the data 
regarding the archived specimens. 
Archived samples will only be used for research and will not be sold; nor will study participants 
receive payment, should samples lead to the development of new products in the future. Any 
future study requesting these samples must first be reviewed and approved by the IRB of each 
applicable institute. With concurrence from these parties, archived specimens will be shipped to 
the MHRP and collaborating laboratories for assays. 
After completion of laboratory work to meet with the study objectives, some of the leftover 
samples, including plasma, serum and/or PBMCs, will be provided to the Bio-Medical Resources 
Center, Department of Medical Sciences, Thai MoPH. As per the MoPH guidelines, the volume 
of blood and white blood cells that will be sent for storage at the Bio-Medical Resources Center 
is up to the researcher’s consideration, but it should be as much as possible to benefit Thailand. 

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The samples will only include the blood products that are collected at any timepoint after the 
participant received the vaccines or placebo injections. As this is for the benefit of Thailand, 
samples from all participants will be sent to the repository as availability permits. 

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11. 
PHARMACOVIGILANCE, SAFETY AND ADVERSE EVENT 
REPORTING 
11.1. 
Specification of Safety Endpoints 
11.1.1. 
Demographic/Medical History 
Gender, date of birth, level of education, occupation and baseline medical history of participants 
will be recorded. 
11.1.2. 
Vital Signs 
Body temperature, pulse, respiratory rate, and blood pressure will be measured both pre- and at 
about 30 minutes post-vaccination. 
11.1.3. 
Physical Examination 
For the purpose of assessing AEs, symptoms and directed medical examination will be 
performed based on the medical judgment of the investigator. In addition, participants will be 
asked to record their temperature and complete a diary card at home in the evening after the 
vaccination and each day for the next 7 days. Study staff will provide both thermometers and 
rulers to participants and will train participants on how to correctly use the thermometer, read the 
temperature, and measure any swelling or redness. 
11.1.4. 
Laboratory Assessments 
11.1.4.1. Hematology 
Complete blood cell count (with differential) will be performed on whole blood as per the SOE 
(Table 3). 
11.1.4.2. Blood Chemistry 
Creatinine and alanine amino-transferase will be tested on plasma or serum according to the 
SOE. 
11.1.4.3. Urinalysis 
Urinalysis by dipstick for blood, protein, and glucose will be performed as per the SOE. If 
dipstick testing indicates a hematuria level of 1+ at screening or enrollment visits or ≥1+ at any 
post-injection follow-up visits where dipstick testing is performed, the urine sample will undergo 
microscopic analysis. Results from microscopic analysis will be used to confirm a participant’s 
eligibility and to allow for the assessment of severity (see Section 11.3.1), if the result indicate 
an adverse event. The results will also allow for the identification of non-exclusionary, 
pre-existing conditions at baseline in comparison to post-injection laboratory results for the 
assessment the product safety. Per Section 11.2.1, any post-injection changes in laboratory 
results (i.e., a worsening of a pre-existing condition post-injection) or a clinically significant 
change in lab values will be considered an adverse event. 

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11.1.4.4. HIV Diagnostic Algorithm 
Diagnostic HIV testing will utilize a sequence of validated tests that will differentiate between 
vaccine induced seroreactivity and true HIV infection. Information to the study staff of each 
vaccine trial site will not include the results of specific tests but will state only HIV "infected" or 
"not infected", or that repeat testing is needed (as in the case of need for a verification specimen). 
Report of results will be delayed to at least 10 days from blood collection so that the timing of 
HIV test reporting does not compromise the double-blind nature of the trial, as a result returned 
immediately after EIA testing could signal the clinical team that this specimen did not require the 
Western blot or immunochromatographic testing which would be needed if vaccine had induced 
an antibody response. 
An HIV-1 EIA assay will be utilized throughout the course of the study. If the EIA is reactive, 
the test will be repeated in duplicate. If repeatedly reactive, an HIV Western blot or 
immunochromatographic test will be performed. If the Western blot or immunochromatographic 
test is positive, HIV nucleic acid testing (NAAT) will be performed on the plasma specimen 
stored at –70°C or lower. If the NAAT is positive, a diagnosis of HIV infection is suspected. 
Notification from the lab that a verification specimen is required is sent to the CRC at the 
research site and the participant is called back for counseling and repeat blood draw. A second 
blood specimen (verification specimen) will be obtained for complete repeat HIV diagnostics. If 
the second plasma specimen is positive both serologically and by NAAT, a diagnosis of HIV 
infection is considered established. If the verification specimen is not positive (negative or 
indeterminate), the participant will be counseled that one additional blood collection (second 
verification specimen) for retesting will be necessary. If results of this repeat verification 
specimen are positive, infection is established; if not positive, the participant will be informed 
that he/she is not HIV infected and will return to the protocol’s regular visit schedule. This 
process is outlined below in the HIV Testing Algorithm in Figure 2. 

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Figure 2: 
HIV Testing Algorithm 
 
*If the first verification specimen is found to be not HIV positive (negative or indeterminate), a second verification 
specimen will be collected and fully tested. If this repeat verification specimen is also negative, the participant is 
diagnosed as HIV not infected; if positive, the diagnosis of HIV infection is considered established. 
 

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11.1.4.4.1. Laboratory assays 
HIV EIA and HIV Western blot or immunochromatographic test: Either Thai or U.S. FDA-
approved kits maybe used.  
HIV nucleic acid tests: plasma collected in a suitable anti-coagulant (EDTA or ACD) will be 
assayed for HIV NAAT using at least one nucleic acid test platform. Supplemental nucleic acid 
tests may also be incorporated. 
Samples that are Western Blot or immunochromatographic test indeterminate may also be 
subject to supplemental Nucleic Acid testing at the discretion of the PI or designee.  
11.1.4.5. Pregnancy Screen 
Pregnancy tests will be performed on all female participants at screening; prior to the 
vaccination; at Visits 4, 5, and 6; and prior to optional procedures (mucosal secretion collection, 
leukapheresis, sigmoid biopsy, and inguinal lymph node biopsy) as indicated in the SOE. No 
vaccine or optional procedures will be performed if the pregnancy test is positive. 
11.2. 
Adverse Event Reporting 
The following terms, as defined by 21 CFR 312.32, apply to IND safety reporting. 
11.2.1. 
Adverse Event and Suspected Adverse Reaction 
An AE is any untoward medical occurrence in a clinical investigation participant administered a 
pharmaceutical product and does not necessarily have a causal relationship with this product. An 
AE can therefore be any unfavorable and unintended sign (including an abnormal laboratory 
finding), symptom, or disease temporally associated with the use of an investigational product, 
whether or not related to the investigational product. (International Conference on 
Harmonization (ICH) E6) 
An AE is considered to be any adverse change or exacerbation from a baseline condition that 
occurs following the administration of an investigational product whether or not the event is 
considered related to the investigational product. Examples of this include but are not limited to 
the following: 
• Adverse changes including new signs and symptoms, intercurrent illness modifying the 
clinical course, or the worsening of a baseline condition including the increased 
frequency of an event or an increased intensity of a condition. 
• Concomitant disease with onset or increased severity after the start of investigational 
product administration. 
• A new pattern in a pre-existing condition, occurring after the receipt of investigational 
product that may signal a clinically meaningful change. 
• Clinically significant changes in laboratory values. 
A suspected adverse reaction is any AE for which there is a reasonable possibility that the 
investigational product caused the AE, with “reasonable possibility” meaning there is evidence 
to suggest a causal relationship between the investigational product and the AE. This implies a 

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lesser degree of certainty about causality than adverse reaction, which means any AE caused by 
an investigational product. 
11.2.2. 
Solicited Adverse Event 
A solicited AE is a predetermined event, identified in the Investigator’s Brochure, which may 
reflect safety concerns related to the investigational product. AEs that will be solicited and 
assessed for this study include localized post-vaccination reactions such as erythema, induration, 
pain and tenderness, swelling and limitation of leg movement, and systemic reactions such as 
fever, tiredness, chills, myalgia, arthralgia, headache, nausea, and rash. These reactogenicity 
events will be recorded 30 minutes post-vaccination and during the 7 days following the 
vaccination. Any local or systemic reactogenicity events beyond 7 days will be reported as an 
AE. 
11.2.3. 
Serious Adverse Event or Serious Suspected Adverse Reaction 
As defined in 21 CFR 312.32, an AE or suspected adverse reaction is considered “serious” if, in 
the view of either the investigator or sponsor, it results in any of the following outcomes:  
• Death 
• Life-threatening AE 
• Inpatient 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 AE or suspected adverse reaction is considered “life-threatening” if, in the view of either the 
investigator or sponsor, its occurrence places the participant at immediate risk of death. It does 
not include an AE or suspected adverse reaction that, had it occurred in a more severe form, 
might have caused death. 
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 participant 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. 
11.2.4. 
Unexpected Adverse Event or Unexpected Suspected Adverse Reaction 
An AE 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. An 
unexpected AE for this study refers to an adverse vaccine experience that has not been 
previously observed (not included in the investigator brochure) rather than from the perspective 
of such experience not being anticipated from the pharmacological properties of the vaccine. 
Expected Events are described in the Investigator Brochure and the Clinical MOP. All other 
events would be considered as unexpected. 

