Randomized, Double-Blind, Placebo-Controlled, Single-Center, Phase 1 Study in Healthy Volunteers to Evaluate the Safety and Immunogenicity of AGS-v PLUS, a Universal Mosquito-Borne Disease and Mosquito Control Vaccine
Phase: PHASE1
Conditions: Mosquito-Borne Infectious Diseases
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

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Page 1 of 67 
 
 
 
 
Randomized, Double-Blind, Placebo-Controlled, Phase 1  
Study in Healthy Volunteers to Evaluate the Safety and  
Immunogenicity of AGS-v PLUS, a Universal Mosquito-Borne  
Disease Vaccine  
  
 
 
  
Sponsored by  
Office of Clinical Research Policy and Regulatory Operations (OCRPRO) 
Division of Clinical Research (DCR) 
National Institute of Allergy and Infectious Diseases 
National Institutes of Health 
  
Protocol #: HP-00084004 
IND # 17213  
  
Vaccine Manufactured by  
SEEK  
  
Principal Investigator  
Matthew B. Laurens, MD, MPH  
 
Co-Investigator 
Matthew J. Memoli, MD, MS  
Version Number and Date  
Version 4.0  
01 May 2020 

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TEAM ROSTER  
Principal Investigator: 
 
 
 
 
 
 
 
 
 
Co-Investigator 
Matthew B. Laurens, MD, MPH  
Associate Professor  
Center for Vaccine Development and Global Health 
(CVD) 
University of Maryland School of Medicine  
685 W Baltimore St, Room 480 
Baltimore, MD 21201  
Tel: 410-706-5462  
Email: mlaurens@som.umaryland.edu  
 
 
Matthew J. Memoli, MD, MS 
Director, LID Clinical Studies Unit 
Viral Pathogenesis and Evolution Section 
Laboratory of Infectious Diseases 
National Institute of Allergy and Infectious Diseases 
National Institutes of Health 
MSC 3203 33 North Dr 
Bethesda, MD 20892-3203 
UNITED STATES 
Email: memolim@niaid.nih.gov 
Phone: 301-443-5971 
 
Associate Investigators: 
DeAnna Friedman-Klabanoff, MD 
University of Maryland School of Medicine 
Tel 410-706-5328 
Email: dfriedman@som.umaryland.edu 
 
Mark A. Travassos, MD, MS  
Assistant Professor 
University of Maryland School of Medicine  
Tel: 410-706-5328  
Email: mtravass@som.umaryland.edu  
  
Andrea A. Berry, MD 
Assistant Professor 
University of Maryland School of Medicine 
Tel: 410-706-5328 
Email: aberry@som.umaryland.edu 
 
Kirsten E. Lyke, MD 
Professor 
University of Maryland School of Medicine 
Tel: 410-706-5328 
Email: klyke@som.umaryland.edu 
 
Miriam K. Laufer, MD, MPH 
Professor 

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University of Maryland School of Medicine 
Tel: 410-706-5328 
Email: mlaufer@som.umaryland.edu 
 
Amed Ouattara, PhD 
Research Associate 
University of Maryland School of Medicine 
Tel: 410-706-5328 
Email: amouattara@som.umaryland.edu 
 
IRB Contact: 
Human Research Protections Office 
University of Maryland Institutional Review Board 
Tel: 410-706-5037 
Fax: 410-706-4189 
Email: HRPO@umaryland.edu 
 
Sponsor Medical Monitor: 
H. Preston Holley, MD, FACP, FIDSA [C] 
Sponsor Medical Monitor 
Clinical Monitoring Research Program Directorate 
(CMRPD) 
Frederick National Laboratory for Cancer Research 
Leidos Biomedical Research 
Tel: 301-228-4005 
Email: Preston.Holley@nih.gov 
 
Study Statistician: 
Sally Hunsberger, PhD  
Mathematical Statistician  
Biostatistics Research Branch  
National Institute of Allergy and Infectious  
Diseases / NIH / U.S. DHHS  
Tel: 240-669-5257  
Email: sallyh@niaid.nih.gov  
 
Study Staff CVD: 
Lisa Chrisley, RN 
Tel: 410-706-6156 
Email: lchrisle@som.umaryland.edu 
 
Faith Pa’ahana-Brown, RN 
Tel: 410-706-6156 
Email: fpbrown@som.umaryland.edu 
 
Collaborators: 
Jesus G. Valenzuela, PhD  
LMVR, VMBS, NIAID  
Tel: 301-761-5070  
Email: jvalenzuela@niaid.nih.gov 
 
Olga Pleguezuelos, PhD 
PepTcell Ltd (t/a SEEK) 

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Central Point, 45 Beech Street 
London, EC2Y 8AD, United Kingdom 
Tel: +44 020 7153 6601   
Email: olga.pleguezuelos@seekacure.com 
 
 

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TABLE OF CONTENTS 
TEAM ROSTER........................................................................................................................................... 2 
TABLE OF CONTENTS.............................................................................................................................. 5 
LIST OF TABLES ........................................................................................................................................ 8 
LIST OF FIGURES...................................................................................................................................... 8 
LIST OF APPENDICES .............................................................................................................................. 8 
LIST OF ABBREVIATIONS ........................................................................................................................ 9 
PROTOCOL SUMMARY .......................................................................................................................... 11 
PRÉCIS ...................................................................................................................................................... 14 
1  
Background Information and Scientific Rationale........................................................................... 15 
2  
Study Objectives ............................................................................................................................... 25 
2.1  
Primary Objective ...................................................................................................................... 25 
2.2  
Secondary Objectives ............................................................................................................... 25 
2.3  
Exploratory Objectives .............................................................................................................. 25 
3  
Study Design ..................................................................................................................................... 26 
3.1  
Description of the Study Design ............................................................................................... 26 
3.2  
Study Endpoints ......................................................................................................................... 27 
4  
Study Enrollment ............................................................................................................................... 27 
4.1 
Inclusion Criteria ........................................................................................................................ 28 
4.4  
Exclusion Criteria ....................................................................................................................... 28 
4.5  
Justification for Exclusion of Pregnant Women and Children (Special Populations) ........... 30 
4.6 
Reasons for Withdrawal ............................................................................................................ 30 
4.7 
Handling of Withdrawals ........................................................................................................... 30 
4.8 
Termination of Study ................................................................................................................. 31 
5  
Study Agents/Interventions .............................................................................................................. 31 
5.1 Description....................................................................................................................................... 31 
5.1.1 AGS-v PLUS............................................................................................................................. 31 
5.1.2 Adjuvants .................................................................................................................................. 31 
5.1.3 Placebo ..................................................................................................................................... 32 
5.2 Study Agents Storage and Stability............................................................................................... 32 
5.3  
Preparation ................................................................................................................................. 32 
5.4  
Dosing and Administration ........................................................................................................ 32 
5.5 
Modification of Study Intervention/Investigational Product for a Participant......................... 33 
5.6  
Disposal and Drug Accountability of AGS-v PLUS ................................................................. 33 
5.6.1 Product Accountability ............................................................................................................. 33 
5.6.2 Accountability of Study Supplies ............................................................................................ 34 
5.6.3 Retention of Samples .............................................................................................................. 34 
5.7  
Concomitant Medications and Procedures.............................................................................. 34 
6  
Study Schedule ................................................................................................................................. 34 
6.1 
Screening (Days -60 to 0) (outpatient visit) ............................................................................. 35 
6.2  
Enrollment/Baseline and First Vaccination (Day 1) (outpatient visit) .................................... 36 
6.3 
Day 8 (+2 days) (phone call)..................................................................................................... 36 
6.4 
Day 22 (+/- 3 days) (outpatient visit) ........................................................................................ 36 
6.5 
Day 29 (+2 days) (phone call) .................................................................................................. 37 
6.6  
Day 43 Mosquito Feeding (+7 days) (outpatient visit) ............................................................ 37 
6.7  
Day 45 (+/- 1 day) (outpatient visit) .......................................................................................... 38 
6.8  
Mosquito Feeding follow-up (Day 50 + 2 days)....................................................................... 38 
6.9  
Telephone Safety Follow-up (Day 181 +14 days, and Day 366 +14 days) .......................... 39 

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7  
Study Procedures/ Evaluations ........................................................................................................ 40 
7.1  
Clinical and Laboratory Evaluations ......................................................................................... 40 
7.1.1 Laboratory Testing of Collected Samples for Primary and Secondary Endpoints ............. 40 
7.2  
Reactogenicity............................................................................................................................ 41 
7.3 
Mosquito Bite Assessment........................................................................................................ 41 
8  
Potential Risks and Benefits............................................................................................................. 41 
8.1  
Risks of Placebo administration ............................................................................................... 41 
8.2  
Risks of AGS-v PLUS ± adjuvant vaccination ......................................................................... 41 
8.3  
Risks of adjuvant........................................................................................................................ 43 
8.4  
Risks of blood draw ................................................................................................................... 44 
8.5  
Risks of Aedes aegypti and Aedes albopictus Mosquito Feeding ......................................... 44 
8.6 
Risk of Skin Biopsies ................................................................................................................. 44 
8.6  
Potential Benefits ....................................................................................................................... 45 
9  
Research Use of Stored Human Samples, Specimens, or Data .................................................. 45 
10  Data Sharing Plan ............................................................................................................................. 46 
10.1  
Sharing of Human Data ......................................................................................................... 46 
11  Remuneration Plan ........................................................................................................................... 46 
12  Assessment of Safety ....................................................................................................................... 46 
12.1  
Documenting, Recording, and Reporting AEs..................................................................... 46 
12.2  
Definitions ............................................................................................................................... 47 
12.3  
Investigator Assessment of AEs ........................................................................................... 49 
12.3.1 Severity ................................................................................................................................... 49 
12.3.2 Causality ................................................................................................................................. 50 
12.4  
Investigator Reporting Responsibilities to the Sponsor ...................................................... 50 
12.4.1 AEs.......................................................................................................................................... 50 
12.4.2 SAEs ....................................................................................................................................... 51 
12.4.3 Unanticipated Problems ........................................................................................................ 51 
12.4.4 Pregnancy .............................................................................................................................. 51 
12.5  
Investigator Reporting Responsibilities to the University of Maryland, Baltimore (UMB) 
IRB 
 ................................................................................................................................................. 51 
12.5.1 Reporting Protocol Deviations .............................................................................................. 51 
12.5.2 Special Reporting Situations................................................................................................. 52 
12.5.3 Expedited Reporting to the UMB IRB................................................................................... 52 
12.5.4 Waiver of Reporting Anticipated Protocol Deviations, Expected UPnonAEs and Deaths 
to the UMB IRB.................................................................................................................................. 52 
12.5.5 Annual Reporting to the UMB IRB........................................................................................ 52 
12.6  
Follow-Up of AEs and SAEs ................................................................................................. 52 
12.7  
Sponsor’s Reporting Responsibilities................................................................................... 53 
12.8  
Pausing Rules for an Individual Participant ......................................................................... 53 
12.8.1 Reporting a Pause ................................................................................................................. 53 
12.8.2 Resumption of a Paused Study ............................................................................................ 53 
12.9  
Halting Rules for the Protocol ............................................................................................... 54 
12.9.1 Reporting a Study Halt .......................................................................................................... 54 
12.9.2 Resumption of a Halted Study .............................................................................................. 54 
12.10 Study Discontinuation................................................................................................................. 54 
12.11 Premature Withdrawal of a Participant ..................................................................................... 55 
12.12 Replacement of a Participant .................................................................................................... 55 
12.13 Safety Oversight ......................................................................................................................... 55 

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12.13.1 Safety Review and Communications Plan (SRCP) .......................................................... 55 
12.13.2 Sponsor Medical Monitor .................................................................................................... 56 
12.13.3 Data and Safety Monitoring Board ..................................................................................... 56 
13  Site Monitoring Plan .......................................................................................................................... 56 
14  Statistical Considerations ................................................................................................................. 57 
14.1  
Study Hypothesis ................................................................................................................... 57 
14.2  
Sample Size Justification and Analysis Plan ....................................................................... 57 
14.3  
Analysis Plan .......................................................................................................................... 59 
14.3.1 Interim 
Analysis ....................................................................................................................... 60 
15  Ethics/Protection of Human Participants ......................................................................................... 60 
15.1  
Informed Consent Process.................................................................................................... 60 
15.2  
Participant Confidentiality ...................................................................................................... 61 
16  Data Handling and Record Keeping ................................................................................................ 61 
16.1  
Data Capture and Management ........................................................................................... 61 
16.2  
Record Retention ................................................................................................................... 61 
SCIENTIFIC REFERENCES.................................................................................................................... 62 
Appendix A: Schedule of Procedures/Evaluations ................................................................................. 64 
Appendix B: Blood Volumes for Specimen Collection ........................................................................... 65 
Appendix C: Toxicity Tables ..................................................................................................................... 66 
Appendix D: UMB IRB reporting guidelines ............................................................................................ 67 
 
 

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LIST OF TABLES 
 
Table 1. Probability of observing at least 1 SAE for samples sizes of 10 and 40 with different true 
SAE rates................................................................................................................................................... 58 
Table 2. Probability of observing at least 2 SAE’s for samples sizes of 10 and 40 with different true 
SAE rates................................................................................................................................................... 59 
 
 
 
LIST OF FIGURES 
 
Figure 1. Mosquito survival. ..................................................................................................................... 17 
Figure 2. Effect of immunization with salivary gland protein fractions on mosquito fecundity............ 18 
Figure 3. IFN response in mice with AGS-mix (AGS-v) or vehicle ...................................................... 19 
Figure 4. Total Ig and IgG2a AGS-mix (AGS-v)-specific antibody responses in mice measured by 
ELISA. ........................................................................................................................................................ 20 
Figure 5. Percentage of animals succumbing to Plasmodium infection transmitted by mosquito bite 
21 days post-vaccination with two doses of a Non Relevant Peptide (NRP) mix or AGS-v 15 days 
apart. .......................................................................................................................................................... 21 
Figure 6. IFN- secretion by splenocytes from mice vaccinated twice with AGS-v PLUS formulated in 
different adjuvants (Montanide ISA-51, Alhydrogel® and Adju-phos). .................................................. 22 
Figure 7. IgG antibody titers against AGS-v PLUS or the single peptides in the serum (1:400) of 
mice vaccinated with Placebo or AGS-v PLUS formulated in Montanide ISA-51, Alhydrogel® or 
Adju-phos adjuvants at pre-vaccination (day 0) and post-vaccination (day 42). Y axis=corrected 
absorbance, X-axis= vaccination received. ............................................................................................ 23 
Figure 8. IgG antibody titers against AGS-v of mice vaccinated with Placebo or AGS-v PLUS 
formulated in Montanide ISA-51, Alhydrogel® or Adju-phos adjuvants at pre-vaccination (day 0), 21 
days after first dose (day 21) and 21 days after the second dose (day 42). Y axis=corrected 
absorbance, X-axis= vaccination received. ............................................................................................ 24 
Figure 9. Study schedule .......................................................................................................................... 35 
 
 
 
LIST OF APPENDICES 
 
Appendix A: Schedule of Procedures/Evaluations ................................................................................. 64 
Appendix B: Blood Volumes for Specimen Collection ........................................................................... 65 
Appendix C: Toxicity Tables ..................................................................................................................... 66 
Appendix D: UMB IRB reporting guidelines ............................................................................................ 67 
 
 

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LIST OF ABBREVIATIONS  
ACT  
  
artemisinin-based combination therapy  
AE  
  
adverse event  
AR  
  
adverse reaction  
BMI  
BTRIS   
body mass index 
Biomedical Translational Research Information System  
CFR  
  
Code of Federal Regulations  
CRF  
case report form 
CSO  
Clinical Safety Office  
CSU 
CVD  
  
Clinical Studies Unit  
Center for Vaccine Development and Global Health (University of Maryland School 
of Medicine) 
DCR  
Division of Clinical Research  
DSMB 
Data and Safety Monitoring Board 
ECG  
electrocardiogram  
ELISA  
enzyme-linked immunosorbent assay  
EP 
European Pharmacopeia 
FACS 
fluorescence-activated cell sorting 
FDA  
  
U.S. Food and Drug Administration  
FLU-v 
influenza vaccine 
FMOC 
9-fluorenylmethoxycarbonyl 
GCP  
Good Clinical Practice  
HIV  
  
human immunodeficiency virus  
HIV-v 
human immunodeficiency virus vaccine 
HLA 
human leukocyte antigen 
HRPP  
Human Research Protection Program  
ICH  
  
International Council for Harmonisation of Technical Requirements for 
Pharmaceuticals for Human Use 
IFA  
  
Incomplete Freund’s adjuvant  
IFN- 
interferon gamma 
IgE  
  
immunoglobulin E 
IgG 
immunoglobulin G 
IgM 
immunoglobulin M 
IND  
  
Investigational New Drug application  
IP  
  
investigational product  
IRB  
  
institutional review board  
IV  
  
intravenous  
kDa 
kilodalton 
LID  
  
Laboratory of Infectious Diseases  
MHC 
MTT 
major histocompatibility complex 
Dimethylthiazol 
NIAID  
National Institute of Allergy and Infectious Diseases  
NIH  
  
National Institutes of Health  
OCRPRO  
Office of Clinical Research Policy and Regulatory Operations  
OHRP  
Office for Human Research Protections  
PBMC  
peripheral blood mononuclear cell  
PI  
  
principal investigator  
SAE  
  
serious adverse event  
SAR  
  
suspected adverse reaction  
SERF  
Safety Expedited Report Form  
SRCP  
Safety Review and Communications Plan  

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SUSAR  
TEAE 
serious and unexpected suspected adverse reaction 
Treatment Emergent Adverse Event  
Th1  
  
Type 1 T helper cells  
Th2 
Type 2 T helper cells 
UMB 
University of Maryland, Baltimore 
UP  
  
Unanticipated Problem  
UPnonAE  
Unexpected Problem that is not an AE  
USP  
  
United States Pharmacopeia  
WBC 
white blood cells 
WFI  
  
water for injection  

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PROTOCOL SUMMARY  
Full Title:  
Randomized, 
Double-Blind, 
Placebo-Controlled, 
Single-
center, Phase 1 Study in Healthy Volunteers to Evaluate the 
Safety and Immunogenicity of AGS-v PLUS, a Universal 
Mosquito-Borne Disease and Mosquito Control Vaccine  
Short Title: 
AGS-v PLUS Mosquito Vax  
IND Sponsor:  
Office of Clinical Research Policy and Regulatory Operations 
Division of Clinical Research 
National Institute of Allergy and Infectious Diseases National 
Institutes of Health 
Laboratory of Infectious Diseases 
Clinical Studies Unit  
Conducted by: 
Center for Vaccine Development and Global Health 
Principal Investigator: 
Matthew B. Laurens, MD, MPH 
Sample Size for Data 
Analysis: 
50 participants 
Accrual Ceiling for 
Vaccinees / Placebo 
Recipients: 
60 participants   
Study Population: 
Healthy volunteers 18 to 50 years of age 
Accrual Period: 
2 months 
Study Design: 
This is a randomized, double-blind, placebo-controlled, 
single-center, Phase 1 study of AGS-v PLUS administered 
as 2 vaccinations before a non-infected Aedes aegypti and 
Aedes albopictus mosquito feeding. 
 
Study Duration: 
The study will begin approximately in April 2019 and will 
require approximately 14 months to complete. The length of 
individual subject participation is about 12 months from 
enrollment. The study will be unblinded once the data from 
procedures up to day 50 related to the primary and 
secondary endpoints have been collected for all participants. 
After unblinding, two follow-up safety calls will be carried out 
on all participants on days 181 and 366. 
Study Agents: 
1012 µg of AGS-v PLUS (50 nmol of each of the five peptides 
in the formulation) non-adjuvanted, adjuvanted in Montanide 
ISA-51 or in Alhydrogel®. 
Study groups: 
Group 1. saline placebo (day 1 and day 22). 
Group 2. AGS-v PLUS non-adjuvanted (day 1 and day 22). 
Group 3. AGS-v PLUS + adjuvant Montanide ISA-51 (on day 
1) and saline placebo on day 22.   
Group 4. AGS-v PLUS + adjuvant Montanide ISA-51 (on day 
1 and day 22). 

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Group 5. AGS-v PLUS + Alhydrogel® adjuvant (on day 1 and 
day 22). 
Intervention 
Description: 
Individuals will be vaccinated subcutaneously with vaccine or 
placebo 21 days apart. 
Primary Objectives: 
1. To determine the safety and tolerability of AGS-v PLUS 
when administered as two doses alone, or with Montanide 
ISA-51 (single and two doses), or with Alhydrogel® (two 
doses) versus saline placebo (two doses). 
2. To measure specific antibody and cellular responses in 
participants receiving two doses of AGS-v PLUS alone, or 
with Montanide ISA-51 (single and two doses), or with 
Alhydrogel® (two doses) versus saline placebo (two doses) 
from pre-vaccination to post-vaccination.  
 
 Secondary Objectives: 
1. To measure changes to the antibody and cellular immune 
responses after clean Aedes aegypti and Aedes albopictus 
mosquito feeding on participants receiving two doses of 
AGS-v PLUS alone, or with Montanide ISA-51 (single and 
two doses), or with Alhydrogel® (two doses) versus saline 
placebo (two doses).  
2. To evaluate the effect of the AGS-v PLUS on Aedes 
aegypti and Aedes albopictus mosquito survival and 
fecundity after feeding on participants receiving two doses of 
AGS-v PLUS alone, or with Montanide ISA-51 (single and 
two doses), or with Alhydrogel® (two doses) versus saline 
placebo (two doses).  
 
Exploratory Objectives:  
1. To evaluate reactions at the bite sites after mosquito 
feeding on participants receiving two doses of AGS-v PLUS 
alone, or with Montanide ISA-51 (one or two doses), or with 
Alhydrogel® (two doses) versus saline placebo (two doses).   
2. To evaluate the effect of incubating Zika virus coated in 
vector saliva with peripheral 
blood mononuclear cells 
(PBMCs) and/or serum collected from individuals receiving 
two doses of AGS-v PLUS alone, or with Montanide ISA-51 
(single and two doses), or with Alhydrogel® (two doses) 
versus receiving saline placebo (two doses). 
3. To evaluate cellular immune responses to Aedes aegypti, 
Aedes albopictus and Anopheles gambiae mosquito salivary 
gland proteins in participants receiving two doses of AGS-v 
PLUS alone, or with Montanide ISA-51 (single and two 
doses), or with Alhydrogel® versus saline placebo (two 
doses) will be assessed from pre-vaccination (day 1) to post-
vaccination (day 43) and post-mosquito feeding (day 50). 

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Primary Endpoints: 
1. Incidence and severity of treatment emergent adverse 
events (AEs) and serious adverse events (SAEs).  
2. Geometric mean titer and fold increase in serum AGS-v 
PLUS specific immunoglobulin G (IgG), immunoglobulin M 
(IgM) and immunoglobulin E (IgE) titers from day 1 to day 43.  
3. Geometric mean concentration and fold increase in Type 
1 and Type 2 T-helper cell (Th1 and Th2) cytokine responses 
(from day 1 to day 43) after in vitro exposure of PBMCs with 
AGS-v PLUS antigens.  
Secondary Endpoints: 
1. Geometric mean titer and the fold increase in AGS-v PLUS 
specific IgG, IgM and IgE titers seven days after mosquito 
feeding (day 50) compared to day 1 and day 43 titers. 
2. Geometric mean concentration and fold increase in AGS-
v PLUS specific Th1 and Th2 cytokine responses in the 
supernatants of PBMCs (day 50 compared to days 1 and 43) 
after in vitro exposure with AGS-v PLUS. 
3. Survival and fecundity in Aedes aegypti and Aedes 
albopictus female mosquitos after feeding on vaccinated 
participants. 
 
Exploratory Endpoints: 
1. Reactions to the mosquito bites by size of wheals/papules, 
redness of the flare, swelling and itching 30 min post-
mosquito feeding (day 43), after 2 days (day 45) and after 7 
days (day 50). 
2. Early innate immune response to the mosquito bites in 
AGS-v PLUS vaccinated participants compared to placebo 
from biopsies taken from the bite site 2 days post-feeding. 
3. In vitro viability of Zika virus coated in mosquito saliva after 
incubation with serum and/or PBMCs from vaccinated 
participants. 
4. Geometric mean concentration and fold increase in Th1 
and Th2 cytokine responses (from day 1 to day 43, and from 
day 43 to day 50) after in vitro exposure of PBMCs with 
mosquito saliva from Aedes aegypti, Aedes albopictus and 
Anopheles gambiae. 
 
 
 
 

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 PRÉCIS  
 
Mosquito-borne diseases continue to cause significant morbidity and mortality worldwide despite ongoing 
control efforts. In 2015, there were over 200 million cases of malaria worldwide, causing nearly half a 
million deaths, with most of the deaths occurring among children under 5 years of age1. Mosquitos also 
transmit arboviruses, including dengue, yellow fever, West Nile virus, chikungunya, Rift Valley fever, 
Japanese encephalitis, and Zika virus. The outbreak of Zika virus in 2015 in Central and South America, 
as well as the Caribbean, serves as a reminder of how quickly these viruses can spread and how difficult 
they can be to control.  
AGS-v, the first mosquito-borne disease vaccine already tested in a Phase 1 trial, contained four peptide 
antigens. AGS-v PLUS was formulated by adding a fifth peptide to AGS-v. With this formulation, the 
manufacturer aims to increase the breadth of protection and increase the humoral responses compared 
to AGS-v. In this protocol, we plan to perform a Phase 1 study of a novel, improved universal mosquito-
borne disease vaccine. Through modulation of the immune system after a mosquito feeding, this vaccine 
targets the vector saliva and may provide prophylaxis against multiple arboviral and protozoal diseases. 
In addition, the vaccine potentially leads to a reduced mosquito lifespan after feeding therefore also 
reducing transmission of these diseases.  
In this protocol, we aim to demonstrate that the safety of AGS-v PLUS vaccine is similar to SEEK’s first 
mosquito-borne disease vaccine, AGS-v, and other peptide-based vaccines (influenza vaccine [FLU-v] 
and human immunodeficiency virus vaccine [HIV-v]) that have shown good safety profiles in previous 
Phase 1 and Phase 2 trials. We also aim to demonstrate that vaccination changes how the immune 
system reacts to mosquito saliva after a controlled clean Aedes aegypti and Aedes albopictus mosquito 
feeding. In 2015 and 2016, large outbreaks of Zika virus occurred in the Americas, resulting in an increase 
in travel-associated cases in US states, widespread transmission in Puerto Rico and the US Virgin 
Islands, and limited local transmission in Florida and Texas. In 2017, the number of reported Zika virus 
disease cases in the United States started to decline. Outbreaks of chikungunya have occurred in 
countries in Africa, Asia, Europe, and the Indian and Pacific Oceans. In late 2013, Chikungunya virus 
was found for the first time in the Americas on islands in the Caribbean. There is a risk that the virus will 
be imported to new areas by infected travelers and will be transmitted person-to-person by local Aedes 
mosquitoes.  
A successful, universal mosquito-borne disease vaccine offers the benefit of targeting emerging 
mosquito-borne diseases as well as the many established infections such as dengue, yellow fever and 
malaria that make dealing with this newly emerging epidemic a challenge.  
 

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1  
Background Information and Scientific Rationale  
  
Mosquito-borne diseases continue to cause significant morbidity and mortality worldwide despite ongoing 
vector control efforts. In 2015, there were over 200 million cases of malaria worldwide, causing nearly 
half a million deaths, with most of the deaths occurring among children under 5 years of age1. Mosquitos 
also transmit arboviruses, including dengue, yellow fever, West Nile virus, chikungunya, Rift Valley fever, 
Japanese encephalitis, and Zika virus. It is difficult to calculate the true burden of these infections. The 
symptomology can include asymptomatic infection or acute febrile illness, but can also include acute and 
severe complications including hemorrhagic fever and death as well as long-term sequelae that can last 
years. Billions of people are at risk of some of these infections globally with millions affected every year. 
The recent outbreak of Zika virus that first emerged in Brazil and now includes several countries in Central 
and South America, the Caribbean, and the U.S. serves as a reminder of how quickly these vector-borne 
viruses can spread and how difficult they can be to control. Chikungunya outbreaks have been reported 
in Africa, Asia and Europe and recently in 2013; the first outbreak in America was reported in the 
Caribbean islands. 
In large parts of Africa, South America and Asia, an estimated 3.2 billion people are at risk of contracting 
malaria1, and arboviral infections occur worldwide without discrimination between developed and 
developing nations. Current prevention programs follow a two-pronged approach simultaneously 
controlling mosquito populations by spraying with insecticides and minimization of suitable habitats and 
reducing transmission by the provision of insecticide-treated mosquito nets to the population. For malaria, 
this approach, along with artemisinin-based combination therapy (ACT), has proven highly successful in 
locations such as Zanzibar2, but the effective implementation requires continuous investment by and 
collaboration from international organizations, local governments, healthcare systems, and the people 
living in those communities. Unfortunately, this approach has not always been possible to sustain, as 
exemplified by failed eradication efforts in the 1950s-1960s3.  
Efforts in malaria and arboviral elimination continue to include the development of an effective vaccine 
that can both prevent disease in a healthy individual (prophylactic vaccine) and block disease 
transmission from an infected individual to the insect vector (transmission blocking vaccine). Most 
attempts at developing a malaria vaccine have centered on the prophylactic application, but unfortunately 
they have shown little efficacy. Even the most successful recent candidate vaccine, RTS, S/AS01 
(RTS,S) by GlaxoSmithKline, had vaccine efficacy of 27-46% in children and infants, respectively4. Even 
if the RTS,S vaccine is licensed, there will be a recognized need for the continued implementation of 
current malaria prevention programs5.  
In this protocol, we propose to perform a Phase 1 study of an improved, universal mosquito-borne disease 
vaccine manufactured by SEEK based on the salivary proteins from the vector and not in the pathogen 
it carries. SEEK sought to create an ideal malaria and arboviral disease vaccine that addresses both 
prophylactic and transmission-blocking components. SEEK’s approach in developing such a vaccine is 
defined by the following three principles:  
  

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• 
Because of complex and variable pathogen life cycle and antigenic make up, the vaccine 
prophylactic effect is not aimed at destroying the parasite itself, but rather at suppressing 
development of the essential physiologic environment that leads to successful human infection.  
• 
Because several mosquito species are capable of pathogen transmission, 
the vaccine 
transmission-blocking effect is not aimed at killing the vector on contact, but rather at preventing 
the very process of blood feeding which is essential for successful vector procreation.  
• 
Because most nations where malaria and arboviral diseases are endemic have limited healthcare 
delivery systems and budgets, the vaccine must be economically viable as a large-scale product 
entirely manufactured synthetically from widely available raw components, and must not require 
complex storage and delivery systems.  
Female mosquitoes have an absolute physiological need for blood-feeding to produce viable eggs and a 
healthy offspring. As they probe the skin for blood with their proboscis they salivate into the bite site 
inoculating parasites (i.e. Plasmodium) or arbovirus (i.e., dengue virus, Zika virus) into their human host.  
Mosquito 
saliva 
contains 
a range of molecules 
including 
vasodilators, 
anticoagulants 
and 
immunomodulators whose purpose is to prevent the disruption of feeding by the host6. Mosquito saliva 
has been shown to inhibit T and B cell proliferation7 and downregulate the expression of interferon gamma 
(IFN-)8, a soluble Type 1 T helper cell (Th1) pro-inflammatory immune mediator known to have in vivo 
therapeutic and prophylactic effects against other arthropod-transmitted diseases such as leishmaniasis9. 
Interestingly, mosquito saliva also triggers a non-inflammatory Type 2 T helper cell (Th2) host immune 
response that causes the characteristic irritation and allergic reaction associated with mosquito bites10. 
With time and continuous exposure, individuals become desensitized to mosquito bites due to a reduction 
in Th2 cytokine mediators, thus allowing for a more effective development of pro-inflammatory Th1 
responses11. Moreover, exposure to bites from uninfected mosquitoes reduces the development of 
Plasmodium yoelii in its murine host and that this protection is associated with a shift to a Th1 response 
at the bite site after repeated exposure to mosquito saliva12.  
Research has shown that mosquito-injected sporozoites can remain at the bite site for at least 5 minutes, 
enter general circulation by 15 minutes with most gone from the initial bite site by 60 minutes13,14,15. During 
this time, it has been suggested that Plasmodium can evade the immune system and travel away from 
the bite site to the liver via the lymphatic system by infecting activated and non-activated macrophages16-
18. Activated macrophages can and do kill parasites that infect them (e.g., Leishmania and Plasmodium), 
but non-activated macrophages are unable to do so. Macrophage activation is characteristic of a Th1 
response and is inhibited by the Th2 response triggered by mosquito saliva. This same process of 
downregulation of Th1 cytokines by modulation of the host immunity towards a Th2 response at the bite 
by the mosquito saliva has been reported to facilitate the transmission of chikungunya virus19, and 
arboviruses in general20.  
Beyond its direct effect on the parasite in the human host, an immune response to saliva has been shown 
to reduce malaria transmission. A monoclonal antibody raised against a 100 kilodalton (kDa) mosquito 
salivary gland protein significantly reduced the invasion by Plasmodium yoelii sporozoites of salivary 
glands in mosquitoes that fed on blood containing that antibody21. There is also evidence that antibodies 
against mosquito midgut and haemolymph antigens reduce the survival and fecundity of mosquitoes that 
fed on blood containing those antibodies22,23. Therefore, the antibodies against salivary antigens may 

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block transmission of these infections not only by reducing mosquito salivary gland invasion, but also by 
reducing the population of the mosquito vector.  
Based on the above scientific argument, SEEK’s approach to developing a vaccine against mosquito-
borne pathogens based on identification of suitable antigenic targets in mosquito saliva capable of 
inducing, upon vaccination of the mammalian host, a strong anti-mosquito saliva Th1 response would (a) 
prevent pathogen infection and (b) block disease transmission by preventing the arthropod vector’s 
salivary gland invasion and/or reducing vector survival and/or fecundity.  
As indicated earlier, SEEK manufactures this vaccine by an entirely synthetic procedure. However, 
mosquito saliva contains a multitude of proteins with molecular weights up to and above 100 kDa. 
Synthetic manufacturing of large proteins by 9-fluorenylmethoxycarbonyl (FMOC) chemistry is currently 
unfeasible. Therefore, SEEK first had to identify suitable vaccine targets in saliva for manufacturing. This 
process of target identification started with the simple separation of Anopheles gambiae salivary gland 
lysate proteins into fractions according to their molecular weight (i.e. <20 kDa, 20-40 kDa, 40-80 kDa and 
>80 kDa). These fractions were used to immunize mice upon which uninfected A. gambiae mosquitoes 
were allowed to feed and the survival and fecundity of these mosquitoes analyzed. As shown in Figure 
1, immunization with several mosquito salivary gland proteins fractions significantly reduced mosquito 
survival. The fraction containing proteins below 20 kDa proved to be the most effective, killing 80% of the 
mosquito population after only 6 days post-feeding. Fractions containing proteins between 20 and 40 kDa 
and over 80 kDa also reduced mosquito longevity, but were less effective than the <20 kDa protein 
fraction. As Plasmodium can complete its mosquito life-cycle stage in between 7 to 10 days, the ability 
of the <20 kDa protein fraction to kill 80% of mosquitoes within 6 days of feeding is important from the 
transmission blocking point of view as infected mosquitoes will die before they can transmit disease.  
 