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11.2.5. 
Other Adverse Events 
All other AEs (those that do not fall under the categories of solicited, an SAE or unexpected) that 
are identified by site staff, study physicians or the PI, will be documented in the participant’s 
clinic records and entered in the study CRFs, including AESIs as listed in Appendix H.  
11.3. 
Relationship to Investigational Product 
The investigator must assign a relationship of each AE to the receipt of the investigational 
product. The investigator will use clinical judgment in conjunction with the assessment of a 
plausible biologic mechanism, a temporal relationship between the onset of the event in relation 
to receipt of the investigational product, and identification of possible alternate etiologies 
including underlying disease, concurrent illness or concomitant medications. The relationship of 
the vaccination to an AE will be determined based on the following definitions:  
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 participant’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 participant’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. 
11.3.1. 
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 one of the following categories: mild, 
moderate, severe, or potentially life-threatening. Refer to the DAIDS grading scale in 
Appendix B for further guidance in the assignment of severity. The criteria below may be used 
for any symptom not included in the grading scale. Any grade 4 (potentially life-threatening) or 
grade 5 (death) AE must be reported as an SAE. 
The severity reported on the AEs form will reflect only the highest severity for continuous days 
an event occurred. 
Mild (Grade 1): Does not interfere with routine activities; minimal level of discomfort. 
Moderate (Grade 2): Interferes with routine activities; moderate level of discomfort. 
Severe (Grade 3): Unable to perform routine activities; significant level of discomfort. 

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Potentially life-threatening (Grade 4): Hospitalization, ER visit, or urgent intervention 
indicated for potentially life-threatening event. 
Death (Grade 5): Death. 
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 participant/event outcome or action criteria 
usually associated with events that pose a threat to a participant’s life or functioning. Seriousness 
(not severity) serves as a guide for defining regulatory reporting obligations.  
11.4. 
Independent Safety Monitor 
The Independent Safety Monitor is a physician with relevant expertise whose primary 
responsibility is to provide independent safety monitoring in a timely fashion in order to assure 
the safety of research participants, regulatory compliance, and data integrity. The Independent 
Safety Monitor is required to review and provide an unbiased written report for all SAEs and 
participant deaths to the ORA PSSO within 24 hours of their awareness of the event. The report 
provided must include, at a minimum, a brief summary of the Independent Safety Monitor’s 
review of the event and event outcome, relationship of the event to the investigational product, 
and alternate etiology if the event is deemed unrelated to the investigational product. 
11.5. 
Protocol Safety Review Team 
The Protocol Safety Review Team (PSRT) will review all AEs (including reportable AEs) on a 
regular and expedited basis, as needed. In addition, the PSRT will review aggregate safety data 
reports on a weekly basis until 4 weeks after all participants have completed the vaccination, 
then the safety reviews will occur monthly. The PSRT will review safety events that potentially 
meet study pause criteria on an expedited basis and will decide if vaccinations should be paused 
for participant safety purposes. The PSRT may pause vaccination at any time but will use as a 
guide, based upon an abundance of data supporting safety of these vaccinations, the study pause 
criteria listed in Section 7.6.1. The PSRT will review the events that led to a study pause and will 
make the decision to consult the SMC, resume, amend or close the study. Vaccinations will 
resume only if PSRT and SMC (if consulted) reviews of the events that led to the pause result in 
a recommendation to permit further vaccinations. Screening and follow-up visits will continue 
uninterrupted during a vaccination pause. 
The PSRT will include the following:  
• Sponsor’s Medical Expert/Protocol Chair or designee 
• Site PIs or designees 
• Sponsor Representative or designee 
• ORA PVG Physician (only required for study pause reviews) 
• Independent Safety Monitor 
Additional PSRT participants may include, as needed, the following: 
• Associate Investigators  
• Clinical research nursing staff 

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• Laboratory directors  
• Data management and regulatory staff 
• Site investigators 
PSRT conference calls or meetings will require, at a minimum, the participation of the Sponsor’s 
Medical Expert/Protocol Chair or designee, at least one site PI or designee, a representative of 
the sponsor (may include ORA PVG Physician as necessary), an ORA PVG Physician (only 
required for study pause reviews), and the Independent Safety Monitor. If the Independent Safety 
Monitor is not available for the PSRT conference call/meeting, they must provide input by email 
and then be immediately emailed the outcome of the PSRT discussion. The IRBs will be notified 
of the decisions taken by the PSRT if they involve suspensions, clinical holds (voluntary or 
involuntary), or terminations of this research in accordance with Section 11.8.2.2. The 
USAMRDC ORA will be responsible for safety reporting to the U.S. FDA. 
11.6. 
Safety Monitoring Committee Reviews 
The SMC for this study will be comprised of an independent group of experts who review safety 
data during the clinical trial. The SMC will meet annually to review blinded study data, after the 
first eight participants are enrolled across Groups 1 and 2 to determine whether it is safe to 
continue enrollment into those groups, after all participants are enrolled in Groups 1 and 2 to 
determine whether it is safe to start enrollment into Groups 3 and 4, and after the first eight 
participants are enrolled across Groups 3 and 4 to determine whether it is safe to continue 
enrollment into those groups. Enrollment will continue unrestricted after any safety concerns 
raised by the SMC are addressed and the Sponsor gives their approval for the continuation of 
enrollment. Screening and follow-up visits will continue uninterrupted during a vaccination 
pause. In addition to the scheduled meetings, the SMC will meet at the request of the PSRT to 
evaluate the next steps during study safety pauses and/or to provide other recommendations 
regarding the safe conduct of the study.  
After all SMC reviews, the SMC Administrator will provide the SMC recommendations to the 
Sponsor’s Representative. The Sponsor’s Representative will then decide whether to continue, 
modify, or suspend the study. The USAMRDC ORA PSSO will communicate the final decision 
to the investigators, who in turn will notify the IRBs and regulatory authorities as appropriate. 
The sponsor will notify the FDA of the final decision, as appropriate. 
11.7. 
Recording Adverse Events 
Section 7.3.3 presents procedures to request unblinding of a participant’s randomization code 
due to a medical emergency or serious medical condition. 
11.7.1. 
Methods/Timing for Assessing, Recording, and Analyzing Safety Endpoints 
AEs, solicited AEs, and SAEs will be assessed at all study visits, documented in the source 
records, and recorded on the CRFs using accepted medical terms and/or the diagnoses that 
accurately characterize the event. When a diagnosis is known, the AE term recorded on the CRF 
will be the diagnosis rather than a constellation of symptoms. The investigator will assess all 
AEs for seriousness, relationship to investigational product, severity, and other possible 
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The timeframe for the collection of AEs and SAEs begins at the time of administration of 
investigational product and persists through the end of the study. When an event has not resolved 
by study closure, it will be documented on the AE CRF as “not recovered/not resolved”. 
11.7.2. 
Duration of Follow-Up of Participants After Adverse Events 
Non-clinically significant AEs still ongoing at the end of the study will be listed as “not 
recovered/not resolved”. SAEs ongoing at the end of the study will be followed to resolution 
(return to baseline status) or stabilization of the condition with the probability that it will become 
chronic. All SAE outcomes will be reported to the USAMRDC ORA PSSO using the SAE 
Report Form. This form should include both a start date and, when available, a resolution date to 
indicate when the AE met serious criteria and when it resolved, stabilized, or was determined 
chronic. 
Investigators are not obligated to actively seek SAEs in former participants; however, if a SAE, 
considered to be related to the investigational product is brought to the attention of the 
investigator at any time following completion of the study, the event will be reported to the 
sponsor’s safety office as defined in Section 11.8.1.1. 
11.8. 
Reporting Adverse Events 
The PI, co-investigators, site investigators and site staff will exercise due diligence in 
ascertaining, accurately recording and promptly entering data on the CRF for all AEs for all study 
participants. As data becomes available from the participant, the clinics and the laboratories, AEs 
will be recorded and entered on the CRF by the site staff. Site investigators will review this AE 
source data in a timely manner and will determine the severity of the events and their relation to 
the study vaccines. Site investigators are encouraged to contact the USAMRDC ORA PSSO for 
consultation regarding AEs. 
The PI will report all AEs to the USAMRDC ORA PSSO and the IRBs (see Table 5) in the 
appropriate safety, annual, and/or final reports. The study site will provide data files to the 
sponsor’s representative for preparation of annual and final reports to the U.S. FDA.  
11.8.1. 
Reporting Serious and Unexpected Adverse Events 
Contact information for reporting SAEs is provided in Table 5. 
Table 5: 
Study Contacts for Reporting Serious and Unexpected Adverse Events 
Sponsor’s Safety Office 
US Army Medical Research & Development Command 
Office of Regulated Activities 
Product Safety Surveillance Office 
ATTN: FCMR-ORA 
1430 Veterans Drive 
Fort Detrick, MD 21702-5009 
Fax: +1 301 619 7790 
Telephone: +1 301 619 1005 
Email: usarmy.detrick.medcom-usamrmc.mbx.sae-
reporting@health.mil 
 

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Institutional Review Board 
Ministry of Public Health Ethical Committee 
Office of the Secretary, the Ethical Review Committee of 
Research in Human Subjects 
3rd Floor, Department of Medicine Services Building 
Tiwanon Road, Nonthaburi 11000, Thailand 
Telephone: +66 2 591 8251, +66 2 590 6171-2 
Fax: +66 2 591 8251 
Email: ecmoph@gmail.com 
 
Royal Thai Army Medical Department IRB 
5th floor, Phramongkutklaovejvithaya Building  
Phramongkutklao Medical School  
315 Rajvithi Road,  
Bangkok 10400, Thailand  
Telephone: +66 2 354 7600, Ext 94270; +66 2 763 4297 
Fax: +66 2 354 9011  
Email: irbrta@yahoo.com 
 
Ethics Committee of the Faculty of Tropical Medicine  
Mahidol University c/o Research and Academic Services  
4th Floor, The 60th Anniversary of His Majesty the King’s 
Accession of the Throne Building  
420/6 Rajvithi Road,  
Bangkok 10400, Thailand  
Telephone: +66 2 354 9100-4 extension 1349, 1525  
Fax: +66 2 306 9126  
Email: tmectropmed@mahidol.ac.th 
 