 
 
Figure 1. Mosquito survival.  
Graph representing the mosquito % survival after feeding on mice immunized with different salivary gland protein fractions 
(vehicle (control), <20 kDa, 20-40 kDa, 40-80 kDa and >80 kDa). Mosquito salivary glands were dissected and their protein 
content separated by SDS-PAGE according to their molecular weight (MW). Proteins within the MW ranges of interest were 
recovered from the gel and used to immunize mice. Fresh A. gambiae mosquitoes fed on these mice and were monitored for 8 
days post-feed for survival (N=150 mosquitoes per group). 

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In addition, immunization with the <20 kDa fraction, and the 20-40 kDa fraction significantly (p<0.05) 
reduced the number of eggs produced, eggs laid, larvae arising from eggs and mature adult offspring by 
the surviving mosquitoes (Figure 2).  
          
 
Figure 2. Effect of immunization with salivary gland protein fractions on mosquito fecundity.  
Vehicle (control), <20KDa, 20-40KDa, 40-80KDa and >80KDa. 
 
Such reduction in the number of viable offspring would have a significant effect on the number of vectors 
available for disease transmission at any one time and hence in the overall disease transmission rate.  
From this point, SEEK proceeded to identify the proteins present in the fractions containing proteins <20 
kDa and between 20 and 40 kDa from data available in the GeneBank sequence repository and by 2D-
SDS-PAGE analysis of these fractions. Most of these proteins were still too large to be chemically 
synthesized and hence further work was required to identify reactive domains in these proteins that could 
successfully be manufactured and still retain the desired immunogenicity. This stage of the process was 
carried out using a computer algorithm developed by SEEK. The algorithm identifies and categorizes T-
cell epitopes within a protein based on analysis of the structural affinity of a peptide for a given major 
histocompatibility complex/human leukocyte antigen (MHC/HLA) allele and the reactivity of this complex 
to T-cells. Any small regions (25+ amino acids) within a protein identified by the algorithm as containing 
multiple T-cell epitopes were cross-referenced against all available human proteomic data (GeneBank 
and Swiss-Prot). Any region showing significant sequence similarity was discarded to minimize the risk 
of inducing an autoimmune challenge following immunization. This algorithm and approach have already 
been successfully applied to the identification of conserved reactive T-cell regions in both influenza virus24 
and human immunodeficiency virus (HIV). The identified sequences constitute two independent 
candidate vaccine preparations that have been tested clinical trials (NCT01071031, NCT01181336, 
NCT03180801, NCT02962908, NCT01226758).  

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The selection of reactive T-cell regions is based on SEEK’s interest in inducing a specific Th1 response 
to the salivary proteins. As indicated earlier in this document, a switch from a Th2 to a Th1 immune 
response to mosquito vector saliva is associated with resistance and natural immunity to malaria 
infection.  
Several reactive regions in salivary proteins of <40 kDa each containing multiple T-cell epitopes were 
identified and synthesized by FMOC chemistry. A cocktail of these small protein-sequences (all less than 
8 kDa), identified as AGS-v (or AGS-mix), were used to immunize mice and to establish the type of 
induced immune response. While multiple peptides may not be needed, the rationale is that the vaccine 
was developed to be active against a range of hematophagous arthropod vectors (such as Anopheles 
and Aedes mosquitos). As shown in Figure 3, immunization with AGS-v induced a strong IFN- response 
from immune cells upon recall with the antigen compared to immunization with a control mix of irrelevant 
peptides. 
 
 
 
 
Immunization with AGS-v was also found to induce a strong antibody response to the vaccine candidate 
(Figure 4), characterized by a significant contribution by antibodies of an IgG2a isotype. Both IFN- and 
IgG2a are considered primary mediators of a Th1 response and hence immunization with AGS-v can and 
does induce the desired Th1 response in mice.  
 
 
             Figure 3. IFN response in mice with AGS-mix (AGS-v) or vehicle 

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Having established that immunization with AGS-v induces a Th1 response in mice, SEEK wanted to 
establish whether this response was able to reduce the rate of malaria infection in a mouse model.  
Traditional experimental models of malaria infection rely on intravenous (IV) injection of large numbers 
of parasites to successfully establish infection in the mouse. It has been reported that these models do 
not reflect the natural process of infection by mosquito bite in which parasite numbers several orders of 
magnitude lower than those used in the experimental models are capable of successfully inducing 
infection25. Interestingly, it has been suggested by the same authors that the systematic use of these 
models could account for the continued failure of experimental vaccines, which having been shown to be 
very effective in such models, did not induce any significant protection when tested in real-life infection 
conditions. It is for this very reason and the nature of the vaccine candidate that SEEK chose to test the 
product in a natural model of infection (i.e. via the bite of infected mosquitoes).  
Preliminary studies using a natural model of infection involving A. gambiae infected with P. yoelii 
nigeriensis showed that mice vaccinated with AGS-v were more resilient to infection than mice vaccinated 
with an irrelevant peptide control mix (Figure 5). AGS-v was found to reduce infection rates 4-fold 
compared to the control group.  
 
 
 
 
 
 
 
  
Antibodies response to AGS-mix 
-0.5 
0 
0.5 
1 
1.5 
2 
2.5 
3 
3.5 
1:100 
1:200 
1:400 
Mouse sera dilution 
Abs 450nm 
AGS-mix immunised total Ig 
AGS-mix immunised IgG2a 
vehicle immunised total Ig 
vehicle immunised IgG2a 
Figure 4. Total Ig and IgG2a AGS-mix (AGS-v)-specific antibody responses in mice measured by ELISA. 

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Further experiments in C57BL6, BALBc, and CD1 were performed to measure immunogenicity. All had 
good responses to the vaccine after two weeks, and antibodies to AGS-v were raised to the vaccine 
antigens. T-cell responses were seen two weeks after vaccination.  
SEEK carried out clinical trials with two additional vaccines developed using the same technology as with 
AGS-v and AGS-v PLUS. Based on their experience with influenza vaccine (FLU-v) and human 
immunodeficiency virus vaccine (HIV-v) in clinical trials it is clear that a combination of four to five 
peptides allows the immune system to mount a response without peptides competing with each other. 
These studies have also assisted to determine an appropriate dose. For these vaccines, 50 nmol of each 
peptide proved to be safest and most immunogenic dose and replicated the ratio nmol/Kg in mouse 
studies. In phase 1 studies, a single dose of 25 nmol or 50nmol of FLU-v or HIV-v with and without 
adjuvant were given to approximately 10 participants in each arm. In the HIV-v phase 1 trial, both doses 
induced a significant and sustained antibody response in 60% of volunteers but only the higher 50nmol 
dose produced a significant vaccine-specific cellular response in approximately 50% of volunteers. In 
FLU-v phase 1 trial, both adjuvanted doses produced a significant IFN- response in more than 80% of 
volunteers in a dose-dependent manner. Three FLU-v phase 2 studies have confirmed that a single dose 
of 50nmol FLU-v adjuvanted in Montanide ISA-51 is the safest and most immunogenic dose after over a 
hundred participants have received this dosing regimen.  
In preclinical toxicology studies, a single 50nmol of each peptide dose of AGS-v administered 
subcutaneously showed no tolerability concerns in New Zealand White Rabbits. In this test, a single dose 
of subcutaneous AGS-v emulsified in Montanide ISA-51 adjuvant or in solution in water was compared 
to the vehicle placebos. All animals survived the test and appeared active and healthy during the study. 
A single subcutaneous injection of AGS-v in suspension caused minimal irritation comparable to vehicle 
control with full clinical resolution. Adjuvanted AGS-v caused minimal to mild irritation with a slightly lower 
irritation incidence when compared to vehicle control.  
Figure 5. Percentage of animals succumbing to Plasmodium infection transmitted by mosquito bite 
21 days post-vaccination with two doses of a Non Relevant Peptide (NRP) mix or AGS-v 15 days 
apart. 

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The National Institute of Allergy and Infectious Diseases (NIAID) – National Institutes of Health (NIH), in 
collaboration with SEEK, has performed a phase 1 clinical study testing the safety, immunogenicity and 
in vitro efficacy of AGS-v administered as two doses non-adjuvanted or adjuvanted in Montanide ISA-51 
compared to placebo (NCT 03055000). Investigators identified no safety concerns to date. 
SEEK has manufactured an improved AGS-v vaccine named AGS-v PLUS. This new formulation 
contains the exact four peptides included in AGS-v with the addition of a fifth peptide that provides higher 
antibody titer and increases the breadth of protection as the protein of origin is found in the saliva of many 
mosquitoes such as Anopheles gambiae, Anopheles darling, Culex quinquefasciatus, Aedes aegypti and 
Aedes albopictus. 
Preclinical immunogenicity data studies show that AGS-v PLUS is capable of inducing strong Th1 
responses measured as IFN- secretion after in vitro exposure of splenocytes from vaccinated mice to 
the vaccine antigens (Figure 6). Even when AGS-v PLUS is formulated with an adjuvant biased towards 
inducing Th2 responses such as Alhydrogel®, a strong IFN- secretion is detected. 
 
 
Figure 6. IFN- secretion by splenocytes from mice vaccinated twice with AGS-v PLUS formulated in 
different adjuvants (Montanide ISA-51, Alhydrogel® and Adju-phos). 
 
 
AGS-v PLUS is also capable of inducing high titers of antigen-specific IgG antibodies (Figure 7) when 
administered as two subcutaneous doses 21 days apart formulated in different adjuvants; an oil in water 
emulsion (Montanide ISA-51) or as a suspension with alum based adjuvants (Alhydrogel® and Adju-
phos). 
 

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Figure 7. IgG antibody titers against AGS-v PLUS or the single peptides in the serum (1:400) of mice 
vaccinated with Placebo or AGS-v PLUS formulated in Montanide ISA-51, Alhydrogel® or Adju-phos 
adjuvants at pre-vaccination (day 0) and post-vaccination (day 42). Y axis=corrected absorbance, X-axis= 
vaccination received. 
 
As seen in Figure 7, the fifth peptide in the AGS-v PLUS formulation, AGS-20, induces strong antibody 
responses with all adjuvants tested confirming that addition of this peptide will increase the overall 
antibody responses to AGS-v PLUS. 
In addition to testing two doses of AGS-v PLUS in the different formulations, replicating the groups from 
the SPITT study (Phase 1 study with AGS-v), the vaccine will also be tested as a single dose in Montanide 
ISA-51. Animal data showed that ISA-51 was the only adjuvant tested that induced an increase in the 
antibody response after a single dose (Figure 8). A single-dose vaccine will always have better 
compliance than one that requires two doses. 
 

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Figure 8. IgG antibody titers against AGS-v of mice vaccinated with Placebo or AGS-v PLUS formulated in 
Montanide ISA-51, Alhydrogel® or Adju-phos adjuvants at pre-vaccination (day 0), 21 days after first dose 
(day 21) and 21 days after the second dose (day 42). Y axis=corrected absorbance, X-axis= vaccination 
received. 
 
In summary, the data presented here supports the validity of SEEK’s scientific hypothesis that 
immunization with mosquito salivary antigens can induce a Th1 specific response to provide both 
prophylactic and transmission blocking immunity. Other groups have shown the ability of vaccination with 
salivary proteins from sand flies to reduce infection in diseases such as Leishmaniasis26,27. SEEK’s 
vaccine is composed entirely of synthetic products that do not require complex heterologous expression 
systems for manufacture and hence can be readily scaled up to accommodate for large scale demand. 
The composition and application of these synthetic products are the subject of a patent by SEEK.  
The AGS-v PLUS preparation is a vaccine candidate capable of acting on both the prophylactic and 
transmission blocking areas of malaria and arbovirus infection control. Moreover, it is a universal vaccine 
as it does not target a single pathogen but the vectors that transmit multiple pathogens, both viral and 
protozoal. These vectors are hematophagous arthropods and are dependent on the immunomodulatory 
activities of arthropod saliva to achieve successful infection of the host20. This is particularly relevant 
since many hematophagous arthropods such as Aedes aegypti transmit more than one pathogen (e.g. 
dengue, Zika and West Nile virus). 
In this protocol we plan to demonstrate that the safety of this vaccine is similar to SEEK’s other peptide 
based vaccines (AGS-v, FLU-v and HIV-v) that have been found to have very good safety profiles in 
Phase 1 and Phase 2 trials. We also aim to demonstrate immunomodulation after a controlled feed with 
clean Aedes aegypti and Aedes albopictus mosquitoes. With the current status of Zika and chikungunya 
in the Americas and the threat of it spreading to the U.S. and the rest of the world, a successful universal 
mosquito-borne disease vaccine offers the benefit of targeting this emerging disease as well as the many 
established infections such as dengue, yellow fever and malaria.  
  

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2  
Study Objectives 
 
2.1  
Primary Objective  
The primary objectives of the study are:  
1. To determine the safety of and tolerability of AGS-v PLUS when administered as two doses 
alone, or with Montanide ISA-51 (single and two doses), or with Alhydrogel® (two doses) 
versus saline placebo (two doses).  
2. To measure specific antibody and cellular responses in participants receiving two doses of 
AGS-v PLUS alone, or with Montanide ISA-51 (single and two doses), or with Alhydrogel® 
(two doses) versus saline placebo (two doses) from pre-vaccination to post-vaccination.  
A previous study tested 2 doses of AGS-v, 21 days apart, as non-adjuvanted or in Montanide. 
For continuation, AGS-v plus will be tested with the same formulation as AGS-v but also with 
a different adjuvant, Alhydrogel®. In addition, a single dose of AGS-v plus in Montanide will be 
tested as animal data showed that only Montanide can trigger an antibody response to AGS-
v after a single dose. In order to keep the blinding, participants in this group will receive saline 
on day 22. 
2.2  
Secondary Objectives  
The secondary objectives of this study are:   
1. To measure changes to the antibody and cellular immune responses after clean Aedes 
aegypti and Aedes albopictus mosquito feeding on participants receiving two doses of AGS-
v PLUS alone, or with Montanide ISA-51 (single and two doses), or with Alhydrogel® (two 
doses) versus saline placebo (two doses).  
2. To evaluate the effect of the AGS-v PLUS on Aedes aegypti and Aedes albopictus mosquito 
survival and fecundity after feeding on participants receiving two doses of AGS-v PLUS alone, 
or with Montanide ISA-51 (single and two doses), or with Alhydrogel® (two doses) versus 
saline placebo (two doses).  
 
2.3  
Exploratory Objectives  
 
1. To evaluate reactions at the bite sites after mosquito feeding on participants receiving two 
doses of AGS-v PLUS alone, or with Montanide ISA-51 (one or two doses), or with Alhydrogel® 
(two doses) versus saline placebo (two doses).   
2. To evaluate the effect of incubating Zika virus coated in vector saliva with peripheral blood 
mononuclear cells (PBMCs) and/or serum collected from individuals receiving two doses of 
AGS-v PLUS alone, or with Montanide ISA-51 (single and two doses), or with Alhydrogel® 
(two doses) versus receiving saline placebo (two doses). 
3. To evaluate cellular immune responses to Aedes aegypti, Aedes albopictus and Anopheles 
gambiae mosquito salivary gland proteins in participants receiving two doses of AGS-v PLUS 
alone, or with Montanide ISA-51 (single and two doses), or with Alhydrogel® versus saline 

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placebo (two doses) will be assessed from pre-vaccination (day 1) to post-vaccination (day 
43) and post-mosquito feeding (day 50). 
3  
Study Design  
 
3.1  
Description of the Study Design  
This is a randomized, double-blind, placebo-controlled, single-center, Phase 1 study of AGS-v PLUS 
administered at Days 0 and 21 before Aedes aegypti and Aedes albopictus mosquito challenge at Day 43. 
Participants will be randomized 1:1:1:1:1 to:  
• 
Group 1: Saline placebo (N=10) on day 1 and day 22. 
• 
Group 2: 1012 µg (50 nmol each peptide) unadjuvanted AGS-v PLUS vaccine (N=10) on day 1 
and day 22. 
• 
Group 3: 1012 µg (50 nmol each peptide) Montanide ISA-51 adjuvanted AGS-v PLUS vaccine 
(N=10) on day 1 and saline placebo on day 22. 
• 
Group 4: 1012 µg (50 nmol each peptide) Montanide ISA-51 adjuvanted AGS-v PLUS vaccine 
(N=10) on day 1 and day 22. 
• 
Group 5: 1012 µg (50 nmol each peptide) Alhydrogel® adjuvanted AGS-v PLUS vaccine (N=10) 
on day 1 and day 22. 
 