The Institutional Review Board, 
Faculty of Medicine, Chulalongkorn University 
1873, 3rd Floor, Ananthamahidol building, 
Rama IV Rd., Patumwan, 
Bangkok, Thailand 
Telephone: +66 2 256 4493 
Fax: +66 2 255 4493 
Email: medchulairb@chula.ac.th 
 
Walter Reed Army Institute of Research IRB 
503 Robert Grant Avenue 
Silver Spring, MD 20910, USA 
Telephone: +1 301 319 9940 
Email: usarmy.detrick.medcom-wrair.mbx.hspb@health.mil 
 
Thai FDA  
IND Section, Medicine Regulation Division (Bureau of Drug 
Control),  
Food and Drug Administration 
Ministry of Public Health 
88/24 Tiwanond Road, Nonthaburi 
11000, Thailand  
Telephone: +66 2 590 7061 
 

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Note: No email address. The report will be submitted via 
http://thaihpvc.fda.moph.go.th or hard copy 
USAMRDC Office of Human and 
Animal Research Oversight 
Office of Human Research Oversight 
Office of Human and Animal Research Oversight (OHARO) 
US Army Medical Research and Development Command,  
ATTN: FCMR-RPH 
504 Scott Street 
Fort Detrick, MD 21702-5012 
Fax: +1 301 619 7803 
Telephone: +1 301 619 2165 
Email: usarmy.detrick.medcom-usamrmc.other.hrpo@health.mil 
 
11.8.1.1. Reporting to the Sponsor 
The investigator will report SAEs to the USAMRDC ORA PSSO according to the requirements 
in Table 6. Further, the investigator should comply with relevant study site SOPs on reporting 
AEs judged to be related to the investigational product and SAEs. 
The information that the investigator will provide to the USAMRDC ORA PSSO includes, but is 
not limited to the following: 
• Investigation new drug application number, sponsor study number 
• Name of the investigational product 
• Investigator name and contact number  
• Subject identification number 
• Serious adverse event term, description 
• Onset date 
• Date(s) of investigational product administration 
• Severity 
• Relationship to the investigational product 
• Participant’s current status 
• Admission/Discharge Summary 
• Consultations 
• Medical record progress notes including pertinent laboratory/diagnostic test results 
When reporting via email, the subject line should be formatted as follows: 
Initial: SAFETY REPORT – IND # 27011, Sponsor Study #S-19-01, Subject# _____, Event 
term: _____ 
Follow-Up: SAE Case Number provided by PSSO (e.g., US-000123), SAFETY REPORT – 
IND # 27011, Sponsor Study #S-19-01, Subject# _____, Event term: _____ 
Table 6: 
Sponsor’s AE Reporting Requirements 
Event 
Report 
Notification Time 
Notification Method 
SAEs 
SAE Report* 
Within 24 hours of event awareness 
Email: 
usarmy.detrick.medcom-
Pregnancy 
Pregnancy Report 
Within 24 hours of event awareness 

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Events of Special 
Interest 
SAE/Events of Special 
Interest Report* 
Within 24 hours of event awareness 
usamrmc.mbx.sae-
reporting@health.mil 
 
If email is not available, 
the information can be 
faxed or called in: 
Fax: +1 301 619 0197 
Phone: +1 301 619 1005 
AE-related 
Withdrawals 
Contact USAMRDC 
ORA PSSO 
Within 5 business days of event 
awareness 
Safety-related 
Deviations 
Contact USAMRDC 
ORA PSSO 
Within 5 business days of event 
awareness 
*Submission of the event report to USAMRDC ORA PSSO must not be delayed 
In order to comply with regulations mandating sponsor notification of specified SAEs to the U.S. 
FDA within 7 calendar days, investigators must submit additional information as soon as it is 
available. The sponsor’s representative will report unexpected SAEs associated with the use of 
the drug to the U.S. FDA as specified at 21 CFR 312.32 (c).  
Investigators must follow all relevant regulatory requirements as well as specific policy 
regarding the timely reporting of SAEs to the USAMRDC OHARO. 
There may be instances when copies of medical records for certain cases are requested by the 
sponsor’s safety office. In this case, all participant identifiers, with the exception of the subject 
number, must be redacted on the copies of the medical records before submission to the 
sponsor’s safety office. 
Reporting to the sponsor’s safety office does not fulfill the investigator’s duty to report all SAEs 
to the IRBs. The PI is responsible for notifying the IRBs (see Table 5) and the USAMRDC 
OHARO. 
11.8.1.2. Reporting to the IRBs 
All SAEs related to participation in the study and all deaths will be promptly (within 48 hours) 
reported to the IRBs and written reports will be submitted to the IRBs within 10 working days. 
The WRAIR HSPB will report all related SAEs and all participant deaths to USAMRDC 
OHARO OHRO as per USAMRDC Command Policy 21. Follow up reports will be submitted as 
additional information becomes available. 
A summary of the non-serious AEs and SAEs (both related and unrelated) that occurred during 
the reporting period will be included in the continuing review report (CRR) to the IRBs/ECs. 
Investigators are required to forward safety information provided by the sponsor’s representative 
to the IRBs in a timely manner. 
11.8.2. 
Additional Immediately Reportable Events 
11.8.2.1. Unanticipated Problems Involving Risks to Subjects or Others 
Unanticipated problems involving risks to subjects or others (UPIRTSO) are any incident, 
experience, or outcome that meets all of the following criteria: 
• Is unexpected (in terms of nature, severity, or frequency) given 
o the procedures that are described in the protocol, investigator’s brochure, or 
informed consent document; and 
o the characteristics of the subject population 
• Is related or possibly related to participation in the study 

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• Suggests that the protocol places participants or others at a greater risk of harm than was 
previously known or recognized 
These events encompass a broader category of events than SAEs and may include issues such as 
problems with loss of control of participant data or the investigational product; adverse 
psychological reactions; or breach of confidentiality. 
The principal investigator will determine whether a given incident, experience or outcome 
constitutes a UPIRTSO and will promptly (within 48 hours) report UPIRTSOs to the IRBs and to 
the sponsor. The Principal Investigator will then submit a written report within 10 working days 
to the IRBs.  
Follow up reports should be submitted as soon as additional information becomes available. A 
summary of UPIRTSOs will also be included in the continuing review report submitted to the 
IRBs. The WRAIR HSPB will report UPIRTSOs to the USAMRDC OHARO OHRO as per 
USAMRDC Command Policy 21. 
11.8.2.2. Study Pause or Termination 
The IRBs will be immediately notified of any suspensions, clinical holds (voluntary or 
involuntary), or terminations of this research by any of the IRBs, the PSRT, the institution, the 
sponsor, or regulatory agencies. WRAIR HSPB will forward the notification to USAMRDC 
OHARO OHRO as per USAMRDC Command Policy 21. 
11.8.2.3. Pregnancy 
Each pregnancy must be reported to the USAMRDC ORA PSSO within 24 hours of 
identification by completing and submitting the Pregnancy Report Form by email or fax. 
Pregnancies must be reported within 48 hours of becoming aware of the event to the IRBs. 
WRAIR HSPB will forward the notification to USAMRDC OHARO OHRO as per USAMRDC 
Command Policy 21. 
Participants who become pregnant after Day 0 will be followed to term, and the following 
information will be reported on the Pregnancy Report: type and date of delivery, Apgar scores, 
and health status of the mother and child including the child’s gender, head circumference, 
gestational age at delivery, height and weight.  
Complications and or abnormalities should be reported including any premature terminations. A 
pregnancy is reported as an AE or SAE only when there is suspicion that the investigational 
product may have interfered with the effectiveness of contraception or there was a serious 
complication in the pregnancy including a spontaneous abortion or an elective termination for 
medical rationale. Otherwise, pregnancy will not be categorized as an adverse event. 
11.8.2.4. Participant Status Change: Incarceration 
When a previously enrolled participant becomes a prisoner and the relevant research protocol 
was not reviewed and approved by the IRB in accordance with the requirements of DODI 
3216.02 section 3.9.c, to include prisoners as research participants, the PI shall promptly notify 
the IRBs. WRAIR HSPB will forward the notification to USAMRDC OHARO OHRO as per 
USAMRDC Command Policy 21. 

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11.8.2.5. AE-related Withdrawal of Consent 
Any AE-related withdrawal of consent during the study must be reported by email or fax to 
USAMRDC ORA PSSO within 5 business days of event awareness. The IRBs will be notified of 
the withdrawal in a follow-up report as per Section 11.8.1.2. 
11.8.2.6. Pending Inspections/Issuance of Reports 
The knowledge of any pending compliance inspection/visit by the U.S. 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, U.S. 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 IRBs and the sponsor’s representative. The WRAIR HSPB 
will report knowledge of any pending inspections/audits by regulatory agencies to the 
USAMRDC OHARO OHRO as per USAMRDC Command Policy 21. 
11.8.3. 
IND Annual Report to the U.S. FDA 
The sponsor’s representative will notify the PI of the due date of the annual report with sufficient 
time for the PI to assemble and submit all of the required clinical study information to the 
sponsor’s representative. 
The Sponsor and Sponsor’s representative (USAMRDC) will be responsible for the preparation 
of the detailed annual synopsis of clinical activity, including AEs, for submission to the U.S. 
FDA. Each annual report will summarize IND activity for one year beginning approximately 
3 months before the IND U.S. FDA anniversary date. Similar information will also be submitted 
to Thai FDA on an annual basis. 
11.8.4. 
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 U.S. FDA.  

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12. 
STATISTICS 
12.1. 
Analysis Overview 
This study intends to descriptively characterize safety and immune responses following delayed 
boosting of individuals previously vaccinated with a late boost of AIDSVAX®B/E with or 
without ALVAC in the RV306 protocol. Statistical analysis considers targeted enrollment into 
the following 5 groups: 
• Placebo (n=16) 
• Group 1: IHV01 and A244/AHFG Full Dose (n=16) 
• Group 2: IHV01 and A244/AHFG Fractional Dose (n=16) 
• Group 3: IHV01 and A244/AHFG Full Dose + ALFQ (n=16) 
• Group 4: IHV01 and A244/AHFG Fractional Dose + ALFQ (n=16) 
Collapsed comparisons of interest include full dose vs. fractional dose (Groups 1+3 vs. Groups 
2+4) and effect of ALFQ (Groups 1+2 vs. Groups 3+4). The placebo group is included primarily 
for purposes of blinding safety and immunogenicity assessments, including blinded samples for 
the set of immunology assays. The main interest is in determining the safety of IHV01 (FLSC), 
A244/AHFG and ALFQ as well as whether immunologic differences can be detected between 
the 4 antigen-containing regimens. Response rates and, where applicable, titer level distributions 
with appropriate summary statistics will be estimated for each immunologic assay performed. 
The study will evaluate a series of cellular, humoral and mucosal immune assays. In many cases, 
trial specimens will be used to further evaluate the practicality of sampling, specimen processing 
and storage procedures combined with assay performance characteristics. As this study will 
enroll only participants who had completed the RV306 study, participant data from RV306 will 
be used in conjunction with data collected in this study to provide a more in-depth analysis of the 
effect of delayed boosting.  
12.2. 
Final Analysis Plan 
12.2.1. 
Analysis Populations 
All participants who received the active vaccination or placebo, and for whom any post-dose 
data is available, will be included in the safety population. The immunogenicity population will 
consist of all participants who received the vaccination or placebo and have at least one 
measured post dose blood sample collected. 
12.2.2. 
Demographics and Baseline Characteristics 
Participant demographics and baseline characteristics will be summarized. The number of 
participants who enroll in the trial, and the number and percentage of participants who complete 
each assessment, will be presented. The percentage of participants who withdraw from the trial 
or discontinue the study drug, and reasons for withdrawal or discontinuation, will be 
summarized. 

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12.2.3. 
Safety Analysis 
AEs will be listed individually and summarized by body system and preferred terms within a 
body system for each treatment group. Serious and/or unexpected AEs will also be discussed on 
a case-by-case basis. For the tabulation of the AEs by body system, a participant will be counted 
only once in a given body system. For example, a participant reporting nausea and diarrhea will 
be reported as one participant, 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 
participants within the body system reporting AEs. AEs will also be described by time period 
(i.e., overall and within 28 days of vaccination).  
The occurrence of local and systemic reactogenicity symptoms, AEs and SAEs will be assessed 
as the proportions of participants experiencing such safety events along with 95% CIs. A key 
comparison of interest is between participants receiving vaccination with and without ALFQ 
(i.e., Groups 1+2 vs. Groups 3+4). Between group and collapsed comparisons will be performed 
using a chi-square (or exact test as appropriate).  
12.2.4. 
Immunogenicity Analysis 
The primary immunogenicity endpoint is the magnitude of immune response to the endpoint titer 
of plasma IgG, the same variable assessed in RV306. Immune response over time (durability) 
will be assessed by the positive incremental area under the curve (AUC) based on a graph with 
log endpoint titer on the y-axis and visit day on the x-axis. Summary statistics at specific time 
points of interest will also be provided using the mean and standard deviation (or median and 
IQR as appropriate). Specific comparisons of interest include overall effect of the vaccine (all 
active vs. placebo), the effect of ALFQ (Groups 1 + 2 vs. Groups 3 + 4), and the effect of 
fractional dosing (Groups 1+3 vs. Groups 2 + 4). Primary comparisons will use a 2-sample t-test 
with an unadjusted alpha of 0.05. Further exploratory comparisons may be performed and 
adjusted for multiplicity as appropriate. 
Secondary immunogenicity endpoints will entail primarily descriptive analyses that mirror 
similar assessments being performed in the RV306 trial, as described in Section 10. In general, 
continuous outcomes will be assessed using summary statistics such as the mean, median, 
standard deviation and IQR. Binary responses will be described using frequencies and 
percentages. Changes from baseline may be assessed within groups using paired t-tests (or 
Wilcoxon signed rank as appropriate) or McNemar’s test for binary outcomes. Changes over 
time between groups may be evaluated using longitudinal models with unstructured covariance 
matrices where possible or covariance strictures selected as appropriate for the observed data. 
12.2.5. 
Missing Values and Outliers 
All attempts will be made to collect all data per protocol. No imputation will be performed for 
missing values. Outliers will not be excluded from the primary analyses. Outliers identified 
during the analysis will be discussed in the analysis report. 
12.2.6. 
Subgroup Analysis 
Gender is a key covariate of interest. While the study is not specifically powered to detect gender 
differences, safety and immunogenicity analyses may also be described by gender to evaluate 
potential gender specific differences. 

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12.2.7. 
Clinical Laboratory Data Analyses 
Clinical laboratory values, including change from baseline, will be summarized by dose cohort 
and control group. The values will be graded according to the toxicity scale (Appendix B) and, if 
clinically significant, reported as AEs. 
Descriptive summary statistics (mean, SD, median, minimum, and maximum) for clinical 
laboratory data at admission and each applicable post-dosing visit, including changes from the 
baseline value collected at admission, will be calculated. If multiple baseline values are obtained, 
only the most recent value will be analyzed. For change-from-screening summaries, participants 
with an undefined change from screening, due to missing data, will be excluded. Clinical 
significance of abnormalities will be indicated in the listings. 
12.3. 
Planned Enrollment and Sample Size Consideration 
The primary purpose of the study is to evaluate the safety of a single dose of IHV01 and 
A244/AHFG with or without ALFQ. Table 7 depicts adverse event rates and associated 95% 
exact Clopper-Pearson confidence intervals in each potential grouping of interest, given 0, 1, 5, 
10, or 15 participants with observed events. If we were to observe zero events within a single 
active study group, (n=16), the upper limit of a 2-sided exact 95% CI would be 20.6% and true 
event rates above this could be ruled out at the α=0.025 level.  
Table 7: 
Exact 95% Clopper-Pearson CI for the AE Rate in Each Study Group 
Group 
Sample 
Size 
Number of Participants with an Event(s) Observed 
0 
1 
5 
10 
15 
All Active 
Groups 
64 
0% 
(0%, 5.6%) 
1.6% 
(0%, 8.4%) 
7.8% 
(2.6%, 17.3%) 
15.6% 
(7.8%, 26.9%) 
23.4% 
(13.8%, 35.7%) 
Fractional 
Dose (Groups 
2+4) 
32 
0% 
(0%, 10.9%) 
3.1% 
(0.1%, 16.2%) 
15.6% 
(5.3%, 32.8%) 
31.2% 
(16.1%, 50%) 
46.9% 
(29.1%, 65.3%) 
Full Dose 
(Groups 1+3) 
ALFQ 
(Groups 3+4) 
No ALFQ 
(Groups 1+2) 
Any 
Individual 
Analysis 
Group 
16 
0% 
(0%, 20.6%) 
6.2% 
(0.2%, 30.2%) 
31.2% 
(11%, 58.7%) 
62.5% 
(35.4%, 84.8%) 
93.8% 
(69.8%, 99.8%) 
While not primary, assessment of immunogenicity is a secondary objective. Table 8 depicts the 
minimum detectable differences in standard deviations for a 2-sample t-test of the IgG antibody 
measures at an alpha of 0.05 for key comparisons of interest. We assume that the 
immunogenicity measures will be normally distributed after log transformation, similar to 
RV306. As per the table below, collapsed comparisons provide adequate power to quantify our 
primary endpoints. For example, we would have an 80% power to detect a 1.02 standard 

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deviation difference between the mean log IgG titer or mean AUC of individual active groups at 
the α=0.05 level with a 2-sample t-test. Based on the relatively tight standard deviations 
observed in RV306, we can reasonably estimate that the log IgG endpoint titer in Group 1 will 
have a standard deviation ranging from about 0.5 to 1.0 depending on the time point. 
Table 8: 
Minimal Detectable Differences, in Standard Deviations, for a 2-Sample 
T-Test (2 sided, α=0.05) 
Comparison 
Sample sizes for 
comparison groups 
Power 
Group 1 
Group 2 
80% 
85% 
90% 
All Active vs. Placebo 
64 
16 
0.79 
0.85 
0.92 
Fractional Dose (Groups 2+4) vs. Full Dose (Groups 1+3) 
32 
32 
0.71 
0.76 
0.82 
ALFQ (Groups 3+4) vs. No ALFQ (Groups 1+2) 
Between Individual Analysis Groups 
16 
16 
1.02 
1.09 
1.18 
12.4. 
Level of Significance to be Used 
Five percent level two-sided tests will be used when comparing safety and primary 
immunogenicity outcomes. Secondary and exploratory outcomes will control for multiplicity as 
appropriate for each analysis. 
12.5. 
Interim Analyses 
Interim analyses may be completed to assess early effects on cytokine production prior to 
introduction into other trials and to ensure the safety of study participants. These data will be 
assigned a blinded unique identifier separate from subject ID to ensure maintenance of blinding. 
The PI will ensure concurrence from USAMRDC ORA as outlined in Section 17 prior to 
presentation or publication of data from interim analyses. Any analyses conducted prior to the 
end of the scheduled follow-up visits will not compromise the integrity of the trial in terms of 
participant retention or safety or immunogenicity endpoint assessments. In particular, early 
unblinded analyses by treatment assignment require careful consideration and will be made 
available on a need to know basis only. 
12.6. 
Statistical Criteria for the Termination of the Trial 
There are no statistical criteria for the purpose of study termination in this clinical trial. 
12.7. 
Procedures for Reporting Deviations from the Statistical Plan 
Any deviation(s) from the statistical plan as indicated in the protocol will be described in an 
amendment to the protocol or the clinical study report. The protocol amendment will be 
submitted to all IRBs as per Section 15.1.2.  
12.8. 
Accounting for Missing, Unused, and Spurious Data 
Non-analyzable data will be documented in the deviations. 