A placebo control will be used to establish the frequency and magnitude of changes in endpoints that 
may occur in the absence of active vaccination. Randomization will be used to minimize bias in the 
assignment of participants to vaccination groups, to increase the likelihood that known and unknown 
participant attributes (e.g., demographic and baseline characteristics) are evenly balanced across 
vaccination groups, and to enhance the validity of statistical comparisons across vaccination groups. 
Blinding of participants and the study team will be used to reduce potential bias during data collection 
and evaluation of endpoints.  
The study will be unblinded once all the data for the primary and secondary endpoints and safety data 
up to day 50 have been collected. Otherwise, the blind should be broken only if specific emergency 
treatment/course of action would be dictated by knowing the vaccination status of the participant. In such 
cases, the investigator may in an emergency determine the identity of the vaccine by opening the sealed 
code. It is recommended that the investigator contact the sponsor or its designee if possible to discuss 
the particular situation, before breaking the blind. In the event the blind is broken, the sponsor must be 
informed as soon as possible. Accidental unblinding (e.g. if the investigator sees the investigational 
product [IP] administration logs) must be reported within 1 working day to the Sponsor, who will advise 
on the corrective steps to be taken. The date and reason for the unblinding must be documented in the 
source document.  
  

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3.2  
Study Endpoints  
Primary:   
1. Incidence and severity of treatment emergent adverse events (AEs) and serious adverse 
events (SAEs).  
2. Geometric mean titer and fold increase in serum AGS-v PLUS specific immunoglobulin G 
(IgG), immunoglobulin M (IgM) and immunoglobulin E (IgE) titers from day 1 to day 43. 
3. Geometric mean concentration and fold increase in Th1 and Th2 cytokine responses (from 
day 1 to day 43) after in vitro exposure of PBMCs with AGS-v PLUS antigens. 
 
Secondary: 
1. Geometric mean titer and fold increase in AGS-v PLUS specific IgG, IgM and IgE titers seven 
days after mosquito feeding (day 50) compared to day 1 and 43.  
2. Geometric mean concentration and fold increase in AGS-v PLUS specific Th1 and Th2 
cytokine responses in the supernatants of PBMCs  (day 50 compared to days 1 and 43) after 
in vitro exposure with AGS-v plus. 
3. Survival and fecundity in Aedes aegypti and Aedes albopictus female mosquitoes after 
feeding on vaccinated participants. 
 
Exploratory: 
1. Reactions to mosquito bites by size of wheals/papules, redness of the flare, swelling and 
itching 30 min post-mosquito feeding (day 43), after two days (day 45) and after 7 days (day 
50). 
2. Early innate immune response to the mosquito bites in AGS-v PLUS vaccinated participants 
compared to placebo from biopsies taken from the bite site 2 days post-feeding. 
3. In vitro viability of Zika virus coated in mosquito saliva after incubation with serum and/or 
PBMCs from vaccinated participants.  
4. Geometric mean concentration and fold increase in Th1 and Th2 cytokine responses (from 
day 1 to day 43, and from day 43 to day 50) after in vitro exposure of PBMCs with mosquito 
saliva from Aedes aegypti, Aedes albopictus and Anopheles gambiae. 
 
 
4  
Study Enrollment 
 
Information regarding study materials will be communicated to potential participants who have previously 
participated in vaccine trials or expressed willingness to participate at the enrollment sites and have 
signed an authorization form that they are willing to be contacted for future studies. This has been done 
with institutional review board (IRB) approval. Educational meetings will be offered to the public both 
within and outside the university system for the purposes of informing individuals of the study. Every effort 

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will be made to achieve a balanced participant population while avoiding the enrollment of vulnerable 
populations. Careful screening will be conducted to avoid enrollment of individuals with substance abuse 
or psychiatric difficulties. Efforts to retain study participants include, but are not limited to: development 
of professional rapport with potential participants, obtaining valid contact and backup contact information 
from potential participants, and telephone reminder calls and/or email reminders for upcoming study 
visits. 
 
The Center for Vaccine Development and Global Health (CVD) will be the site for participant enrollment 
and inoculation. Mosquito feedings will occur in the Malaria Challenge Suite, where studies of controlled 
human malaria infection take place. Outpatient follow-up will be conducted at the CVD outpatient clinic. 
 
4.1 
Inclusion Criteria  
1. Healthy women and men who are ≥18 and ≤50 years of age.  
2. Willingness to complete all study visits and comply with all study requirements.  
3. A male participant is eligible for the study if he agrees to practicing abstinence or using a condom 
with spermicide plus an acceptable form of contraception (see inclusion criteria 4) being used by 
any female partner from 4 weeks before study start to 12 weeks after the second vaccine 
administration.  
4. A female participant is eligible for this study if she is not pregnant or breast feeding and 1 of the 
following:  
• 
Of non-child bearing potential (i.e., women who have had a hysterectomy or tubal ligation, or 
are postmenopausal, as defined by no menses in ≥1 year).  
• 
Of childbearing potential but agrees to practice effective contraception or abstinence for 4 
weeks before study initiation and 12 weeks after the second vaccine administration. 
Acceptable methods of contraception include a female partner who is the sole sexual partner 
of the female participant, a male partner who is sterile and is the sole sexual partner of the 
female participant, or a male partner who uses a condom with spermicide plus 1 or more of 
the following: 1) implants of levonorgestrel; 2) injectable progestogen; 3) an intrauterine 
device with a documented failure rate of <1%; 4) oral contraceptives; and 5) double barrier 
method including diaphragm.  
5. Willing to have samples stored for future research.  
6. Agrees to abstain from alcohol intake for 24 hours before each study visit.  
7. Agrees to not donate blood or blood products throughout the study.  
8. Score >70% on comprehension quiz at screening 
 
4.4  
Exclusion Criteria  
1. Participant has any underlying or current medical condition, which, in the opinion of the 
Investigator, would interfere with the participation in the study.  

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2. Individual with body mass index (BMI) ≤18 and ≥40.  
3. Participants who have a clinically significant (as determined by the PI or designee) baseline Grade 
1 or greater toxicity, or any Grade 2 or greater toxicity (regardless of clinical significance) by the 
toxicity table.  
4. Receipt of blood or blood products including immunoglobulin within 3 months before enrollment.  
5. Receipt of any unlicensed drug within 3 months or 5.5 half-lives (whichever is greater) before 
enrollment.  
6. Receipt of any unlicensed vaccine within 6 months before enrollment.  
7. Participated in study NCT03055000 testing safety and immunogenicity of AGS-v. 
8. Self-reported or known history of alcoholism or drug abuse within 6 months before enrollment.  
9. Self-reported or known history of psychiatric or psychological issues that require treatment and are 
deemed by the PI or designee to be a contraindication to protocol participation.  
10. History of a previous severe allergic reaction with generalized urticaria, angioedema, anaphylaxis 
or anaphylactoid reaction.  
11. Any condition or event that, in the judgment of the PI or designee, is a contraindication to protocol 
participation or impairs the volunteer’s ability to give informed consent.  
12. Known allergy to any vaccine component, including adjuvants. 
13. History of severe immunization reaction.  
14. Severe allergic reaction to mosquito bites (anaphylaxis)  
15. Have taken oral or parenteral (including intra-articular) corticosteroids of any dose within 
30 days before study vaccination 
16. Have taken high-dose inhaled corticosteroids* within 30 days before each study vaccination 
17. Received or plan to receive a licensed, live vaccine within 30 days before or after the study 
vaccination 
18. Received or plan to receive a licensed, inactivated vaccine within 14 days before or after study 
vaccination 
19. Serologic evidence of infection with HIV, hepatitis B virus, or hepatitis C virus 
20. Ongoing chronic skin condition, or acute skin condition at the time of vaccination or mosquito 
feeding, except for mild eczema. 
21. History of keloid formation after previous biopsies, lacerations, abrasions, surgeries, or other 
skin procedures (e.g., cosmetic piercings) that are deemed by the PI or designee to be a 
contraindication to protocol participation. 

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22. Pregnancy, breastfeeding, or planning to become pregnant up to one month after mosquito 
feeding. 
* 
High-dose 
defined 
per 
age 
as 
using 
inhaled 
high 
dose 
per 
reference 
chart 
https://www.nhlbi.nih.gov/sites/default/files/media/docs/asthma_qrg_0_0.pdf 
  
Co-enrollment Guidelines: Co-enrollment in other trials is restricted and discouraged, but may take 
place with the approval of the PI and after study staff notification. Limitations of total blood volume 
collection between the two studies must conform with NIH limits on blood collection. 
4.5  
Justification for Exclusion of Pregnant Women and Children (Special Populations)  
In this study, an investigational vaccine will be administered to the participants. Therefore, children, 
pregnant women, and other individuals at high risk of complications will be excluded as the risk to these 
individuals may be increased. In addition, the investigational vaccine has not undergone preclinical 
reproductive safety testing.  
  
Participants younger than 18 years of age will be excluded from the study. Because there are insufficient 
data regarding dosing or AEs available in adults to judge the potential risk in children, the study is of 
“greater than minimal risk” and does not meet the criterion of 45 Code of Federal Regulations (CFR) 46, 
Subpart D, governing the participation of children in research.  
4.6 
Reasons for Withdrawal 
Participants are free to withdraw from the study at any time. Participants will be encouraged to remain in 
the study to be followed for safety purposes.  
 
A study participant will be discontinued from participation in the study for any one of the following reasons: 
• 
Request by participant to terminate participation; 
• 
Loss to follow-up; 
• 
Request of primary care provider; 
• 
At the request of the IRB/Ethics committee, NIH, NIAID, or the U.S. Food and Drug 
Administration (FDA); 
• 
The participant’s well-being, based on the opinion of the investigator. 
Because a true intent-to-treat analysis requires the inclusion of all participants enrolled to the extent 
possible, this requires an intent-to-treat design in which all participants are followed according to the pre-
specified schedule with primary, and perhaps secondary, outcome assessments, regardless of 
compliance, adverse effects, or other post-enrollment observations— participant refusal excepted. 
 
4.7 
Handling of Withdrawals 
If voluntary withdrawal occurs, the participant will be asked for permission to continue scheduled 
evaluations, complete an end-of-study evaluation, and be given appropriate care under medical 

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supervision until the symptoms of any adverse event resolve or until the participant’s condition becomes 
stable.  
It is vital to collect safety data on any participant discontinued because of an AE or SAE, and every effort 
will be made to undertake protocol-specified safety follow-up procedures. Participants will follow early 
termination procedures if withdrawn. 
 
4.8 
Termination of Study 
The study may be discontinued due to the development of laboratory toxicities, at the discretion of the 
FDA or Division of Microbiology and Infectious Diseases (DMID), or due to natural disaster. 
 
 
5  
Study Agents/Interventions 
 
5.1 Description 
5.1.1 AGS-v PLUS 
AGS-v PLUS contains five peptides, or drug substances: AGS-6, AGS-20, AGS-30, AGS-31, and AGS-
35. Each peptide consists of 32 to 44 amino acids. They are chemically synthesized and available as 
acetate salts. The peptides contain multiple T-cell reactive regions capable of inducing Th1 responses.  
  
A solution of the five drug substances (50 nmol net peptide/g of each drug substance) is prepared by 
completely dissolving the drug substances in 50% acetic acid. The five solubilized peptides are mixed to 
make up AGS-v PLUS bulk solution. This mix is filter-sterilized before filling glass vials automatically with 
the sterile AGS-v PLUS bulk solution by weight which results in 1012 g of AGS-v PLUS solution per vial. 
The vials are then lyophilized to remove the water and acetic acid used in the compounding whereupon 
a relatively neutral pH, suitable for human injection, will be achieved after reconstitution. The vials are 
sealed and stored at -20oC. 
  
Bottle labels bear the appropriate label text, including the caution statement: "Caution: New Drug – 
Limited by Federal (or United States) law to investigational use," as required by governing regulatory 
agencies. Any addition or modification to the label apart from the variable data must be authorized by the 
drug supplier.  
5.1.2 Adjuvants  
The adjuvants used are Montanide ISA-51 and Alhydrogel®. 
 
Montanide ISA-51, manufactured by Seppic (France), is a mixture of oil and water that when combined 
with an antigen (such as a vaccine) it boosts the response to that antigen. It is comprised of two raw 
materials Drakeol 6 VR as a mineral oil and mannide monooleate as the surfactant28. Drakeol 6 VR is a 
mineral oil with United States Pharmacopeia/European Pharmacopeia (USP/EP) monograph. It is a 
mixture of several hydrocarbons with different length obtained from petroleum. Drakeol 6 VR stays at the 
injection site and is progressively eliminated by competent-cells such as macrophages. It can also be 
partially metabolized into fatty acids, triglycerides, phospholipids, or sterols. 30% of the mineral oil 
disappears during the first month and the majority of the oil found outside the injection site is in the liver 

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and fatty tissues in the form of phospholipids and fatty acids28. Mannide monooleate is a non-ionic 
surfactant based on oleic acid and sugar. Oleic acid is a distribution of various fatty acids with a 
predominant species C18'. 30-40% of mannide monooleate is removed from the injection site after 
24 hours. After 3 months, 30% of the surfactant still remains28. Montanide ISA 51 is highly purified and 
avoids having impurities and high levels of fatty acids that have led to toxicities with other versions of 
Incomplete Freund’s adjuvant (IFA) in the past. It also uses a different ratio of emulsifier to oil resulting 
in a more consistent and controllable emulsion. It has been used in multiple vaccine trials including those 
for malaria28.  
 
Alhydrogel® (Croda Denmark) is a sterilized, aluminium hydroxide wet gel suspension which has been 
tested free of pyrogenicity. It tolerates re-autoclavation, but is destroyed if frozen. When the pH is 
maintained at 5-7, which is normal during vaccine production, Alhydrogel® has a positive charge so it 
readily adsorbs negatively charged antigens (e.g. proteins with acidic isoelectric points kept at neutral 
pH). Alhydrogel® improves attraction and uptake of antigen by antigen presenting cells (APCs) by 
generating particles of antigen adsorbed to aluminium salts. This improves Th2 antibody production 
specific to the antigen. Alhydrogel® adjuvant has been used in multiple commercial vaccine formulations29 
with a 40+ year history of safe and effective use. 
 
5.1.3 Placebo  
The placebo contains sterile saline for injection only.  
 
5.2 Study Agents Storage and Stability  
The vials of water for injection (WFI), 0.09 normal saline, Montanide ISA-51 and Alhydrogel® should be 
stored at room temperature (15-30⁰C).  
AGS-v PLUS must be stored at ≤-20oC (±5 oC). AGS-v PLUS is expected to be stable for at least 
24 months at this temperature. Stability testing will be performed to cover the duration of the trial. The 
four peptide formulation (AGS-v) was tested for stability up to 6 months at +25oC 60%RH, 12 months at 
+5oC and 18 months at -20oC. AGS-v was stable at all conditions tested. Montanide ISA-51, Alhydrogel®, 
saline ampoules and WFI ampoules should be stored at room temperature (15-30°C).  
No study drug can be dispensed beyond the expiration date.  
 
5.3  
Preparation  
• 
Please see pharmacy manual for specifics regarding study product preparation. 
  
5.4  
Dosing and Administration  
Participants will be randomized to one of the following vaccination regimens:  
  
• 
Group 1: two doses (days 1 and 22) saline (0.5 mL) 

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• 
Group 2: two doses (days 1 and 22) non-adjuvanted AGS-v PLUS (50 nmol each peptide) as a 
suspension in 0.5 mL WFI. 
• 
Group 3: one dose (day 1) of AGS-v PLUS (50 nmol each peptide) in 0.25 mL of adjuvant 
Montanide ISA-51 emulsified in 0.25 mL of WFI and one dose of saline placebo (0.5 mL) on 
day 22 
• 
Group 4: two doses (days 1 and 22) of AGS-v PLUS (50 nmol each peptide) in 0.25 mL of adjuvant 
Montanide ISA-51 emulsified in 0.25 mL of WFI. 
• 
Group 5: two doses (days 1 and 22) of AGS-v PLUS (50 nmol each peptide) as a suspension in 
0.375 mL of saline and 0.125 mL of Alhydrogel®. 
 
All doses will be administered by subcutaneous injection in the fatty tissue of the triceps.  
  
The person administering the vaccine will be blinded to the type of formulation by an opaque label placed 
over the syringe by the pharmacy team. Trained and licensed study staff will administer vaccine.  
  
Vaccine administration will be performed following these instructions as detailed in the CVD SOPs.  
  
Detailed instructions on how to administer a subcutaneous injection can be found in CVD SOPs and in 
the Pharmacy manual.  
 
5.5 
Modification of Study Intervention/Investigational Product for a Participant 
The study product will be administered to enrolled participants according to the study schedule. There is 
no planned modification of study product administration for participants.  
  
5.6  
Disposal and Drug Accountability of AGS-v PLUS 
5.6.1 Product Accountability  
The University of Maryland Medical Center Investigational Drug Service will be responsible for recording 
the receipt of all vaccine supplies and for ensuring the supervision of the storage and allocation of these 
supplies. When a shipment is received, an assigned qualified person verifies the quantities received and 
the accompanying documentation and returns the acknowledgment of receipt to the drug supplier.  
  
Drug administration will be recorded in the source documents, in the electronic case report forms (CRFs) 
and in the Drug Administration Record form. The latter includes the participant identification, quantity 
(volume) and date of administration. The containers from which the vaccine was administered to the 
participants will be retained for dose confirmation.  
  
At the end of the study, delivery records of study vaccine will be reconciled with used / unused stocks 
and appropriate forms will be completed, to verify that all used, unused or partially used supplies have 
been returned or destroyed and that no study supplies remain in the Investigator’s possession.  
  

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All unused vaccine supplies, partially used and empty containers will be returned or destroyed after the 
study monitor has completed final accountability inspection and in agreement with the drug supplier 
(SEEK).  
 