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13. 
DIRECT ACCESS TO SOURCE DATA/DOCUMENTS 
Participants will be identified on eCRFs and source documents by a unique participant 
identification number. No personal identifier will be used in any publication or communication 
used to support this research study. The participant identification number will be used if it 
becomes necessary to identify data specific to a single participant. Clinical and research records 
may be reviewed by representatives of USAMRDC OHARO OHRO, WRAIR IRB, MoPH EC, 
RTA IRB, Chulalongkorn IRB, Mahidol FTM EC, U.S. FDA, Thai FDA, the US DoD, the 
sponsor, the sponsor’s representative and other regulatory agencies as part of their 
responsibilities for insuring the protection of research participants. Personal identifiers will be 
removed from photocopied medical and research records. 
13.1. 
Study Monitoring 
Study monitoring will be executed under contract by experienced, independent study monitors 
with monitoring oversight and regulatory compliance falling under the responsibility of the 
USAMRDC ORA. Prior to initiating the monitoring services, 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 participants and the investigators’ 
compliance with the protocol and GCPs. 
Monitoring visits by the independent study monitors will be scheduled to take place during the 
study at appropriate intervals, and after the last participant has completed the study. A report of 
monitoring observations will be provided to the PI (for corrective actions), USAMRDC ORA, 
and the product manager. 
13.2. 
Audits and Inspections 
Authorized representatives of the sponsor, the U.S. FDA, Thai FDA, the independent ethics 
committee or institutional review board 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.  
The investigator should contact the sponsor’s representative, WRAIR HSPB/IRB and OHARO 
OHRO immediately if contacted by a regulatory agency about an inspection. 
13.3. 
Institutional Review Board 
The PI must obtain approval from all IRBs (Table 5) prior to initiation of the study. Initial IRB 
approval, and all materials approved by the IRBs, including the participant consent form and 
recruitment materials, must be maintained by the investigator and made available for inspection. 

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The PI will be responsible for preparing and submitting CRRs per institution and IRB 
requirements (see Section 15.1.4). The PI or a designee will transmit the approved final study 
report to the IRBs as soon as the documents are available. 

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14. 
QUALITY CONTROL AND QUALITY ASSURANCE 
To ensure compliance with GCP and all applicable regulatory requirements, the sponsor’s 
representative may conduct quality assurance audits. Refer to Section 13.2 for more details 
regarding the audit process. 
Auditing of the clinical trial may be conducted at any time during the study to ensure continued 
compliance with regulations, policies and procedures. Auditing will be undertaken, as needed, by 
independent personnel designated by USAMRDC ORA. Audit findings will be documented in a 
formal audit report that will detail the conduct of the audit and summarize the observations 
noted. 

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15. 
ETHICS 
15.1. 
Ethics Review 
The study is based on suitably performed laboratory and animal experimentation and will be 
conducted under a protocol reviewed by all applicable IRBs (see Table 5) and USAMRDC 
OHARO OHRO. The study is to be conducted by scientifically and medically qualified persons. 
The IRB will determine whether the benefits of the study are in proportion to the risks. The 
rights and welfare of the participants will be respected; the physicians conducting the study will 
ensure that the hazards do not outweigh the potential benefits; the results to be reported will be 
accurate; participants 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. 
15.1.1. 
Review/Approval of Study Protocol 
Before this clinical study can be initiated, the study protocol and other required documents will 
be submitted to the following departments in the order listed for review and/or approval, with the 
concurrent reviews by the U.S. FDA and Thai FDA: 
• Integrated Product Team 
• AFRIMS Scientific Review Committee 
• Sponsor’s Protocol Review Board (PRB) 
• WRAIR IRB, RTA IRB, Faculty of Tropical Medicine Ethical Review Board, 
Mahidol University, Chulalongkorn IRB, MoPH EC, and TSHAVD 
• Office of Human and Animal Research Oversight, Office of Human Research 
Oversight (OHARO OHRO) 
• Commander, WRAIR, if applicable 
• Sponsor’s Representative (acting for The Office of the Surgeon General of the Army) 
• USAMRDC Commanding General, if applicable 
Enrollment in this protocol may not begin until all approvals have been obtained and the formal 
authorization letter is received by the PI from the sponsor’s representative. 
15.1.2. 
Protocol Modifications 
All modifications to the protocol and supporting documents (informed consent, recruitment 
materials, etc.) must be reviewed and approved prior to implementation. Any protocol 
amendment will be agreed upon and approved by the sponsor’s representative, via submission to 
the PRB, prior to submission to the IRBs (see Table 5) and prior to implementation of said 
change or modification. Any modification that could potentially increase risk to participants 
must be submitted to the U.S. FDA prior to implementation.  
Substantive modifications to the protocol and any modifications that could potentially increase 
risk to participants must also be submitted to the USAMRDC OHARO OHRO for approval prior 
to issuance of the WRAIR Commander Approval Authorization. The USAMRDC OHARO 
OHRO defines a substantive modification as a change in PI, change or addition of an institution, 
elimination or alteration of the consent process, change in the IRB of Record, change to the study 
population that has regulatory implications (e.g., adding children, adding active duty population, 

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etc.), significant change in study design (i.e., would prompt additional scientific review) or a 
change that could potentially increase risks to participants. Any changes of the IRB used to 
review and approve the research will be promptly reported to the USAMRDC OHARO OHRO 
and ORA PRB. The WRAIR HSPB will submit protocol amendments and modifications to the 
USAMRDC OHARO OHRO as per USAMRDC Command Policy 21.  
The Informed Consent Form must be revised to concur with any significant amendment that 
directly affects participants and must also be reviewed and approved with the amendment. New 
participants enrolled in the study will be consented with the most recent approved consent form. 
Participants already enrolled in the study will be informed about the revision and asked to re-
consent. This may be accomplished by repeating the consent process with the revised consent 
form with attention given to the changes, or it may be done using an addendum consent that 
states the revision or new information. The new document must be signed, placed in the study 
record, and a copy given to the participant. 
Modifications or updates to the investigational brochures (IBs) will also be submitted as protocol 
amendments to the IRBs for review and approval during the data collection phase of the study 
and for review and acknowledgment during the data analysis phase of the study. 
15.1.3. 
Protocol Deviation Procedures 
A protocol deviation is defined as an isolated occurrence involving a procedure that did not 
follow the study protocol. 
The timeline for reporting protocol deviations to the IRBs is determined by the categorization of 
the deviation: (1) emergent/significant or (2) non-emergent/minor. Protocol deviations arising 
from or leading to unanticipated problems should be reported in the appropriate timeframe 
according to the seriousness of the event as a significant deviation or a minor deviation. The 
unanticipated problem will be submitted as described in Section 11.8.1.2. 
Emergent/significant deviations are departures from the protocol that have a significant impact 
on the welfare or safety of a participant or on the integrity of the study data. Examples: providing 
the wrong lab result to a participant or failure to obtain a scheduled blood draw for multiple 
participants. Changes in protocol procedures may be initiated without prior IRB and sponsor 
approval, only in cases where the change(s) is/are necessary to eliminate an immediate apparent 
hazard. Emergent/significant deviations should be reported promptly (within 48 hours) to the 
IRBs and the sponsor, upon becoming aware of the event, by telephone or email. A written 
report is required to be submitted by the PI to the IRBs and sponsor within 10 working days of 
knowledge of the significant deviation. Deviations will be reported by the WRAIR HSPB to the 
USAMRDC OHARO OHRO as per USAMRDC Command Policy 21. 
Non-emergent/minor deviations are routine departures that typically involve a participant’s 
failure to comply with the protocol. Examples include missing scheduled visits and failing to 
complete a required questionnaire. Minor deviations will be reported to the sponsor and the 
WRAIR IRB in a summary report with the CRRs and the closeout report. 
15.1.4. 
Continuing Review and Closeout Reports 
The PI is responsible for submitting the required CRRs and associated documents to the IRBs for 
review and approval, allowing sufficient time for review and continuation determination prior to 