5.6.2 Accountability of Study Supplies  
All materials supplied are for use only in this clinical study and should not be used for any other purpose.  
The PI is responsible for the IP accountability, reconciliation and record maintenance. In accordance with 
all applicable regulatory requirements, the investigator or designated site staff must maintain IP 
accountability records throughout the course of the study. The investigator or designated site staff will 
document the amount of IP received from the drug supplier (SEEK) and the amount administered to 
participants.  
A Drug Dispensing Log must be kept current and will contain the following information:   
• 
The identification of the participant to whom the drug was dispensed.  
• 
The date(s) of the drug dispensed to the participant.  
  
The inventory must be available for inspection by the study monitor at any point during the study. Drug 
supplies, excluding empty containers, will either be collected at the end of the study by the study monitor, 
returned or destroyed by the investigator as agreed in writing with the drug supplier (SEEK). The PI will 
keep records indicating identification and quantity of each unit disposed of or returned, the method of 
destruction (taking into account the requirements of local law), and the person who disposed of the test 
substance. Such records must be submitted to the drug supplier.  
  
5.6.3 Retention of Samples  
It will be the responsibility of the drug supplier (SEEK) to ensure adequate samples of all study drug are 
retained in accordance with the regulatory guidelines.  
5.7  
Concomitant Medications and Procedures  
At each study visit/contact from screening to study day 366 the study team will question the participant 
about any medication taken starting from 30 days before screening, including herbals, vitamins and 
holistic/naturopathic medications. Concomitant medication, including vaccines and any other medication, 
will be recorded in the CRFs with trade name and/or generic name of the medication, medical indication, 
start and end dates of treatment.  
 
6  
Study Schedule  
This study will take place at the CVD, University of Maryland School of Medicine. All aspects of the 
protocol will be carried out in accordance with CVD University of Maryland guidelines, Good Clinical 
Practice (GCP) and International 
Council for Harmonisation 
of Technical Requirements 
for 
Pharmaceuticals for Human Use (ICH) involving human-participant research. Please see Figure 9 below.  
 

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Figure 9. Study schedule 
    
6.1 
Screening (Days -60 to 0) (outpatient visit) 
• 
Potential participants will be provided with a verbal description of the study (purpose and study 
procedures), and will be asked if they have any questions and to read/sign the consent form. 
The consent form will be signed and potential participants must score >70% on a 
comprehension quiz before the performance of any study procedures. The comprehension quiz 
may be repeated once. 
• 
The study staff will discuss with the potential participant his/her medical history, study eligibility 
criteria and concomitant medication use. 
• 
Vital signs (height, weight, oral temperature, blood pressure, heart rate) will be obtained. BMI 
will be calculated to ensure eligibility. 
• 
Documentation of usual responses to mosquito bites as none (0), mild (1), moderate (2), or 
severe (3).  
• 
A physical exam will be performed. 
• 
For females who are capable of bearing children, a serum pregnancy test will be performed at 
the clinical trial study site at the time of screening regardless of age unless written medical 
demonstration of sterility or amenorrhea (defined as one year with medical evaluation) can be 
provided.  
• 
A blood sample will be collected from an arm vein to screen for health as follows:  
o Hematology: hemoglobin (Hgb), white blood cell count (WBC), and platelet count; 
o Chemistry: glucose (random), alanine aminotransferase (ALT), and creatinine; 
o Serology: HIV, hepatitis C virus (HCV), and hepatitis B surface antigen (HBsAg). 
• 
Medications taken in the previous 30 days will be recorded. 

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6.2  
Enrollment/Baseline and First Vaccination (Day 1) (outpatient visit) 
Randomization will use computer generated randomization codes. These codes will be sent to the 
pharmacy where the unblinded pharmacist will have the key and prepare the appropriate agent for 
administration. All vaccination syringes will be labeled to cover contents so that the blinded staff cannot 
identify the different treatments.    
 
The first vaccine or placebo administration will take place on Day 1. The research team will confirm 
continuing consent with the volunteer before performing any and all procedures. Once it is determined 
that the participant still meets inclusion/exclusion criteria for the research study, participants will receive 
the first of two injections.  
  
 The following procedures will be performed at enrollment:  
• 
Review of medical/concomitant medication history  
• 
Targeted physical exam/assessment as needed  
• 
Vital signs pre-vaccination  
• 
Vital signs 30-45 minutes post vaccination 
• 
Blood collection for routine safety labs (Hemoglobin, white blood cell count, platelets, serum 
creatinine, serum alanine aminotransferase, random glucose) 
• 
serum/whole blood prevaccination for research (as per Appendix B) 
• 
Urine pregnancy test (females of childbearing potential only)  
• 
Review of inclusion/exclusion criteria  
• 
Vaccine or placebo administration  
• 
Vaccine injection site and AE assessment pre-vaccination and  
• 
30-45 minutes post vaccination  
• 
AE memory aid, thermometer, and ruler will be distributed before discharge home  
 
6.3 
Day 8 (+2 days) (phone call) 
• 
Review of medical/medication history  
• 
Review of AEs and AE memory aid from first vaccination 
 
  
6.4 
Day 22 (+/- 3 days) (outpatient visit) 
The second study vaccine administration will take place if the participant continues to meet all inclusion 
and none of the exclusion criteria following these procedures:  
• 
Review of medical/medication history  
• 
Targeted physical exam/assessment as needed 

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• 
Vital signs (temperature, heart rate, blood pressure) 
• 
Vital signs at least 30-45 minutes post vaccination  
• 
Blood collection for routine safety labs (Hemoglobin, white blood cell count, platelets, serum 
creatinine, serum alanine aminotransferase, random glucose) 
• 
Urine pregnancy test (females of childbearing potential only)  
• 
Vaccine or placebo administration  
• 
Vaccine injection site reaction and AE assessment pre-vaccination and at least 30-45 minutes 
post vaccination  
• 
AE memory aid, thermometer, and ruler will be distributed before discharge home if the previous 
set is lost 
 
6.5 
Day 29 (+2 days) (phone call) 
• 
Review of medical/medication history  
• 
Review of AEs and AE memory aid from second vaccination 
• 
Participants will be asked not to apply cream, perfume or deodorant on the day of mosquito 
exposure.  
 
 
6.6  
Day 43 Mosquito Feeding (+7 days) (outpatient visit) 
The following procedures/assessments and testing will be performed:  
• 
Review of medical/medication history and addition of any new information  
• 
Targeted physician assessment and physical exam if needed 
• 
Vital signs (temperature, heart rate, blood pressure)  
• 
Urine pregnancy test (females of reproductive potential only)  
• 
Blood collection for routine safety labs (hemoglobin, white blood cell count, platelets,  
serum 
creatinine, serum alanine aminotransferase, random glucose) 
• 
serum/whole blood for research before mosquito feeding as per Appendix B.  
• 
Mosquito feeding: Starved clean female Aedes aegypti and Aedes albopictus mosquitos will be 
selected from a mosquito colony approved for human feeding studies in Laboratory of Malaria 
and Vector Research (LMVR), NIAID. Clean mosquitoes are referred to as mosquitoes colonized 
in an insectary and that are pathogen free. Clean mosquitoes are raised in an incubator dedicated 
for clinical trials. This ensures these insects are never exposed to human blood after emergence 
as adult mosquitoes up to the time they are utilized for feeding on human volunteers enrolled in 
the clinical trial. The insectary has well-established safety precautions to prevent outside 
mosquitoes from entering the facility. Mosquitoes will be aspirated into a secured Plexiglas 
feeding device and brought to the CVD from the LMVR Insectary.  
• 
There will be 10 participants in each treatment arm. Each participant will be exposed to 
Aedes aegypti on the right arm and Aedes albopictus mosquitoes on the left arm.  

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• 
The feeding site will be wiped clean with mild unscented soap and water and the device 
will be placed on the participant’s arm for 10-20 minutes. The mosquitos will feed through 
a disposable mesh on the bottom of the feeding device. This device permits the 
evaluation of mosquito feeding on the human participant at the end of exposure. In the 
unlikely event of no feeding or poor feeding (only 0-2 mosquitoes fed or probed as noted 
by trained staff for a particular strain), the volunteer may undergo a repeat feed with 5-
10 fresh mosquitoes ONCE.  
• 
Participants will be asked not to apply cream, perfume or deodorant on the day of 
mosquito exposure. Once the mosquitos have fed they will be brought back to the lab for 
further study.  
• 
The PI or study doctor will perform mosquito bite assessments at least 30 min after 
feeding. 
  
• 
AE assessment.  
 
6.7  
Day 45 (+/- 1 day) (outpatient visit) 
Two days after the mosquito feeding the participants will return for a follow-up. The following 
procedures/assessments and testing will be performed:  
• 
Review of medical/medication history and addition of any new information  
• 
Targeted physician assessment and physical exam if needed 
• 
AE assessment 
• 
Mosquito bite assessment 
• 
Collection of 3 skin biopsies: 2 from sites of mosquito bites previously marked by permanent 
marker, and a third from an unbitten area of normal skin 
 
 6.8  
Mosquito Feeding follow-up (Day 50 + 2 days) 
Seven days after mosquito feeding, 
participants 
will return for a follow-up. The following 
procedures/assessments and testing will be performed:  
• 
Review of medical/medication history and addition of any new information  
• 
Vital signs (temperature, heart rate, blood pressure) 
• 
Targeted physician assessment and physical exam if needed 
• 
Whole blood and serum collection for research as per Appendix B 
• 
Blood collection for routine safety labs (Hemoglobin, white blood cell count, platelets, serum 
creatinine, serum alanine aminotransferase, random glucose) 
• 
Mosquito bite assessment by the PI or trained study staff. 
• 
Assessment of biopsy healing 
• 
AE assessment  

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6.9  
Telephone Safety Follow-up (Day 181 +14 days, and Day 366 +14 days)  
 
All participants will be followed for a minimum of 12 months from the time of enrollment in the study. 
However, the study will be unblinded when the safety data up to day 50 and data for primary and 
secondary endpoints have been collected for all participants. Any participant who experiences 
complications due to vaccine or placebo administration will be followed until resolution of such 
complications or appropriate referral to the necessary medical care has been made.  
  
Follow-up visits will take place over a telephone call on days 181 and 366 (+/- 14 days). The study doctor 
will discuss ongoing, unresolved AEs with the participant and if deemed necessary, schedule a clinic visit. 
The procedures/evaluations to be conducted at these visits are shown in Appendix A. The follow-up call 
on day 366 will be the final study intervention unless the participant requires additional follow-up of study-
related complications.  

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7  
Study Procedures/ Evaluations  
 
7.1  
Clinical and Laboratory Evaluations  
See Appendix A for clinical and laboratory procedures performed during the vaccination and mosquito 
feeding visits as well as follow-up portions of the study. See Appendix B for Blood Volumes for Specimen 
Collection. Many of these tests are being performed to monitor safety and ensure that participants are 
healthy enough to undergo vaccination and mosquito feeding. The CVD has established limits for blood 
draws for research purposes, and the planned blood draws are within these limits. 
7.1.1 Laboratory Testing of Collected Samples for Primary and Secondary Endpoints  
o AGS-v PLUS specific IgG, IgM and IgE assay will be performed using enzyme-linked 
immunosorbent assay (ELISA). The time points assayed will be day 1, day 43 and day 50. 
o Th1 and Th2 response assays: PBMCs collected from participants will be incubated with 
AGS-v PLUS and saliva from Aedes aegypti, Aedes albopictus and Anopheles gambiae 
and measurements of Th1 (Interferon-gamma) and Th2 (Interleukin-4) cytokines will be 
performed using ELISA. The time points assayed will be day 1, day 43 and day 50.  
o Biopsies taken on day 45 will be used to assess expression of inflammatory markers. 
o Feeding on human volunteers: 24 hour after emergence from pupae, male and female 
Aedes aegypti and Aedes albopictus mosquitoes will be allowed to mate for 24-48 hours. 
After this period, female mosquitoes will be separated from males. These mosquitoes 
would have only been sugar-fed up until now. Female mosquitoes will be placed in a 
secured Plexiglass feeding device and allowed to feed on the volunteer’s arms on Day 43 
(approximately 21 days after they have completed the vaccination schedule). Each 
participant from each arm will be exposed to the bites of Aedes aegypti on the right arm 
and Aedes albopictus on the left arm. Mosquitoes that had a blood meal will then be 
removed and followed up to assess any changes to their life cycle, i.e., life-span, number 
of eggs laid, number of fertile eggs, number of surviving larvae etc. The mosquito colony 
used in this study will not be fed on human blood before exposure to participants, but on 
non-mammalian blood to minimize the presence of blood borne pathogens.  
o Zika virus killing, in vitro toxicity assay:  
▪ 
Blood samples will be obtained from all volunteers 21 days after vaccination is 
completed (day 43 pre-feeding). PBMCs+sera containing immune cells and antibodies 
generated against AGS-v PLUS will be tested for their ability to kill or inactivate Zika 
virus coated with salivary gland proteins from Aedes aegypti and Aedes albopictus. 
Viability of the virus will be tested by infecting Vero cells with the PBMC/virus 
supernatants. Infectivity of the Zika virus will be assessed by measuring the viability of 
the Vero cells using MTT assay. 
 
▪ 
Zika virus will be obtained from commercial suppliers and will be propagated in vitro.  
 

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 7.2  
Reactogenicity  
On a daily basis during the 7-day period after each injection participants will evaluate and record 
temperature, any local (redness, swelling, and pain) or general symptoms (e.g., fever, chills, headache, 
myalgia, arthralgia, malaise, sweats, fatigue, nausea, vomiting) they experience on the AE memory aid. 
At the day 8 and day 29 visits (phone call) the AE memory aids will be reviewed via phone and study staff 
will record reactogenicity during their discussion with each participant.  
  
The AE memory aid will be completed by the participant in the evening. Participants will be instructed on 
how to complete the card.  
 
7.3 
Mosquito Bite Assessment  
 
Mosquito bites will be assessed by the PI or trained study staff on days 43 (30 minutes after mosquito 
feeding), 45, and 50. Local reaction will be assessed by grading the size in mm of redness and 
swelling/induration. 
  
8  
Potential Risks and Benefits  
 
8.1  
Risks of Placebo administration  
Subcutaneous injection of sterile saline poses minimal risk. The primary risks of placebo administration 
are a local injection-site reaction or infection at the site of injection. Signs of a local injection-site reaction 
include pain, swelling, and redness at the site of injection. These reactions resolve after short period of 
time and pose little risk to the participant. Applying cold packs and administering over the counter pain 
medications if necessary can generally treat these reactions. Signs of infection include pain, redness, 
swelling, edema, and purulent drainage at the site.  
8.2  Risks of AGS-v PLUS ± adjuvant vaccination  
Vaccine reactions must be considered a risk of all vaccines. These include minor and severe vaccine 
reactions. A minor vaccine reaction typically occurs within a few hours of injection and resolve within a 
short period of time posing little risk to the participant. Symptoms include local pain, swelling, and redness 
at the site of injection and may include systemic symptoms like fever, malaise, muscle pain, headache, 
and loss of appetite. Severe reactions usually do not result in long-term problems for the participant, but 
can be disabling. They are rarely life threatening, but can be in <0.5% of individuals. These reactions can 
include severe anaphylactic reactions and seizure. Topical symptomatic therapy may be administered if 
necessary to treat vaccine reactions including cold compresses, and topical anti-pruritic and pain relief. 
Additional standard of care therapy for pain or anaphylaxis will be administered according to CVD 
standards if the reaction becomes severe or life threatening.  
  
The Sponsor has carried out a similar study testing the safety and immunogenicity of AGS-v, a vaccine 
that contains four of the five peptides in AGS-v PLUS (NCT 03055000). No safety concerns were raised 
by the safety review committee during the trial.  
 

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The manufacturer, SEEK, has completed clinical trials in the European Union for the FLU-v and HIV-v 
vaccines. The technology utilized for peptide identification, vaccine development and manufacturing for 
the FLU-v and HIV-v vaccines is the same technology utilized for AGS-v PLUS vaccine. FLU-v, HIV-v 
and AGS-v all utilize Montanide ISA-51 as the adjuvant too. Therefore, we expect similar safety findings 
for AGS-v PLUS as were observed with FLU-v, HIV-v and AGS-v. Below is a brief safety summary for all 
trials above mentioned: 
 
• 
Phase 1 AGS-v 001: Safety issues had been mild in severity. The vaccinations were well tolerated. 
In the unadjuvanted AGS-v group the most common AEs were Grade 1 or 2 injection site pain, 
fatigue, and headache (37.5%, 31.3%, 31.3% of participants respectively). Some participants also 
experienced Grade 1 or 2 systemic reactions of myalgia/feverishness/chills/diaphoresis (6.1-
18.8% of participants). The adjuvanted AGS-v group experienced a higher level of related AEs, 
with the most common being injection site pain, swelling, erythema, and pruritis (52.9%, 47.1%, 
41.2%, and 41.2% respectively). They also experienced some systemic grade1 and 2 AEs such 
as fatigue (41.2%) and feverishness (35.3%), as well as myalgia, chills and diaphoresis (5.9-
29.4%). The placebo group experienced fewer AEs with only injection site pain and erythema in 
25% and 6.3% respectively. They experienced a smaller amount of systemic AEs as well ranging 
from 6.3% to 12.5% for headache, fever, chills, fatigue, and myalgia. The most common AEs on 
Mosquito Feeding Day were Grade 1 erythema (93.8%) and Grade 1 local swelling (93.8%).  
 