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the established continuing review date. Summaries of enrollment and safety reports will be 
provided in the CRR, as they are made available by the sponsor. 
After all study related activities, including data analysis are completed, a closeout report will be 
submitted as required to the same bodies. 
A closeout report will be submitted after 5 years or upon completion of the study, whichever 
occurs first. The WRAIR HSPB will forward CRRs and closeout report to the USAMRDC 
OHARO OHRO as per USAMRDC Command Policy 21. 
15.2. 
Ethical Conduct of the Study 
This study will be conducted in accordance with all applicable IRB/EC, Federal, and DoD 
human research protections requirements; all state and local laws; and the Belmont Principles of 
respect for persons, beneficence, and justice. 
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, the applicable ethical 
principles, local regulatory requirements and the CFR. The PI confirms this by signing this study 
protocol and U.S. FDA Form 1572. 
15.2.1. 
Confidentiality 
The PIs will maintain participant research records at the sites for this study. All participants will 
receive study numbers that are known only to the investigators and study staff. All samples and 
documents (with the exception of the consent forms) will be labeled only with a participant’s 
study number and not personal information. The link between a participant ID number and 
participant identifiable information will be maintained in a secure, locked file cabinet in a locked 
room, which is accessible only to the PI/designees. Clinical and research records may be 
reviewed by representatives of USAMRDC OHARO OHRO, WRAIR IRB, MoPH EC, RTA 
IRB, Chulalongkorn IRB, Mahidol FTM EC, U.S. FDA, Thai FDA, the US DoD, the sponsor, 
the sponsor’s representative, and other regulatory agencies as part of their responsibilities for 
insuring the protection of research participants.  
Every effort will be made to keep the records as confidential as possible within the limits of the 
law. All data and medical information obtained about participants as individuals will be 
considered privileged and held in confidence. By Thai law, participant health information must 
be reported to the Thai government authority if they are diagnosed with any of the following 
communicable diseases: Plague, Smallpox, Crimean-Congo hemorrhagic fever, West Nile fever, 
Yellow fever, Lassa fever, Nipah virus disease, Marburg virus disease, Ebola virus disease, 
Hendra virus disease, SARS, MERS, extensively drug-resistant tuberculosis, and COVID-19. 
This study does not test for these diseases, however knowledge of these diagnoses will prompt 
study staff to file a report to the Thai government authority. If a participant shows signs or 
symptoms of these diseases, then they will be referred for immediate care.  
Research and clinical information relating to participants will be shared with other investigators 
and the scientific community through presentation or publication; however, participants will 
NOT be identified by name or Thai National ID number. Electronic data will be stored for at 
least 2 years after the IND is inactivated. 

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15.2.2. 
Compensation for Participation 
Participants will receive 1,000 Baht for food, time, inconvenience and transportation costs 
associated with each scheduled visit, including post-test counseling visits. Participants will 
receive 1,000 Baht for each unscheduled visit that is necessary to address safety concerns, at the 
Investigator’s or designee’s discretion. 
In addition to the compensation provided for scheduled and unscheduled study visits, 
participants will receive additional compensation for food, time, inconvenience and 
transportation costs associated with the optional procedure(s) that they choose to undergo as 
shown in Table 9 below: 
Table 9: 
Compensation for Participation 
Optional procedure 
Amount 
Semen collection 
1000 Baht 
Rectal sponges/swab 
1000 Baht 
Cervico-vaginal menstrual disc 
1000 Baht 
Vaginal/endocervical cytobrush/swab 
1000 Baht 
Penile swab 
1000 Baht 
Leukapheresis 
2000 Baht 
Sigmoid biopsy 
2000 Baht 
Lymph node biopsy 
2000 Baht 
15.2.3. 
Medical Care for Research-Related Injury 
Participants who experience illness or injury arising from participation in the study should 
contact the study site immediately. The site will either treat and care for the participant directly 
or refer the participant to an appropriate medical facility for care and treatment. Regardless of 
where the participant is treated, the study team will continue to provide health care counseling 
and guidance regarding the appropriate treatment. The cost of medical care for such illness or 
injury will be paid for by a limited set-aside fund and a clinical trials medical insurance policy. 
While it is anticipated that the combination of the set-aside fund and the insurance policy is more 
than enough to pay for the research related injury medical care cost associated with this study, 
there is a limit to the amount of coverage available. The participant’s personal insurance 
coverage schemes will not be used to cover the cost of medical treatment. Other than medical 
care and other payments as stated in this protocol, there is no other compensation available to the 
study participant from this research study; however, participants may pursue their legal right in 
accordance with Thai law to ask for other compensation relating to research related injury. 
15.3. 
Written Informed Consent 
The informed consent process and document will be reviewed and approved by the IRBs and 
sponsor’s representative prior to initiation of the study. The consent document contains a full 
explanation of the possible risks, advantages, and alternate treatment options, and availability of 
treatment in the case of injury, in accordance with 21 CFR 50. The consent document indicates 
that by signature, the participant permits witnessing of applicable study procedures by the 
sponsor’s representative, as well as access to relevant medical records by the sponsor’s 
representative and by representatives of the U.S. and Thai FDAs. The sponsor’s representative 

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will submit a copy of the initial IRB- and sponsor’s representative-approved consent form to the 
U.S. FDA and will maintain copies of revised consent documents that have been reviewed and 
approved by the IRBs. 
A written informed consent document, in compliance with 21 CFR Part 50, 32 CFR Part 219, 45 
CFR 46, and the Belmont Principles will be signed by the participant before any study-related 
procedures are initiated for that participant. This consent document must be retained by the 
investigator as part of the study records. Each participant will receive a copy of the signed 
informed consent document. The investigators or their designees will present the protocol in lay 
terms to individual participants. Questions on the purpose of the protocol, protocol procedures, 
and risks to the participants will then be solicited. Any question that cannot be answered will be 
referred to the PI. No participant should grant consent until questions have been answered to 
his/her satisfaction. The participant should understand that the study product is an investigational 
drug and is not licensed by the U.S. and Thai FDAs for commercial use, but is permitted to be 
used in this clinical research. Informed consent includes the principle that it is critical the 
participant be informed about the principal potential risks and benefits. This information will 
allow the participant to make a personal risk versus benefit decision and understand the 
following:  
• Participation is entirely voluntary,  
• Participants may withdraw from participation at any time, 
• Refusal to participate involves no penalty, and  
• The individual is free to ask any questions that will allow him/her to understand the 
nature of the protocol. 
Should the protocol be modified, the participant consent document must be revised to reflect the 
changes to the protocol. If a previously enrolled participant is directly affected by the change, the 
participant will receive a copy of the revised informed consent document. The approved revision 
will be read, signed, and dated by the participant. A description of this clinical trial will be 
available on http://www.ClinicalTrials.gov. The data stored in this website will be an aggregate 
summary of the trials results and will not contain individual demographic information or 
personal identifiers. 

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16. 
DATA HANDLING AND RECORDKEEPING 
The primary source document for this study will be the participant’s study folder. 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 each site. During the study, all hard copy data collection 
forms will be locked in cabinets accessible only by appropriate study staff when not in use. 
For this study, an 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. No source data will be recorded directly in the eCRF without a prior written 
record of the data. The investigator is ultimately responsible for the accuracy of the data 
transcribed on the eCRF. Data monitoring and management will be performed in the EDC 
database system by the study clinical monitor and BIOPHICS. During the study, de-identified 
data will be periodically transferred in a secure manner from BIOPHICS to EMMES/DCAC for 
interim blinded analyses and PSRT/SMC safety review. After database lock, data will be 
transferred from BIOPHICS to EMMES/DCAC, the PI and the Sponsor in a secure manner. 
A detailed data management plan will be written and approved by the study team and the 
Protocol Chair prior to study start, with approval by the sponsor’s representative in the 
USAMRDC ORA. All updates to the data management plan must be approved before study 
close-out and database lock. 
16.1. 
Inspection of Records 
The sponsor’s representative or designee will be allowed visit the investigation facilities for the 
purpose of monitoring any aspect of the study. The investigator agrees to allow the monitor to 
inspect the drug storage area, investigational product stocks, drug accountability records, 
participant charts, study source documents, and other records relative to study conduct. 
Participants’ health information is used to report results of research to the sponsor’s 
representative and Federal regulators and may be reviewed during study audits for compliance 
with study plans, regulations, and research policies. The consent document indicates that by 
signature, the participant permits access to relevant medical records by the sponsor’s 
representative and by representatives of the US and Thai regulatory authorities. 
16.1.1. 
Retention of Records 
For this study, an eCRF will be used for study data collection and monitoring. Completed, 
monitored eCRFs will be electronically stored with password protection in a secure location by 
the sponsor’s representative or designee. A copy of each completed eCRF will be retained by the 
investigator. If it becomes necessary for the sponsor’s representative or designee or the U.S. or 
Thai FDAs to review any documentation relating to the study, the investigator must permit 
access to such records. The clinical trial information will be entered into a database maintained 
by the National Institutes of Health/National Library of Medicine (NIH/NLM).  

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Federal regulations require that the PI retain a copy of all records that support eCRFs for this 
study (i.e., ICFs, clinical laboratory results, source documents, IP dispensing records) for 
whichever of the following is the shortest:  
• Two years following the date of approval by the U.S. FDA of the IP for the purposes 
that were the subject of the investigation; or  
• Five years following the date on which the results of the investigation were submitted 
to the U.S. FDA in support of, or as part of, an application for a research or marketing 
permit for the IP for the purposes that were the subject of the investigation.  
If the investigation does not result in the submission of data in the support of, or as part of, an 
application for a research or marketing permit, records must be retained for 2 years following 
notification by the Office of the Surgeon General, or his representative, that the entire clinical 
investigation (not merely the PI’s portion) is completed, terminated or discontinued; or for 
2 years following the withdrawal of an IND Application.  
If a PI retires, relocates or for other reasons withdraws from the responsibility of keeping the 
study records, custody must be transferred to a person who will accept this responsibility. The 
Office of the Surgeon General, or his representative, must be notified in writing of the name and 
address of the new custodian.  
The PI will be responsible for retaining sufficient information about each participant (i.e., name, 
address, telephone number, and participant identifier) in the study, so that the sponsor’s 
representative, the WRAIR IRB, MoPH EC, RTA IRB, Chulalongkorn IRB, Mahidol FTM EC, 
the U.S. FDA, Thai FDA, sponsor, employees of USAMRDC, or other regulatory authorities 
may have access to this information should the need arise.  
It is the policy of the USAMRDC that data sheets are to be completed for all participants 
participating in research (Volunteer Registry Data Sheet). The data sheets will be entered into 
this Command’s Volunteer Registry Database. The information to be entered into this 
confidential database includes: 
• Names (first and last name) 
• ID Number 
• Date of birth 
• Contact Information, both permanent and local 
• Study name and study dates, and dates of individual’s participation 
• Serious AE and unexpected AEs related to the vaccines experienced during the time 
of trial participation 
• Details of the product received  
The intent of this data base is twofold: first, to readily answer questions concerning an 
individual’s participation in research sponsored by USAMRDC; and second, to ensure that 
USAMRDC can exercise its obligation to ensure research participants are adequately warned 
(duty to warn) of risks and to provide new information as it becomes available. The information 
will be stored in Thailand for 75 years under the responsibility of Armed Forces Research 

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Institute of Medical Sciences, Thailand in coordination with the Faculty of Tropical Medicine, 
Mahidol University. The database will be kept according to Personal Data Protection Act 
(PDPA) 2019. However, name, surname and ID number will be stored confidentially and 
separately from the volunteer registry database. This database will not be used for research. 

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17. 
PUBLICATION POLICY 
All data collected during this study will be used to support this IND. All data may be published 
in the open medical or military literature with the identity of the participants protected. 
Participant confidentiality will be maintained by exclusion of personally identifiable information 
from the research database and from any publications that result. Anyone desiring to publish or 
present data obtained during the conduct of the study will conform to study site policies and then 
forward the publication for review to the USAMRDC ORA or designee and 
usarmy.detrick.medcom-usamrmc.list.clearances@health.mil prior to submission. 

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18. 
LIST OF REFERENCES 
Ahuka-Mundeke, S., Casey, R. M., Harris, J. B., Dixon, M. G., et al. (2018), 'Immunogenicity of 
Fractional-Dose Vaccine during a Yellow Fever Outbreak - Preliminary Report', N Engl J Med. 
Akapirat, S., Karnasuta, C., Vasan, S., Rerks-Ngarm, S., et al. (2018), 'Characterization of HIV-1 
gp120 antibody specificities induced in anogenital secretions of RV144 vaccine recipients after 
late boost immunizations', PLoS One, 13 (4), e0196397. 
Alam, S. M., Liao, H. X., Tomaras, G. D., Bonsignori, M., et al. (2013), 'Antigenicity and 
immunogenicity of RV144 vaccine AIDSVAX clade E envelope immunogen is enhanced by a 
gp120 N-terminal deletion', J Virol, 87 (3), 1554-68. 
Alving, C. R., Rao, M., Steers, N. J., Matyas, G. R., et al. (2012), 'Liposomes containing lipid A: 
an effective, safe, generic adjuvant system for synthetic vaccines', Expert Rev Vaccines, 11 (6), 
733-44. 
Barouch, D. H. (2008), 'Challenges in the development of an HIV-1 vaccine', Nature, 455 
(7213), 613-9. 
Beck, Z., Matyas, G. R., and Alving, C. R. (2015), 'Detection of liposomal cholesterol and 
monophosphoryl lipid A by QS-21 saponin and Limulus polyphemus amebocyte lysate', Biochim 
Biophys Acta, 1848 (3), 775-80. 
Bonsignori, M., Hwang, K. K., Chen, X., Tsao, C. Y., et al. (2011), 'Analysis of a clonal lineage 
of HIV-1 envelope V2/V3 conformational epitope-specific broadly neutralizing antibodies and 
their inferred unmutated common ancestors', J Virol, 85 (19), 9998-10009. 
Bonsignori, M., Kreider, E. F., Fera, D., Meyerhoff, R. R., et al. (2017), 'Staged induction of 
HIV-1 glycan-dependent broadly neutralizing antibodies', Sci Transl Med, 9 (381). 
Buchbinder, S. P., Mehrotra, D. V., Duerr, A., Fitzgerald, D. W., et al. (2008), 'Efficacy 
assessment of a cell-mediated immunity HIV-1 vaccine (the Step Study): a double-blind, 
randomised, placebo-controlled, test-of-concept trial', Lancet, 372 (9653), 1881-93. 
Chintanaphol, M., Sacdalan, C., Chottanapund, S., Pinyakorn, S., et al. (2018), 'Brief Report: 
Safety and Tolerability of Inguinal Lymph Node Biopsy in Individuals With Acute HIV 
Infection in Thailand', J Acquir Immune Defic Syndr, 79 (2), 244-48. 
Chua, J. V., Davis, C., Husson, J. S., Nelson, A., et al. (2021), 'Safety and immunogenicity of an 
HIV-1 gp120-CD4 chimeric subunit vaccine in a phase 1a randomized controlled trial', Vaccine, 
39 (29), 3879-91. 
DeVico, A., Fouts, T., Lewis, G. K., Gallo, R. C., et al. (2007), 'Antibodies to CD4-induced sites 
in HIV gp120 correlate with the control of SHIV challenge in macaques vaccinated with subunit 
immunogens', Proc Natl Acad Sci U S A, 104 (44), 17477-82. 

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Easterhoff, D., Moody, M. A., Fera, D., Cheng, H., et al. (2017), 'Boosting of HIV envelope CD4 
binding site antibodies with long variable heavy third complementarity determining region in the 
randomized double blind RV305 HIV-1 vaccine trial', PLoS Pathog, 13 (2), e1006182. 
Esparza, J (1993), 'Development of WHO-sponsored sites for HIV vaccine evaluation', AIDS Res 
Hum Retroviruses,  (9), S133-4. 
Flynn, N. M., Forthal, D. N., Harro, C. D., Judson, F. N., et al. (2005), 'Placebo-controlled phase 
3 trial of a recombinant glycoprotein 120 vaccine to prevent HIV-1 infection', J Infect Dis, 191 
(5), 654-65. 
Fouts, T. R., Bagley, K., Prado, I. J., Bobb, K. L., et al. (2015), 'Balance of cellular and humoral 
immunity determines the level of protection by HIV vaccines in rhesus macaque models of HIV 
infection', Proc Natl Acad Sci U S A, 112 (9), E992-9. 
Gamble, Lena J. and Matthews, Qiana L. (2010), 'Current progress in the development of a 
prophylactic vaccine for HIV-1', Drug design, development and therapy, 5, 9-26. 
Genito, C. J., Beck, Z., Phares, T. W., Kalle, F., et al. (2017), 'Liposomes containing 
monophosphoryl lipid A and QS-21 serve as an effective adjuvant for soluble circumsporozoite 
protein malaria vaccine FMP013', Vaccine, 35 (31), 3865-74. 
Hammer, S. M., Sobieszczyk, M. E., Janes, H., Karuna, S. T., et al. (2013), 'Efficacy trial of a 
DNA/rAd5 HIV-1 preventive vaccine', N Engl J Med, 369 (22), 2083-92. 
Haynes, B. F., Gilbert, P. B., McElrath, M. J., Zolla-Pazner, S., et al. (2012a), 'Immune-
correlates analysis of an HIV-1 vaccine efficacy trial', N Engl J Med, 366 (14), 1275-86. 
Haynes, Barton F., Gilbert, Peter B., McElrath, M. Juliana, Zolla-Pazner, Susan, et al. (2012b), 
'Immune-Correlates Analysis of an HIV-1 Vaccine Efficacy Trial', New England Journal of 
Medicine, 366 (14), 1275-86. 
Johnston, M. I. and Fauci, A. S. (2008), 'An HIV vaccine--challenges and prospects', N Engl J 
Med, 359 (9), 888-90. 
Joyce, M. G., King, H. A. D., Naouar, I. E., Ahmed, A., et al. (2021), 'Efficacy of a Broadly 
Neutralizing SARS-CoV-2 Ferritin Nanoparticle Vaccine in Nonhuman Primates', bioRxiv. 
Koff, W. C. (2010), 'Accelerating HIV vaccine development', Nature, 464 (7286), 161-2. 
Ling Munier, C. Mee, Andersen, Christopher R., and Kelleher, Anthony D. (2011), 'HIV 
Vaccines', Drugs, 71 (4), 387-414. 
McElrath, M. J. (1995), 'Selection of potent immunological adjuvants for vaccine construction', 
Semin Cancer Biol, 6 (6), 375-85. 
Medlock, J., Pandey, A., Parpia, A. S., Tang, A., et al. (2017), 'Effectiveness of UNAIDS targets 
and HIV vaccination across 127 countries', Proc Natl Acad Sci U S A, 114 (15), 4017-22. 