• 
Phase 1 FLU-v-001: no deaths occurred and only one SAE was reported with this being 
considered unrelated to FLU-v (appendicitis). There was no apparent increase in adverse events 
(AEs) with a change in dose from the 250 μg or 500 μg (500 ug of FLU-v is the equivalent of 825 
ug of AGS-v as both contain 50 nmol of each peptide and a total of four peptides), but an 
approximate two-fold increase in AEs was seen in the adjuvant versus non-adjuvant groups. This 
latter point is likely responsible for the largely local administration site reaction AEs, and the 
forearm site and 1 ml volume is suspected to be the cause rather than the adjuvant itself. In 
conclusion, no safety or tolerability concerns were identified. 
 
• 
Phase 1b FLU-v-002: all 32 participants reported AEs: 32 mild, 10 moderate and one severe (a 
pre-syncopal episode in the placebo group). There were no deaths or SAEs. The most frequently 
reported AEs were related to the injection site reactogenicity and are consistent with those already 
documented with use of the adjuvant. There were no other clinically significant changes in the 
other safety parameters (complete physical examination, clinical chemistry, hematology and 
urinalysis, vital signs including oral temperature, electrocardiogram [ECG]). 
 
• 
Phase 2b FLU-v003: A total of 175 participants were enrolled to take part in a study carried in the 
Netherlands as part of a large European Consortium (UNISEC). Participants were allocated 
(2:2:1:1) to receive: 2 doses non-adjuvanted FLU-v, 1 dose of FLU-v adjuvanted in Montanide 
ISA-51, 2 doses of non-adjuvanted placebo or 1 dose adjuvanted placebo. The FLU-v vaccine 
formulations were safe and tolerated. A small number of participants mainly in the adjuvanted 
FLU-v group experienced severe reactions at the injection site that resolved with the use of over 
the counter anti-inflammatory drugs and required no additional follow-up by the medical team. 
The majority of solicited AEs that were related to the vaccination were observed at the site of 
injection and those were mild to moderate in intensity. An increase in the number of adverse 

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events was observed with the addition of adjuvant Montanide ISA-51 to the formulation. It is well 
known that emulsions prepared in this adjuvant have a tendency to create indurations at the site 
of injection which slowly disappear as the antigen is processed and the water in oil emulsion is 
degraded. Adjuvanted placebo had considerable more treatment emergent adverse events 
(TEAEs) definitely related to treatment than non-adjuvanted placebo (74.1% vs 18.8% 
participants). Addition of FLU-v to the placebo formulations increased the number of participants 
experiencing TEAEs (74.1 % to 93% of participants in adjuvanted placebo to adjuvanted FLU-v, 
and 18.8% to 34.5% participants in non-adjuvanted placebo to non-adjuvanted FLU-v). This 
indicates that the antigen itself is immunogenic and induces a small inflammatory reaction that 
translates into AEs at the injection site. These AEs were mostly mild to moderate with the 
participants in adjuvanted FLU-v group showing the highest number of these AEs. A total of 20 
participants experienced adverse events classified as severe in intensity with only 7 of them being 
definitely related to the study drug and defined as reactions at the site of the injection (1/58 in 
non-adjuvanted FLU-v, 5/57 in adjuvanted FLU-v, 0/32 in non-adjuvanted placebo and 1/27 in 
adjuvanted placebo). 
 
• 
Phase 2b FLU-v004: 121 participants were randomized (1:1:1) into one of three arms: 2 doses 
adjuvanted placebo (Montanide ISA-51+WFI), two doses adjuvanted FLU-v or one dose 
adjuvanted FLU-v +adjuvanted placebo. Participants were then challenged with an H1N1 
influenza virus. Overall the data showed a good safety profile for the IMP. The single dose FLU-
v showed a better profile than the 2 doses. However, there were no significant issues with the 
single dose arm and no concerns with the 2-dose arm. Injection site reactions were as expected 
and of an ‘acceptable’ level. A greater incidence of local reactions was seen in active arms, which 
may suggest a local reaction to the protein in addition to the adjuvant. The number of definitely 
related AEs in both FLU-v treatment groups was considerably higher than the placebo but of 
similar severity. The main AE was induration at the site of the injection and was considerably 
higher in the vaccine groups than placebo (46.8% and 60.8% compared to 18%). 
 
• 
Phase 1b HIV-v-001: all 59 participants reported an AE. Fatigue and injection site pain were the 
most commonly reported AEs. The majority of AEs were of mild severity (69.7%). There were 
5 SAEs in 3 participants with 3 SAEs occurring in the one participant and only 2 SAEs occurring 
within 30 days of vaccination. None of the SAEs were considered to be related to the study drug. 
No participants died and no participant discontinued the study due to an AE.  
 
8.3  
Risks of adjuvant  
 
Montanide ISA-51 
Clinical experience with Montanide ISA 51 dates back to the 1990s and most trials were related to cancer 
and Acquired Immune Deficiency Syndrome (AIDS). Currently, cancer trials in melanoma, colorectal, 
prostate, cervical, brain cancer and leukemia are ongoing. Frequency of vaccination is often between 2 
and 4 weeks, and the number of injections can reach up to 40. Route of immunization is most often 
subcutaneous and volume of injection can reach up to 3 mL. Most common local reactions are local pain, 
tenderness, erythema and granuloma at the injection site. Less frequently, mild to moderate transient 
indurations and swelling are described. General reactions are mainly ‘flu-like symptoms such as chills, 

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fever and headaches. Lethargy and nausea are also observed. The intensity is usually mild or moderate. 
No biological changes are generally observed.  
 
Aluminium based adjuvants 
Alhydrogel® has been used in many commercial vaccines29. After almost a century, aluminum salts 
maintain their dominance as adjuvants in human vaccines. This reflects the fact that aluminum adjuvants 
are extremely effective at enhancing antibody responses, are well tolerated, do not cause pyrexia and 
have the strongest safety record of any human adjuvants. Hence, aluminum adjuvants remain the gold 
standard against which all new adjuvants need to be compared and any new adjuvant must prove it 
provides better protection, tolerability or safety, or preferably all, when compared to aluminum adjuvant. 
Aluminum adjuvants suffer from a number of minor toxicities potentially explained by its mechanisms of 
action. For example, aluminum induces injection site pain and tenderness that may reflect cell necrosis 
and induction of inflammasome activation and IL1 production which explains why some participants 
develop persistent lumps and granulomas at the injection site. Aluminum adjuvants may also induce 
contact dermatitis to aluminum in a fraction of immunized participants. Aluminum adjuvant-containing 
vaccines can cause post-immunization headache, arthralgia and myalgia, which could reflect alum’s 
propensity to induce IL-1, with IL1 administration to human participants reproducing these symptoms. On 
the positive side, aluminum adjuvants rarely cause severe local reactions and are not normally 
associated with systemic inflammatory problems such as pyrexia29. 
 
 
8.4  
Risks of blood draw  
Risks of blood draw include pain, bruising, bleeding, and rarely fainting or infection. To minimize this risk, 
the skin is cleaned with alcohol before puncture; sterile, unused needles and lancets will always be used; 
and pressure will be held at the puncture site after removal of the needle or lancet. Although the quantity 
of blood drawn would not lead to any ill effects on the participants’ health, some adults feel faint with 
phlebotomy. This risk will be minimized by having trained technicians perform the procedure, and by 
placing participants in a recumbent position if they feel light headed or appear as if they are about to faint. 
Clinicians will be available for evaluation if there is any untoward effect. 
 
8.5  
Risks of Aedes aegypti and Aedes albopictus Mosquito Feeding  
The risk of mosquito feeding is minimal, but participants may suffer pruritis, mild rash, or irritation at the 
site of the bites. This may require topical symptomatic therapy. In rare cases a more severe irritation 
could occur, anaphylactic reaction, or secondary infection at the site of the bite where antibiotics or 
systemic anti-inflammatory medication may be required.  
8.6 
Risk of Skin Biopsies 
From each participant, three 3 mm skin biopsies will be taken, one from the A. aegypti bite (right arm), 
one from the A. albopictus bite (left arm) and the last one from normal unbitten skin (control). Risks of 
biopsy include local pain, bleeding, redness, infection, a scar and possible keloid formation. Oral or 
topical antibiotics and oral analgesics will be used to manage pain and infection as needed. Any non-
routine complications resulting from the procedure will be addressed in consultation with the NIH 

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Clinical Center (CC) Dermatology service. Injection of local anesthetic may cause a minimal burning 
discomfort or bruising at the site of the needle puncture. 
 
8.6  
Potential Benefits  
The vaccine is still under development. There is no direct benefit to the participant. The information 
collected from this study will allow a better understanding of the safety and immunogenicity of AGS-v 
PLUS and may inform further development of this or other universal mosquito-borne disease vaccines.  
 
9  
Research Use of Stored Human Samples, Specimens, or Data  
 
Intended Use: Samples and data collected under this protocol may be used to study aspects of Zika 
virus infection and other mosquito-borne diseases. Genetic testing will not be performed. A separate 
signed informed consent document will be obtained for any other research not described in this protocol.  
Storage: Access to stored samples will be limited using a locked freezer in a locked laboratory. Samples 
and data will be stored using codes assigned by the investigators. Data will be kept in password-protected 
computers or in locked cabinets in locked rooms accessed only by study staff. Only investigators and 
designated study staff will have access to the samples and data.  
Tracking: Samples will initially be stored in laboratories of primary and associate investigators. Samples 
will be tracked using a database located on password-protected computers, which will be maintained by 
the investigators and their designees. Only investigators and their designees will have access to this 
database.  
Disposition at the Completion of the Protocol:  
• 
In the future, other investigators may wish to study these samples and/or data. In that case, IRB 
approval must be sought before any sharing of samples and/or data. Any clinical information shared 
about the samples would similarly require prior IRB approval.  
• 
At the completion of the protocol (termination), samples will be stored at suitable conditions for a 
minimum of 1 year. After this time and in agreement between the Sponsor and vaccine manufacturer, 
SEEK, the samples will either be destroyed, or after IRB approval, transferred to another existing 
protocol.  
• 
At the completion of the protocol (termination), data will be stored for a minimum of 3 years. After 
this time and in agreement between the Sponsor and vaccine manufacturer, SEEK, the data will 
either be destroyed, or after IRB approval, transferred to another existing protocol.  
 
Reporting Loss or Destruction of Samples/Specimens/Data to the IRB:  
• 
Any loss or unanticipated destruction of samples or data (for example, due to freezer malfunction) 
that meets the definition of protocol deviation and/or compromises the scientific integrity of the data 

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collected for the study will be reported to the IRB in accordance with local regulations. The PI will also 
notify the IRB if the decision is made to destroy the samples.  
• 
Additionally, participants may decide at any point not to have their samples stored. In this case, the 
PI will destroy all known remaining samples and report what was done to both the participant and to 
the IRB. This decision will not affect the subject’s participation in this protocol or any other protocols 
at CVD.  
10  
Data Sharing Plan  
 
10.1  Sharing of Human Data 
Human data generated in this study will be shared for future research as follows:  
• 
De-identified data in an NIH-funded or approved public repository.  
• 
Identified data in Biomedical Translational Research Information System (BTRIS).  
• 
De-identified 
or identified data with approved outside collaborators 
under appropriate 
agreements.  
Data will be shared through:  
• 
ClinicalTrials.gov  
• 
BTRIS.  
• 
Approved outside collaborators under appropriate individual agreements.  
• 
Publication and/or public presentations at scientific meetings.  
 
11  
Remuneration Plan  
Participants will be compensated for time undergoing study procedures according to standard 
compensation policy at CVD.  
Study visits will be compensated according to the number of visits the participant completes. Participants 
will only be reimbursed for the protocol visits and interim visits requested by the investigators if medically 
necessary. Remuneration will be provided to the participants as the study visits are completed.  
12  
Assessment of Safety 
  
12.1  Documenting, Recording, and Reporting AEs  
At each contact with the participant, information regarding AEs will be elicited by appropriate questioning 
and examinations and will be:  
• 
Immediately documented in the participant’s medical record/source document,  
• 
Recorded in the study database  
• 
Reported as outlined below (e.g., Investigational New Drug (IND) Sponsor, IRB, FDA).  

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If a diagnosis is clinically evident (or subsequently determined), the diagnosis rather than the individual 
signs and symptoms or lab abnormalities will be recorded as the AE.  
  
All abnormal laboratory findings will be reviewed on a routine basis by the PI or designee to identify 
potential safety signals. An abnormal lab not included on the toxicity table should be assessed in a similar 
fashion to the criteria above.  
 
12.2  Definitions  
  
Adverse Event (AE) 
An AE is any untoward or unfavorable medical occurrence in a human subject, including any abnormal 
sign (for example, abnormal physical exam, or laboratory finding), symptom, or disease, temporally 
associated with the subject’s participation in the research, whether or not considered related to the 
research.  
 
Treatment Emergent Adverse Event (TEAE) 
A TEAE is any untoward or unfavorable medical occurrence in a human subject, including any abnormal 
sign (for example, abnormal physical exam, or laboratory finding), symptom, or disease, that manifested 
after administration of the study treatment, whether or not considered related to the treatment. 
  
Adverse Reaction (AR)  
An AE that is caused by an investigational agent (drug or biologic).  
  
Protocol Deviation 
A protocol deviation is any noncompliance with the clinical trial protocol, Good Clinical Practice (GCP), 
or Manual of Procedures requirements. The noncompliance may be either on the part of the subject, 
the investigator, or the study site staff. As a result of deviations, corrective actions are to be developed 
by the stie and implemented promptly.  
These practices are consistent with ICH E6: 
 
4.5 
Compliance with Protocol, Sec tions 4.5.1, 4.5.2, and 4.5.3 
 
5.1 
Quality Assurance and Quality Control, Section 5.1.1 
 
5.2.0 
Noncompliance, Sections 5.20.1, and 5.20.2. 
 
Suspected AR (SAR)  
An AE for which there is a reasonable possibility that the investigational agent caused the AE. 
‘Reasonable possibility’ means that there is evidence to suggest a causal relationship between the drug 
and the AE. A SAR implies a lesser degree of certainty about causality than AR, which implies a high 
degree of certainty.  
  
Serious Adverse Event (SAE)  
An SAE is an AE that results in 1 or more of the following outcomes:  
• 
Death  

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• 
A life-threatening event (places the subject at immediate risk of death from the event as it 
occurred)  
• 
An inpatient hospitalization or prolongation of an existing hospitalization  
• 
A persistent or significant incapacity or substantial disruption of the ability to conduct normal 
life functions  
• 
A congenital anomaly/birth defect  
• 
A medically important event*  
*Medical and scientific judgment should be exercised in deciding whether expedited reporting is 
appropriate in other situations, such as important medical events that may not be immediately life 
threatening or result in death or hospitalization but may jeopardize the participant or may require 
intervention to prevent one of the other outcomes listed above.  
  
Unexpected Adverse Event (UAE) 
An AE is unexpected if it is not listed in the Investigator’s Brochure or Package Insert (for marketed 
products) or is not listed at the specificity or severity that has been observed. It is the responsibility of the 
IND sponsor to make this determination.  
  
Serious and Unexpected SAR (SUSAR)  
A SUSAR is a SAR that is both serious and unexpected.  
  
Unanticipated Problem (UP)  
A UP is any event, incident, experience, or outcome that is  
1. Unexpected in terms of nature, severity, or frequency in relation to  
a. The research risks that are described in the IRB-approved research protocol and informed 
consent document, Investigator’s Brochure, or other study documents; and  
b. The characteristics of the participant population being studied; and  
2. Possibly, probably, or definitely related to participation in the research; and  
3. Places participants or others at a greater risk of harm (including physical, psychological, 
economic, or social harm) than was previously known or recognized. (Per the IND Sponsor, an 
AE with a serious outcome will be considered increased risk).  
  
Serious Unanticipated Problem 
A UP that meets the definition of a Serious Adverse Event or compromises the safety, welfare or rights 
of subjects or others. 
 
UP that is not an AE (UPnonAE)  
An UP that does not fit the definition of an AE, but which may, in the opinion of the investigator, involve 
risk to the subject, affect others in the research study, or significantly impact the integrity of research 
data. Such events would be considered a non-serious UP. For example, breaches of confidentiality, 
accidental destruction of study records, or unaccounted-for study agent will be reported.  
  

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12.3  Investigator Assessment of AEs  
The investigator will assess all AEs with respect to seriousness (criteria listed above), severity (intensity 
or grade), and causality (relationship to study agent and relationship to research) according to the 
following guidelines.  
  
12.3.1 Severity  
The investigator will grade the severity of each AE according to Appendix C. For items not found in 
Appendix C, the investigator will use the FDA “Toxicity Grading Scale for Healthy Adult and Adolescent 
Volunteers Enrolled in Preventive Vaccine Clinical Trials” September 2007, which can be found at:  . 
https://www.fda.gov/regulatory-information/search-fda-guidance-documents/toxicity-grading-scale-
healthy-adult-and-adolescent-volunteers-enrolled-preventive-vaccine-clinical 
 
  
All lab and vital sign abnormalities found before administration of the vaccine or placebo will be 
documented as a baseline AE and will be assessed for clinical significance and the participant will be 
reassessed for inclusion or exclusion to receive vaccination. After administration of vaccine or placebo, 
all new gradable abnormalities not found at baseline will be reported as AEs.  
  