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Migasena, S., Suntharasamai, P., Pitisuttithum, P., Kitayaporn, D., et al. (2000), 'AIDSVAX 
(MN) in Bangkok injecting drug users: a report on safety and immunogenicity, including 
macrophage-tropic virus neutralization', AIDS Res Hum Retroviruses, 16 (7), 655-63. 
Moody, M. A. (2014), 'Modulation of HIV-1 immunity by adjuvants', Curr Opin HIV AIDS, 9 
(3), 242-9. 
National AIDS Committee (1997), National Plan for Prevention and Alleviation of HIV/AIDS, 
1997-2001 (Bangkok). 
Pitisuttithum, P., Nitayaphan, S., Thongcharoen, P., Khamboonruang, C., et al. (2003), 'Safety 
and immunogenicity of combinations of recombinant subtype E and B human immunodeficiency 
virus type 1 envelope glycoprotein 120 vaccines in healthy Thai adults', J Infect Dis, 188 (2), 
219-27. 
Pitisuttithum, P., Gilbert, P., Gurwith, M., Heyward, W., et al. (2006), 'Randomized, double-
blind, placebo-controlled efficacy trial of a bivalent recombinant glycoprotein 120 HIV-1 
vaccine among injection drug users in Bangkok, Thailand', J Infect Dis, 194 (12), 1661-71. 
Pitisuttithum, P., Choopanya, K., and Rerk-Ngnam, S. (2010), 'HIV-vaccine research and 
development in Thailand: evolution and challenges', Vaccine, 28 Suppl 2, B45-9. 
Rao, M., Onkar, S., Peachman, K. K., White, Y., et al. (2018), 'Liposome-Encapsulated HIV-1 
gp120 Induces Potent V1V2-Specific Antibodies in Humans', J Infect Dis. 
Regules, J. A., Cicatelli, S. B., Bennett, J. W., Paolino, K. M., et al. (2016), 'Fractional Third and 
Fourth Dose of RTS,S/AS01 Malaria Candidate Vaccine: A Phase 2a Controlled Human Malaria 
Parasite Infection and Immunogenicity Study', J Infect Dis, 214 (5), 762-71. 
Rerks-Ngarm, S., Pitisuttithum, P., Nitayaphan, S., Kaewkungwal, J., et al. (2009), 'Vaccination 
with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand', N Engl J Med, 361 (23), 
2209-20. 
Rerks-Ngarm, S., Pitisuttithum, P., Excler, J. L., Nitayaphan, S., et al. (2017), 'Randomized, 
Double-Blind Evaluation of Late Boost Strategies for HIV-Uninfected Vaccine Recipients in the 
RV144 HIV Vaccine Efficacy Trial', J Infect Dis, 215 (8), 1255-63. 
Resik, S., Tejeda, A., Lago, P. M., Diaz, M., et al. (2010), 'Randomized controlled clinical trial 
of fractional doses of inactivated poliovirus vaccine administered intradermally by needle-free 
device in Cuba', J Infect Dis, 201 (9), 1344-52. 
Robb, M. L., Rerks-Ngarm, S., Nitayaphan, S., Pitisuttithum, P., et al. (2012), 'Risk behaviour 
and time as covariates for efficacy of the HIV vaccine regimen ALVAC-HIV (vCP1521) and 
AIDSVAX B/E: a post-hoc analysis of the Thai phase 3 efficacy trial RV 144', Lancet Infect Dis, 
12 (7), 531-7. 
Tian, M., Cheng, C., Chen, X., Duan, H., et al. (2016), 'Induction of HIV Neutralizing Antibody 
Lineages in Mice with Diverse Precursor Repertoires', Cell, 166 (6), 1471-84 e18. 

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UNAIDS (2020), 'Report on the Global HIV/AIDS Epidemic - 2020 Fact Sheet', 
(https://www.unaids.org/sites/default/files/media_asset/UNAIDS_FactSheet_en.pdf). 
Walker, L. M., Phogat, S. K., Chan-Hui, P. Y., Wagner, D., et al. (2009), 'Broad and potent 
neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target', Science, 326 
(5950), 285-9. 
Walker, L. M., Huber, M., Doores, K. J., Falkowska, E., et al. (2011), 'Broad neutralization 
coverage of HIV by multiple highly potent antibodies', Nature, 477 (7365), 466-70. 
Walker, Mary Clare, Fast, Patricia E., Graham, Barney S., Belshe, Robert, et al. (1995), 'Phase 
I/II Preventive Vaccine Trials: Conference Summary', AIDS Research and Human Retroviruses, 
11 (10), 1279-85. 
Zhou, T., Lynch, R. M., Chen, L., Acharya, P., et al. (2015), 'Structural Repertoire of HIV-1-
Neutralizing Antibodies Targeting the CD4 Supersite in 14 Donors', Cell, 161 (6), 1280-92. 
 

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19. 
APPENDICES 
APPENDIX A. STUDY PERSONNEL ROLES AND RESPONSIBILITIES 
Protocol Chair/Sponsor’s Medical Expert: Responsible for overseeing the direction and the 
management of the study and will provide consultative medical and technical expertise for the 
overall conduct of the clinical trial. 
Site-Principal Investigator: Responsible for the study design and will serve as a liaison 
between the sites, vaccine developer and Sponsor, and contribute support to overall project 
management and the analysis and reporting of the study data. The PI will act as the qualified 
physician responsible for all trial site related medical decisions and will take all necessary 
precautions to ensure that the study obtains the proper clearance for all publication and abstracts, 
and maintain a study regulatory file as instructed by the study IND sponsor. 
Associate Investigators: Will oversee the conduct of the study at the clinic, and are responsible 
for local IRB submission and approval, study conduct, and reporting all unanticipated problems 
and AEs to the protocol team and the IRBs. They may conduct the participant study visits, assist 
with AE assessment and reporting, report any findings and study status directly to the study PI, 
and coordinate with the PI in the planning, design, and execution of the study. 
Community Representative: The Community Advisory Board Chairperson will liaise between 
the PI and communities where the participants are recruited and conveys questions, concerns, 
advises and recommendations regarding participants’ participation in the study. 
U.S. Army HIV Vaccine Product Manager: Responsibilities include providing input on 
protocol development and oversight of study operations. The U.S. Army HIV Vaccine Product 
Manager will not have contact with study participants or personal identifiers.  
U.S. MHRP HIV Vaccine Product Manager: Responsible for supervising the manufacturing, 
QC/QA, and stability testing of the vaccine candidates; overseeing the shipment of study 
products to the study sites; providing input on protocol development; and will serve as a liaison 
between MHRP and the study pharmacist. The U.S. MHRP HIV Vaccine Product Manager will 
not have contact with study participants or personal identifiers. 
Laboratory Investigator: Responsible for supervising clinical and research laboratory activities 
for the study. The laboratory investigator will not have contact with study participants or 
personal identifiers. 
Independent Safety Monitor: Responsible for providing independent safety monitoring in order 
to ensure the safety of research participants, regulatory compliance, and data integrity. The 
Independent Safety monitor will provide unbiased written reports to the ORA PSSO for all SAEs 
and participant deaths. The Independent Safety Monitor may also provide assessments on safety 
data reviews and study pauses resulting from safety events that meet halting criteria. 
Study Pharmacist: Responsible for overseeing the import, distribution, inventory, and 
accountability of vaccine, communication with sponsor / MHRP regarding pharmacy related 
issues, training and consultation with the study site pharmacy personnel. 

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Counselors: Counselors can be a physician, nurse or any specifically trained study personnel 
who performs HIV pre- and post-test HIV counseling at clinical study site. 
Consultants: Protocol consultants are responsible for providing input for the study design, 
protocol development, and serve as technical advisors and subject matter experts for study 
execution. Consultants will not have contact with study participants or personal identifiers. 
Data Manager: Responsible for overall data management, periodically providing de-identified 
data to Emmes/DCAC for PSRT and SMC safety reviews, and providing a final data transfer of 
all clinical trial data to Emmes/DCAC, the PI, and the Sponsor no later than 20 business days 
following final database closure including, but not limited to, the data dictionary, formats, 
assignments, and any accompanying memorandums. The media format and transfer 
specifications will be agreed upon with the Sponsor prior to closure. 
Statistician: Will receive coded data from the data manager and use those data to perform all 
investigational data analyses in collaboration with MHRP and the study team. The statistician 
will not have contact with study participants or personal identifiers. 
USAMRDC ORA Product Safety Surveillance Office: The USAMRDC ORA PSSO will be 
responsible for coordinating and integrating the review of safety data regarding OTSG-sponsored 
products. The USAMRDC ORA PSSO will review each SAE report and other immediately 
reportable event reports for medical consistency, accuracy, and completeness and will follow 
each event until it is satisfactorily resolved. The USAMRDC ORA PSSO will also be involved in 
the decision to unblind as may be necessary for participant safety. 

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APPENDIX B. DIVISION OF AIDS (DAIDS) TABLE FOR GRADING 
THE SEVERITY OF ADULT AND PEDIATRIC ADVERSE 
EVENTS 

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APPENDIX C. MAIN INFORMED CONSENT FORM 

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APPENDIX D. OPTIONAL PROCEDURES INFORMED CONSENT 
FORMS 

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APPENDIX E. STORAGE AND FUTURE USE INFORMED CONSENT 
FORM 

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APPENDIX F. WITHDRAWAL OF CONSENT FOR SAMPLE STORAGE 
FORM 

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APPENDIX G. TEST OF UNDERSTANDING AND ANSWER KEY 

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APPENDIX H. ADVERSE EVENTS OF SPECIAL INTEREST (AESI) 
LIST 

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APPENDIX I. GASTROINTESTINAL QUESTIONNAIRES FOR 
MICROBIOME ANALYSIS  

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APPENDIX J. FEMALE GENITAL TRACT (FGT) QUESTIONNAIRE 
(FOR FEMALE PARTICIPANTS) 

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APPENDIX K. DIETARY QUESTIONNAIRES FOR MICROBIOME 
ANALYSIS 

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APPENDIX L. EXTERNAL COLLABORATOR LIST 

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APPENDIX M. UNBLINDING LETTER 
 

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APPENDIX N. VACCINE-INDUCED SEROREACTIVITY LETTER 
 

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APPENDIX O. DIARY CARD 
 

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APPENDIX P. VOLUNTEER VISIT SCHEDULE 
 

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APPENDIX Q. APPOINTMENT/EMERGENCY CONTACT CARD 
 

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APPENDIX R. BRIEFING SLIDES 
 

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APPENDIX S. VOLUNTEER REGISTRY DATA SHEET 

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APPENDIX T. MENSTRUAL DISC SELF-INSERTION INSTRUCTIONS 

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APPENDIX U. PENILE SWAB SELF-COLLECTION INSTRUCTIONS 

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APPENDIX V. INFORMED CONSENT FORM FOR GENETIC TESTING