Severity grading for clinical events that are not found in the FDA Healthy Volunteer Toxicity Table will be 
graded according to the following grading scale:  
  
• 
Grade 1 (Mild)  
Events causing no or minimal interference with daily activity and not requiring medical 
intervention 
• 
Grade 2 (Moderate)  
  
Events causing greater than minimal interference with daily activity but not requiring medical 
intervention  
• 
Grade 3 (Severe)  
  
Events causing inability to perform daily activity and/or requiring medical intervention  
• 
Grade 4 (Potentially Life-Threatening)*  
  
Events causing inability to perform basic self-care functions OR medical or operative 
intervention indicated to prevent permanent impairment, persistent disability, or death 
• 
Grade 5 (Death) 
Events causing death 
 
  
*Note: A severity assessment of “potentially life-threatening” is not necessarily the same as life-
threatening as an "SAE" criterion. The latter means that the event is an immediate threat to life as 
opposed to a potential threat to life.  

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12.3.2 Causality  
Causality (likelihood that the event is caused by the study agent) will be assessed considering the factors 
listed under the following categories:  
  
Definitely Related  
• 
Reasonable temporal relationship  
• 
Follows a known response pattern  
• 
Clear evidence to suggest a causal relationship  
• 
There is no alternative etiology  
  
Probably Related  
• 
Reasonable temporal relationship  
• 
Follows a suspected response pattern (based on similar products)  
• 
No evidence of a more likely alternative etiology  
  
Possibly Related  
• 
Reasonable temporal relationship  
• 
Little evidence for a more likely alternative etiology  
  
Unlikely Related  
• 
Does not have a reasonable temporal relationship  
OR  
• 
Good evidence for a more likely alternative etiology  
  
Not Related  
• 
Does not have a temporal relationship  
OR  
• 
Definitely due to an alternative etiology  
  
Note: Other factors should also be considered for each causality category when appropriate. Causality 
assessment is based on available information at the time of the assessment of the AE. The investigator 
may revise the causality assessment as additional information becomes available.  
 
12.4  Investigator Reporting Responsibilities to the Sponsor  
12.4.1 AEs  
AE data will be submitted to the IND sponsor when requested for periodic safety assessments, review of 
IND annual reports, review of IND safety reports, and preparation of final study reports.  

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12.4.2 SAEs  
All SAEs (regardless of relationship and whether or not they are also UPs) must be reported on the Safety 
Expedited Report Form (SERF) or Red Cap system and sent to the Sponsor Clinical Safety Office (CSO) 
by fax or e-mail attachment, or directly online via secure server. Deaths and immediately life threatening 
SAEs will be reported within 1 business day after the site becomes aware of the event. All other SAEs 
will be reported within 3 business days of site awareness.  
  
CSO CONTACT INFORMATION:  
Clinical Safety Office  
5705 Industry Lane  
Frederick, MD 21704  
Phone: 301-846-5301  
Fax: 301-846-6224  
E-mail: rchspsafety@mail.nih.gov   
12.4.3 Unanticipated Problems  
UPs that are also AEs must be reported to the CSO and sent by fax or e-mail attachment using the NIH 
Problem Report Form no later than 7 calendar days of site awareness of the event. Report Ups that are 
also AEs to the CSO on a SERF or a local IRB UP form. UPs that are not AEs are not reported to the 
Sponsor CSO.  
12.4.4 Pregnancy  
All pregnancies will be reported on the Pregnancy Notification/Outcome Form to the CSO within 1 
business day from site awareness.  
  
Pregnancy outcome data (e.g., delivery outcome, spontaneous or elective termination of the pregnancy) 
will be reported to the CSO within 3 business days of the site’s awareness.  
 Although pregnancy itself is not an AE, events that meet SAE criteria during pregnancy, delivery, or in 
the neonate (e.g., congenital anomaly/birth defect) are reportable on the SERF.  
  
In the event of pregnancy, the following steps will be taken:  
• 
Discontinue the study agent and procedures but continue to follow-up for safety.  
• 
Report to the Data and Safety Monitoring Board (DSMB) and the IRB.  
• 
Advise the research participant to notify the obstetrician of study participation and study agent 
exposure.  
12.5  Investigator Reporting Responsibilities to the University of Maryland, Baltimore (UMB) 
IRB  
12.5.1 Reporting Protocol Deviations  
It is the responsibility of the site principal investigator and personnel to use continuous vigilance to 
identify and report deviations as per UMB IRB reporting guidelines in Appendix D. All deviations must 
be promptly reported to the sponsor per the sponsor’s protocol deviation reporting procedures. 

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All protocol deviations must be addressed in study subject data collection forms. A completed copy of 
the protocol deviation form must be maintained in the Regulatory File, as well as in the subject's chart 
for subject-specific protocol deviations. Protocol deviations must be sent to the UMB IRB per their 
guidelines. The site principal investigator and personnel are responsible for knowing and adhering to 
their IRB requirements. 
 
12.5.2 Special Reporting Situations  
Protocol deviations of interest that may require expedited reporting to the UMB IRB  (as per UMB IRB 
reporting guidelines in Appendix D) and/or safety evaluation include, but are not limited to:  
• 
Overdose of a study drug  
• 
Suspected abuse/misuse of a study drug  
• 
Inadvertent or accidental exposure to a study drug  
• 
Medication error involving a product (with or without participant/patient exposure to the study 
drug, e.g., name confusion)  
  
All protocol deviations should be recorded in the study database.  
12.5.3 Expedited Reporting to the UMB IRB  
SAEs, serious and non-serious UPs, deaths, serious deviations, and serious or continuing non-
compliance will be reported as per UMB IRB reporting guidelines in Appendix D.   
12.5.4 Waiver of Reporting Anticipated Protocol Deviations, Expected UPnonAEs and Deaths to 
the UMB IRB  
Anticipated deviations in the conduct of the protocol will not be reported to the IRB unless they occur at 
a rate greater than anticipated by the study team. Expected AEs will not be reported to the IRB unless 
they occur at a rate greater than that known to occur in vaccine recipients. If the rate of these events 
exceeds the rate expected by the study team, the events will be classified and reported as though they 
are UPs.  
12.5.5 Annual Reporting to the UMB IRB  
Annual reporting to the UMB IRB will be in compliance with UMB IRB guidelines.  
12.6  Follow-Up of AEs and SAEs  
AEs that occur following enrollment of the participant after first vaccination will be followed until the final 
outcome is known or until stable, if chronic, or until the end of the study follow-up period. AEs that have 
not resolved by the end of the study follow-up period will be recorded as “ongoing.” Any participant who 
experiences complications due to vaccine/placebo administration will be followed until resolution of such 
complications or appropriate referral to the necessary medical care has been made.  
  
SAEs that have not resolved by the end of the follow-up period will be followed until final outcome is 
known. If it is not possible to obtain a final outcome for an SAE (e.g., the participant is lost to follow-up), 

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the reason a final outcome could not be obtained will be recorded by the investigator in the study database 
and on the SERF.  
  
SAEs that occur after the Day 366 phone call that are reported to and are assessed by the investigator 
to be possibly, probably, or definitely related will be reported to the CSO, as described above (section 
12.4.2 SAEs).  
12.7  Sponsor’s Reporting Responsibilities  
SUSARs as defined in 21 CFR 312.32 and determined by the IND Sponsor will be reported to FDA and 
all participating investigators as IND Safety Reports.  
  
The IND Sponsor will also submit an IND Annual Report of the progress of the investigation to the FDA 
as defined in 21 CFR 312.33.  
12.8  Pausing Rules for an Individual Participant  
Pausing is the suspension of administration of study agent to a single participant until a decision is made 
whether or not to resume administration of the study agent.  
  
The pausing criteria for a single participant in this study include any of the following:  
• 
A participant experiences an SAE that is possibly, probably, or definitely related to a study 
agent;  
• 
A participant experiences two Grade 3 or greater AEs that are possibly, probably, or definitely 
related to a study agent;  
• 
Any safety issue that the site investigator determines should pause administration of a study 
agent to a single participant.  
  
The CSO, in collaboration with the PI, may also pause for an entire group if a safety concern is identified.  
12.8.1 Reporting a Pause  
If a pausing criterion is met, a description of the AE(s) or safety issue must be reported by the PI or 
designee within 1 business day of recognition of the event to the CSO, the IRB, and the DSMB by fax or 
email.  
12.8.2 Resumption of a Paused Study  
The CSO, in collaboration with the PI and the DSMB, will determine whether or not it is safe to resume 
administration of the study agent to the participant. The PI will notify the IRB of the decision on resumption 
of the study agent.  
  
A participant who does not resume study agent will continue to be followed for safety.  
  

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12.9  Halting Rules for the Protocol  
Halting the study requires immediate discontinuation of study agent administered for all participants and 
suspension of enrollment until a decision is made whether or not to continue enrollment and study agent 
administration.  
  
The halting rules are:  
• 
1 or more participants experience the same or similar SAEs that are possibly, probably, or 
definitely related to the study agent;  
OR  
• 
2 or more of the same or similar AE in different participants that are grade 3 or above and are 
possibly, probably, or definitely related to the study agent;  
OR  
• 
Any safety issue that the PI and/or the CSO determines should halt the study.  
  
The PI and/or CSO will determine if the study should be halted. In addition, the FDA may halt the study 
at any time following review of any safety concerns.  
  
12.9.1 Reporting a Study Halt  
If a halting rule is met, a description of the AE(s) or safety issue must be reported by the PI or designee 
within 1 business day to the Co-Investigator, IND Sponsor, CSO, the IRB, and the DSMB by fax or email.  
12.9.2 Resumption of a Halted Study  
The IND sponsor, in collaboration with the PI and the DSMB will determine if it is safe to resume the 
study. The PI will notify the IRB of the decision on resumption of the study.  
  
Participants who do not resume study agent will continue to be followed for safety.  
 
  
12.10 Study Discontinuation  
Office of Clinical Research Policy and Regulatory Operations (OCRPRO), the study sponsor, the IRB, 
and the FDA have the right to terminate this study at any time. Reasons for terminating the study may 
include, but are not limited to, the following:  
1. The incidence or severity of AEs in this study indicates a potential health hazard to participants  
2. Participant enrollment is unsatisfactory  
3. Data recording is inaccurate or incomplete  
4. Investigators do not adhere to the protocol, or applicable regulatory guidelines in conducting 
the study  
  
The IRB, the NIAID, the FDA, or other government agencies, as part of their duties to ensure that research 
participants are protected may discontinue the study at any time. Subsequent review of serious, 
unexpected and related AEs by the IRB, the DSMB, the sponsor, the FDA, and other regulatory 

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authorities may also result in suspension of further trial interventions/administration of study agent at a 
site. The FDA, other regulatory authorities, and the study sponsor retain the authority to suspend 
additional enrollment and study agent administration for the entire study as applicable. 
   
12.11 Premature Withdrawal of a Participant  
Participants may withdraw before vaccine administration and no further testing or follow-up will be 
performed. The PI or designee will discuss with the participant why she/he wants to prematurely withdraw 
from the study to determine the best course of action for the participant. If the participant would like to 
withdraw after vaccination, clinical laboratory tests and procedures for safety purposes will continue as 
obtainable and at a frequency determined by the PI. If the participant does not return for scheduled follow-
up visits, the study staff will make every reasonable effort to contact the participant by phone, mail, or 
email, or a combination of the latter and reiterate that follow-up visits are strongly encouraged for safety 
reasons.   
  
An individual participant will be withdrawn for any of the following:  
• 
An individual participant’s decision. (The investigator should attempt to determine the reason for 
the participant’s decision.)  
• 
Non-compliance with study procedures to the extent that it is potentially harmful to the participant 
or to the integrity of the study data.  
• 
A change in the participant’s baseline condition after enrollment so that the participant no longer 
meets one or more of the inclusion/exclusion criteria.  
• 
The investigator determines that continued participation in the study would not be in the best 
interest of the participant.  
 
12.12 Replacement of a Participant  
If a participant withdraws or appears ineligible to continue the study before vaccine/placebo 
administration, he/she will be removed and no data will be used in analysis or publication of the study. 
The participant may be replaced in the accrual with a new volunteer who qualifies and consents to the 
study, and who can complete both vaccinations 14-28 days before the planned date of mosquito 
challenge. Safety data from all participants that have withdrawn will be used and included in the safety 
analysis.  
12.13 Safety Oversight  
12.13.1 Safety Review and Communications Plan (SRCP)  
A Safety Review and Communication Plan (SRCP) has been developed for the protocol. The SRCP is 
an internal communications document between the PI and the CSO, which delineates the safety oversight 
responsibilities of the PI, the CSO, and other stakeholders. The SRCP also includes the overall plan for 
conducting periodic safety surveillance assessments.  

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12.13.2 Sponsor Medical Monitor  
A Medical Monitor, representing the IND Sponsor (OCRPRO), has been appointed for oversight of safety 
in this clinical study. The Sponsor Medical Monitor will be responsible for performing routine safety 
assessments in an SRCP.  
12.13.3 Data and Safety Monitoring Board  
The NIAID intramural data and safety monitoring board (DSMB) includes independent experts that do 
not have direct involvement in the conduct of the study and have no significant conflicts of interest as 
defined by NIAID policy. The DSMB will review the study protocol, consent document(s), and 
investigator brochure prior to initiation and twice a year thereafter, or as may be determined by the 
DSMB. 
   
The DSMB may convene additional reviews as necessary. The DSMB will review the study data as 
needed to evaluate the safety, efficacy, study progress, and conduct of the study.  
  
All deaths, SAEs, UPs, pregnancies, and IND safety reports will be reported to the DSMB at the 
same time they are submitted to the IRB and CSO unless otherwise specified herein.  
  
All cases of intentional or unintentional unblinding will be reported to the DSMB not later than one 
business day from the time of study personnel awareness.  
  
The principal investigator will notify the DSMB at the time pausing or halting criteria are met and 
obtain a recommendation concerning continuation, modification, or termination of the study. The 
principal investigator will submit the written DSMB summary reports with recommendations to the 
IRB(s). 
 
13  
Site Monitoring Plan  
According to the ICH-GCP 5.18 and FDA 21 CFR 312.50, clinical protocols are required to be adequately 
monitored by the study sponsor. This study monitoring will be conducted according to the sponsor’s 
clinical monitoring guidelines. Clinical monitors will visit the clinical research site to monitor aspects of 
the study in accordance with the appropriate regulations and the approved protocol. The objectives of a 
monitoring visit will be:  
1) To verify the existence of signed informed consent documents and documentation of the informed 
consent process for each monitored participant;  
2) To verify the prompt and accurate recording of all monitored data points, and prompt reporting of all 
SAEs;  
3) To compare clinical data abstracts with individual participants’ records and source documents 
(participants’ charts, laboratory analyses and test results, physicians’ progress notes, nurses’ notes, and 
any other relevant original participant information); and  
4) To help ensure investigators are in compliance with the protocol. The monitors also will inspect the 
clinical site regulatory files to ensure that regulatory requirements (FDA; University of Maryland, 
Baltimore; Office for Human Research Protections [OHRP]) and applicable guidelines (ICH-GCP) are 
being followed. During the monitoring visits, the investigator (and/or designee) and other study personnel 
will be available to discuss the study progress and monitoring visit.  

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The investigator (and/or designee) will make study documents (e.g., consent forms, clinical data 
abstracts, and pertinent hospital or clinical records) readily available for inspection by the FDA, IRB, site 
monitors, and NIAID staff for confirmation of the study data.  
  
A specific protocol monitoring plan will be discussed with the PI and study staff before enrollment. The 
plan will outline the frequency of monitoring visits based on such factors as study enrollment, data 
collection status, and regulatory obligations.  
 
14  
Statistical Considerations  
 
14.1  Study Hypothesis  
The study hypothesis is that: 
- AGS-v PLUS specific immunoglobulin will increase after vaccination with AGS-v PLUS compared to 
placebo 
-PBMCs collected after vaccination with AGS-v PLUS incubated with AGS-v PLUS antigens will show a 
greater Th1 response as compared to placebo vaccination. Interferon–gamma is the main cytokine 
marker for Th1 responses. 
14.2  Sample Size Justification and Analysis Plan  
The primary objective of this study is to evaluate safety. 
 
For the safety endpoint we are interested in identifying the number of participants with at least one SAE 
that is possibly, probably or definitely related to vaccine (halting rule 1). With 10 participants in each 
group we would have an 80% chance of observing at least one participant with at least one SAE if the 
true underlying rate for that SAE in a vaccinated arm is 0.15. We will have a 10% chance of observing at 
least one SAE if the true underlying rate is .01. If we collapse across the vaccinated arms we would have 
40 participants. With 40 participants we would have a 95% chance of observing at least one participant 
with AEs if the true rate is 0.07. There is a 4% chance of observing at least one SAE out of 40 if the true 
rate is .001. With this sample size we will have a high probability of beginning to see a severe adverse 
event safety signal if one exists. Table 1 shows the probability of observing at least one participant with 
an SAE for various true SAE rates with samples sizes of 10 and 40. 
 
In halting rule 2 we will halt the study if at least 2 or more of the same or similar AE’s (possibly, probably 
or definitely related to vaccine) that are grade 3 or above are observed. With 10 participants in each 
group we would have an 80% chance of observing 2 or more participant with at least one AE if the true 
underlying rate in a vaccinated arm is 0.27. We will have a less than a 1% chance of observing at least 
2 AE if the true underlying rate is .01. If we collapse across the vaccinated arms we would have 40 
participants. With 40 participants we would have an 84% chance of observing at least two participants 
with AEs if the true rate is 0.08. There is a 6% chance of observing at least two AE out of 40 if the true 
rate is .01. With this sample size we will have a high probability of beginning to see an adverse event 
safety signal if one exists. Table 2 shows the probability of observing at least one participant with an SAE 
for various true SAE rates with samples sizes of 10 and 40. 

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Since this is a Phase 1b study we are looking for activity of the vaccine that would indicate the vaccines 
should be studied further. Since we do not wish to expose more participants than necessary to an 
experimental vaccine but we also don’t wish to miss an active vaccine we will design the study to have 
85% power with a one sided type I error rate of 0.1. We will also not adjust for the multiple comparisons 
(4 active arms being compared to placebo).  
  
With 10 participants per arm, there will be at least 85% power to detect a true difference in the primary 
immunogenicity endpoint of 0.95 standard deviations using a one-sided t-test with significance level of 
0.1(R version 3.4.1).  
 
Table 1. Probability of observing at least 1 SAE for samples sizes of 10 and 40 with different true SAE 
rates 
True rate of severe 
adverse events 
  
Probability of observing at least one participant with 
SAE 
(Halting rule 1) 
N=10 
N=40 
.001 
0.01 
0.04 
0.01 
0.10 
 0.33 
0.02 
 0.18 
 0.55 
0.03 
 0.26 
 0.70 
0.04 
 0.34 
 0.80 
0.05 
 0.40 
 0.87 
0.06 
 0.46 
 0.92 
0.07 
 0.52 
 0.95 
0.08 
 0.57 
 0.96 
0.09 
 0.61 
 0.98 
0.1 
 0.65 
 0.99 
0.11 
0.69 
0.99 
0.12 
.72 
0.99 
0.13 
0.75 
0.99 
.14 
 0.78 
 0.99 
0.15 
0.80 
0.99 
 
 

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Table 2. Probability of observing at least 2 SAE’s for samples sizes of 10 and 40 with different true SAE 
rates 
True rate of severe adverse 
events 
 
Probability of observing at least 2AE’s (halting rule 2) 
 
N=10 
N=40 
.01 
<.001 
.06 
.08 
.19 
.84 
.27 
.80 
>.99 
 
 
14.3  Analysis Plan  
T-tests will be used to compare immunogenicity endpoint measurements for the primary endpoint and 
other continuous endpoints between each treatment group and the placebo group. For binary endpoints 
Fishers exact test will be used. AEs will be tabulated by treatment arm. 
 
The fold increase in cellular and humoral responses to AGS-v PLUS from day 1 to days 43 and 50, and 
from day 43 to day 50 will be calculated for each participant. The mean fold increases on each active 
group will be compared to the mean fold increases in the placebo group. The geometric mean antibody 
titers and geometric mean cytokine concentrations to AGS-v PLUS on days 1, 43, and 50 will be 
calculated and compared between the different treatment groups. 
 
The fold increase in cellular responses to mosquito saliva from day 1 to days 43 and 50, and from day 43 
to day 50 will be calculated for each participant. The mean fold increases on each active group will be 
compared to the mean fold increases in the placebo group. The geometric mean cytokine concentrations 
to mosquito saliva on days 1, 43 and 50 will be calculated for each treatment group and compared to 
placebo. 
 
For the mosquito analysis, the following parameters will be compared on the active groups versus placebo 
for each mosquito  (Aedes aegypti and Aedes albopictus): 
-mean number of days mosquitoes survive after feeding on vaccinated participants 
-mean number of eggs laid 
-mean number of mosquitoes emerging from the laid eggs 
 
For the visual assessment of the mosquito bite the following parameters will be calculated for each group 
and active groups will be compared to placebo at three different time points: day 43 (30 min post feeding), 
day 45 and day 50: 
-mean number of visible bites 
-mean redness measured across the widest part (mm) 
-mean swelling measured using the ball point pen method (mm2). Ball point pen measurement of 
induration uses the pen to slide on top of the skin so it halts at the edge of true induration (versus redness) 
 
From the biopsies taken on day 45, the following comparisons will be made: 

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-gene expression: the level of expression of inflammatory cytokines at the bite site compared to normal 
skin (control) will be calculated for each participant for each mosquito bite. The mean will be calculated 
in each group and compared to placebo. 
 
The viability/infectivity of Zika virus after culture with PBMCs and serum from vaccinated participants will 
be assessed by infecting Vero cells with the cell supernatant followed by measuring viability of Vero cells 
using MTT assay. The higher the infectivity of the Zika virus the lower the MTT value will be for Vero 
cells. The fold changes from day 0 to day 43 will be measured for each participant and the mean 
calculated for each group and compared to placebo. 
  
For randomization, the first 10 participants will be randomized into 5 arms with 2 in each arm. An interim 
safety analysis after the first group of 10 participants have received the first immunization and completed 
the Day 8 phone call will be done by the DSMB by teleconference. Recruitment activities will continue 
but vaccination of new participants will be halted until the DSMB review has been completed. Once the 
interim safety analysis is done on the initial enrolment, an additional set of 40 participants will be enrolled 
and randomized using a permuted block randomization with a block size of 10.  
14.3.1 Interim Analysis 
Data for all groups collected up to day 50 will undergo interim analysis for safety and immunogenicity, 
and an interim study report will be generated. Due to the importance of this data, these results may be 
shared with the greater scientific community via meeting abstracts, professional presentations, and 
manuscript submissions to scientific journals. The data to be presented and the authorship will be 
approved by the study investigators, vaccine developer, and sponsor before any official communication. 
 
15  
Ethics/Protection of Human Participants 
  
15.1  Informed Consent Process  
Informed consent is a process where information is presented to enable persons to voluntarily decide 
whether or not to participate as a research participant. It is an ongoing conversation between the human 
research participant and the researchers, which begins before consent is given and continues until the 
end of the participant’s involvement in the research. Discussions about the research will provide essential 
information about the study and include: purpose, duration, experimental procedures, alternatives, risks, 
and benefits. Participants will be given the opportunity to ask questions and have them answered.  
  
The participants will sign the informed consent document before undergoing any procedures. The 
participant may withdraw consent at any time throughout the course of the trial. A copy of the informed 
consent document will be given to the participants for their records. The researcher will document the 
signing of the consent form in the participant’s medical record. The rights and welfare of the participant 
will be protected by emphasizing to them that the quality of their medical care will not be adversely 
affected if they decline to participate in this study.   
  
  

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15.2  
Participant Confidentiality  
All records will be kept confidential to the extent provided by federal, state, and local law. The study 
monitors and other authorized representatives of the sponsor may inspect all documents and records 
required to be maintained by the investigator, including but not limited to medical records. Electronic 
records will be kept in secure electronic systems. Clinical information will not be released without written 
permission of the participant, except as necessary for monitoring by IRB, the FDA, the NIAID, the OHRP, 
or the sponsor’s designee.  
 
16  
Data Handling and Record Keeping  
 
16.1  Data Capture and Management  
Study data will be collected and maintained in the study database and collected directly from participants 
during study visits and telephone calls. Source documents include all recordings of observations or 
notations of clinical activities and all reports and records necessary to confirm the data abstracted for this 
study. Data entry into these systems will be performed by authorized individuals. The Investigator is 
responsible for assuring that the data collected are complete, accurate, and recorded in a timely manner.  
  
Data that may potentially unblind the vaccine assignment (e.g., vaccine preparation/accountability data, 
and vaccine allocation) will be handled with special care to ensure that the integrity of the blind is 
maintained and the potential for bias is minimized. This can include making special provisions, such as 
segregating the data in question from view by the investigators, sponsor clinical team, or others as 
appropriate until the time of database lock and unblinding.  
16.2  Record Retention  
The investigator is responsible for retaining all essential documents listed in the ICH GCP Guidelines and 
21 CFR 312.62. Study records will be maintained at a minimum by the PI for a period of 2 years following 
the date a marketing application is approved for the drug for the indication for which it is being investigated; 
or, if no application is to be filed or if the application is not approved for such indication, until 2 years after the 
investigation is discontinued and FDA is notified.Records will be maintained in compliance with institutional, 
IRB, state, and federal medical records retention requirements, whichever is longest. All stored records 
will be kept confidential to the extent required by federal, state, and local law.  
  
Should the investigator wish to assign the study records to another party and/or move them to another 
location, the investigator will provide written notification of such intent to OCRPRO/NIAID with the name 
of the person who will accept responsibility for the transferred records and/or their new location. 
Destruction or relocation of research records will not proceed without written permission from 
NIAID/OCRPRO.  

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Bhattarai A, Ali AS, Kachur SP, et al. Impact of artemisinin-based combination therapy and insecticide-
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Najera JA, Gonzalez-Silva M, Alonso PL. Some lessons for the future from the Global Malaria Eradication 
Programme (1955-1969). PLoS medicine.  
2011;8(1):e1000412.  
4. 
Rts SCTP. Efficacy and safety of the RTS,S/AS01 malaria vaccine during 18 months after vaccination: a 
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Greenwood B, Targett G. Do we still need a malaria vaccine? Parasite immunology. 2009;31(9):582-586.  
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Ribeiro JM. Role of saliva in blood-feeding by arthropods. Annual review of entomology. 1987;32:463-478.  
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Wasserman HA, Singh S, Champagne DE. Saliva of the Yellow Fever mosquito, Aedes aegypti, modulates 
murine lymphocyte function. Parasite immunology. 2004;26(6-7):295-306.  
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Cross ML, Cupp EW, Enriquez FJ. Differential modulation of murine cellular immune responses by salivary 
gland extract of Aedes aegypti. The American journal of tropical medicine and hygiene. 1994;51(5):690-
696.  
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Silvestre R, Cordeiro-Da-Silva A, Santarem N, Vergnes B, Sereno D, Ouaissi A. SIR2-deficient Leishmania 
infantum induces a defined IFNgamma/IL-10 pattern that correlates with protection. Journal of immunology. 
2007;179(5):3161-3170.  
10. 
Peng Z, Yang M, Simons FE. Immunologic mechanisms in mosquito allergy: correlation of skin reactions 
with specific IgE and IgG antibodies and lymphocyte proliferation response to mosquito antigens. Annals 
of allergy, asthma & immunology : official publication of the American College of Allergy, Asthma, & 
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16. 
Vanderberg JP, Stewart MJ. Plasmodium sporozoite-host cell interactions during sporozoite invasion. 
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30. 
National 
Institutes 
of 
Health: 
Giving 
a 
subcutaneous 
injection 
2015; 
http://www.cc.nih.gov/ccc/patient_education/pepubs/subq.pdf, 2016.  

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Appendix A: Schedule of Procedures/Evaluations   
Study Phase >  
Screen 
Injec
tion 
1 
Follow
-up 
call 
Injecti
on 2 
Follow
-up 
call 
Feedin
g 
Follow
-up 
Follow-
up 
Follow
-up 
Follow
-up 
call 
Study Day > 
DAYS 
-60 to 
0 
D1 
D8 
D22 
D29 
D43 
D45 
D50 
D181 
D366 
Outpatient Visit  
X 
X 
 
X 
 
X 
X 
X 
 
 
Comprehension quiz 
X 
 
 
 
 
 
 
 
 
 
Written Consent  
X 
 
 
 
 
 
 
 
 
 
Medical/Medication History  
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Physician Assessment and PE (targeted and as 
needed after screening) 
X 
X 
 
X 
 
X 
X 
X 
 
 
Review of inclusion/exclusion criteria and 
review of consent  
X 
X 
 
X 
 
 
 
 
 
 
Vital signs (temperature, heart rate, blood 
pressure) 
X 
X 
 
X 
 
X 
 
X 
 
 
Vital signs, Vaccine injection site and AE 
assessment pre-vaccination and 30-45 min post-
vaccination  
 
X 
 
X 
 
 
 
 
 
 
Provision of memory aid, ruler and thermometer 
 
X 
 
X 
 
 
 
 
 
 
Review of AEs 
 
 
X 
X 
X 
X 
X 
X 
 
 
Review of Memory Aid 
 
 
X 
 
X 
 
 
 
 
 
Telephone Call to Assess  
Symptoms and AEs 
 
 
X 
 
X 
 
 
 
X 
X 
Pregnancy test (serum/urine)‡  
X 
X 
 
X 
 
X 
 
 
 
 
Vaccine administration  
 
X 
 
X 
 
 
 
 
 
 
Randomization  
 
X 
 
 
 
 
 
 
 
 
Mosquito Feeding  
 
 
 
 
 
X 
 
 
 
 
Mosquito Bite Assessment 
 
 
 
 
 
X 
X 
X 
 
 
Safety Labs: hemoglobin, white blood cells, 
platelets, serum creatinine, serum alanine 
aminotransferase, random glucose 
X 
X 
 
X 
 
X 
 
X 
 
 
Screening Labs: HIV, hepatitis B and C  
X 
 
 
 
 
 
 
 
 
 
Serum and Whole Blood Collection  
 
X 
 
 
 
X 
 
X 
 
 
Skin Biopsy 
 
 
 
 
 
 
X 
 
 
 
Skin Biopsy inspection 
 
 
 
 
 
 
 
X 
 
 
 † 
Vital Signs: participants must be sitting for a minimum of 5 minutes before these procedures being 
performed; vital signs include blood pressure, heart rate, temperature. Weight and height will be taken at 
the screening visit only.   
‡ 
Serum pregnancy testing at the screening and urine pregnancy testing for all other visits. On Days 1, 22, 
43, study vaccination or mosquito feeding will not be initiated until negative results are available. Females 
of childbearing potential only.  
  

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Appendix B: Blood Volumes for Specimen Collection  
 
 
 
 
Study 
Schedule/Procedures 
Volume Screening 
Day 1 
prevaccination 
Day 22 
prevaccination 
Day 43 
(pre-
mosquito 
feeding) 
Follow-up Day 
50 
Serum creatinine, serum 
alanine aminotransferase, 
hepatitis BsAg, hepatitis C 
Ab & b-hCG, Pregnancy, 
if needed 
8.5 mL 
per blood 
draw 
8.5 mL 
x 
x 
x 
x 
HIV 
3.5 mL 
3.5 mL 
X 
X 
X 
X 
Complete blood count 4 mL per 
blood 
draw 
4 mL 
4 mL 
4 mL 
4 mL 
4 mL 
Serum creatinine, serum 
alanine aminotransferase 
5 mL per 
blood 
draw 
x 
5 mL 
5 mL 
5 mL 
5 mL 
Serum for Immunology 
studies 
4mL 
x 
4mL 
x 
4ml 
 
4mL 
Whole blood for 
Immunology studies 
50 mL 
x 
50 mL 
x 
50 ml 
50 mL 
Daily Volume (mL) 
 
16 mL 
63 mL 
9 mL 
63 mL 
63 mL 
Cumulative Volume 
 
 
79 mL  
88 mL  
151 mL 
214 mL 

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Appendix C: Toxicity Tables  
 
Vital Signs 
Vital Signs 
Mild (Grade 1) 
Moderate (Grade 
2) 
Severe (Grade 3) 
Potentially Life Threatening 
(Grade 4) 
Fever  (C) * 
          
38.0-38.4 
100.4-101.2 (F) 
38.5-38.9 
101.3-102.1(F) 
39.0-40.0 
102.2 – 104.0 (F) 
>40.0 
>104.0(F) 
Hypertension (systolic) 
mm Hg ** 
141-150 
151-160 
>160 
ER visit or hospitalization for 
malignant hypertension 
Hypertension (diastolic) 
mm Hg 
91-95 
96-100 
>100 
ER visit or hospitalization for 
malignant hypertension 
Hypotension (systolic) mm 
Hg 
85-89 
80-84 
<80 
ER visit or hospitalization for 
hypotensive shock 
Bradycardia – beats per 
minute 
50-54 
or 45-50 if 
baseline <60 
45-49 
or 40-44 if baseline 
<60 
<45 
or <40 if baseline 
<60 
ER visit or hospitalization for 
arrhythmia 
Tachycardia – beats per 
minute 
101-115 
116-130 
>130 
or ventricular 
dysrhythmia 
ER visit or hospitalization for 
arrhythmia 
*  
Oral temperature; no recent hot or cold beverages or smoking 
**  Assuming seated position, 5 minutes at rest conditions, not sleeping subjects, measurements on the same arm and several concordant 
results 
Hematology 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially Life 
Threatening (Grade 4) 
WBC Decreased –cell/mm3 
2,500 – 3,500 
1,500 – 2,499 
1,000 – 1,499 
<1,000 
WBC Increased –cell/mm3 
11,001 – 15,000 
15,001 – 
20,000 
20,001 – 25,000 
>25,000 
Hgb g/dL (Female) 
11.0 – 11.5 
9.5 – 10.9 
8.0 – 9.4 
<8.0 
Hgb g/dL (Male) 
12.0 – 12.5 
10.0 – 11.9 
8.5 – 9.9 
<8.5 
Platelets Decreased –cell/mm3 
120,000 – 
130,000 
100,000 – 
119,999 
25,000 – 99,000 
< 25,000 
 
 
 
 
 
Chemistry 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially Life 
Threatening (Grade 4) 
ALT Increased IU/L 
46 – 105 
106 – 175 
176 - 450 
>450 
Creatinine mg/dL (Increased) (Female) 
>ULN – 1.7 
1.8 – 2.0 
2.1 – 2.5 
>2.5 or requires dialysis 
Creatinine mg/dL (Increased) (Male) 
>ULN – 1.7 
1.8 – 2.0 
2.1 – 2.5 
>2.5 or requires dialysis 
Glucose-hypoglycemia mg/dL 
65-67 
55-64 
45 - 54 
<45 
Glucose-hyperglycemia random- mg/dL 
140-159 
160-200 
>200 
Insulin requirements or 
hyperosmolar coma 

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Appendix D: UMB IRB reporting guidelines