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

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Randomized, Double-Blind, Placebo-Controlled, Phase 1 
Study in Healthy Volunteers to Evaluate the Safety and 
Immunogenicity of AGS-v, a Universal Mosquito-Borne 
Disease Vaccine  
 
NIAID Protocol Number 
17-I-0059 
 
 
Sponsored by 
Office of Clinical Research Policy and Regulatory Operations (OCRPRO), DCR, NIAID, 
NIH 
 
IND # 17213 
 
Vaccine Manufactured by 
SEEK 
 
Principal Investigators 
Matthew J. Memoli, MD, MS 
 
Version Number and Date 
Version 5.0 
February 09, 2018 
 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 2 of 62 
TEAM ROSTER 
 
Principal Investigators 
 
Matthew J. Memoli, MD, MS 
Director, Clinical Studies Unit 
LID, NIAID 
Building 33, Room 3E19A 
Bethesda, MD 20892 
Tel: 301-443-5971 
Email: memolim@mail.nih.gov 
 
 
 
 
 
Associate Investigators 
 
 
 
 
Jesus G. Valenzuela, PhD 
LMVR, VMBS, NIAID 
Tel: 301-761-5070 
Email: jvalenzuela@niaid.nih.gov 
 
Jessica Manning, MD, MS 
LID, NIAID 
Tel: 301-496-7196 
Email: jessica.manning@nih.gov 
 
Shaden Kamhawi, PhD 
LMVR, VMBS, NIAID 
Tel: 301-761-5070 
Email: skamhawi@niaid.nih.gov 
 
Luiz Fabiano Oliveira, MD PhD 
LMVR, VMBS, NIAID 
Tel: 301-496-4018 
Email: LOliveira@niaid.nih.gov 
 
Iliano V. Coutinho-Abreu, PhD 
LMVR, VMBS, NIAID 
Tel: 301-761-3977 
Email:iliano.vieiracoutinhoabreugomes2@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 
 
 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 3 of 62 
Sponsor Medical Monitor 
H. Preston Holley, MD 
Medical Monitor 
Leidos Biomedical Research, Inc. 
5705 Industry Lane 
Frederick, MD 21704 
Tel: 301-228-4005 
Email: horace.holley@nih.gov 
  
Study Staff NIH 
Holly Ann Baus, RN, MSN 
LIR, NIAID 
Tel: 301-761-6800 
Email: holly.baus@nih.gov 
 
 
 
Susan M. Reed, BA, CRC 
LID, NIAID 
Tel: 301-451-3354 
 
 
Rani S. Athota, BS, MPH 
LID, NIAID 
Tel: 301-594-0803 
 
 
 
Unblinded Study Staff NIH  
    Alison Han, MD, MS 
     LID, NIAID 
     Tel: 301-480-5722 
     Email: alison.han@nih.gov 
 
     Lindsay M. Czajkowski, MSN, NP-C 
     LID, NIAID 
Tel: 301-451-7705 
 
 
 
Collaborators 
 
Kathie-Anne Walters, PhD 
Institute for Systems Biology 
401 Terry Ave N 
Seattle, WA 98109 
Tel: 206-732-1377 
Email: kathie.walters@systemsbiology.org 
 
 
 
 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 4 of 62 
TABLE OF CONTENTS 
TEAM ROSTER ................................................................................................................ 2 
TABLE OF CONTENTS .................................................................................................... 4 
LIST OF TABLES .............................................................................................................. 6 
LIST OF TABLES .............................................................................................................. 6 
LIST OF FIGURES ........................................................................................................... 6 
LIST OF ABBREVIATIONS .............................................................................................. 7 
PROTOCOL SUMMARY .................................................................................................. 8 
1 
BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE ..................... 12 
2 
STUDY OBJECTIVES ......................................................................................... 21 
2.1 
Primary Objective ................................................................................................ 21 
2.2 
Secondary Objectives ......................................................................................... 21 
3 
STUDY DESIGN ................................................................................................. 22 
3.1 
Description of the Study Design .......................................................................... 22 
3.2 
Study Endpoints .................................................................................................. 22 
4 
STUDY POPULATION ........................................................................................ 23 
4.1 
Rationale for Participant Selection ...................................................................... 23 
4.2 
Recruitment Plan ................................................................................................. 24 
4.3 
Inclusion Criteria ................................................................................................. 24 
4.4 
Exclusion Criteria ................................................................................................ 25 
4.5 
Justification for Exclusion of Pregnant Women and Children (Special 
Populations) ........................................................................................................ 26 
5 
STUDY AGENTS/INTERVENTIONS .................................................................. 26 
5.1 
Agent 1: AGS-v, AGS-v + Adjuvant, or Placebo ................................................. 26 
5.1.1 
Formulation, Packaging and Labeling ..................................................... 26 
5.1.2 
Study Agent Storage and Stability ........................................................... 27 
5.1.3 
Adjuvant .................................................................................................. 27 
5.1.4 
Placebo ................................................................................................... 27 
5.1.5 
Preparation .............................................................................................. 27 
5.2 
Dosing and Administration .................................................................................. 28 
5.3 
Disposal and Drug Accountability of AGS-v ........................................................ 29 
5.3.1 
Product Accountability ............................................................................. 29 
5.3.2 
Accountability of Study Supplies ............................................................. 30 
5.3.3 
Retention of Samples .............................................................................. 30 
5.4 
Concomitant Medications and Procedures ......................................................... 30 
6 
STUDY SCHEDULE ........................................................................................... 30 
6.1 
Randomization and Blinding ............................................................................... 31 
6.2 
Randomization and Vaccine or Placebo Administration (Day 0) ......................... 31 
6.3 
Day 7 (+/- 2 days) ............................................................................................... 32 
6.4 
Day 14 (+/- 2 days) ............................................................................................. 32 
6.5 
Day 21 (+/- 2 days) ............................................................................................. 33 
6.6 
Day 28 (+/- 2 days) ............................................................................................. 33 
6.7 
Day 35 (+/- 2 Days) ............................................................................................. 33 
6.8 
Day 42 Mosquito Feeding (+14 days) ................................................................. 34 
6.9 
Day 44 Telephone Call (+/- 1 day) ...................................................................... 35 
6.10 
Follow-up ............................................................................................................. 35 
7 
STUDY PROCEDURES/ EVALUATIONS .......................................................... 35 
7.1 
Clinical and Laboratory Evaluations .................................................................... 35 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 5 of 62 
7.1.1 
Laboratory Testing of Collected Samples for Primary and Secondary 
Endpoints ................................................................................................ 35 
7.2 
Reactogenicity ..................................................................................................... 37 
8 
POTENTIAL RISKS AND BENEFITS ................................................................. 37 
8.1 
Risks of Placebo administration .......................................................................... 37 
8.2 
Risks of AGS-v ± adjuvant vaccination ............................................................... 37 
8.3 
Risks of adjuvant ................................................................................................. 39 
8.4 
Risks of blood draw ............................................................................................. 40 
8.5 
Risks of Aedes aegypti Mosquito Feeding .......................................................... 41 
8.6 
Potential Benefits ................................................................................................ 41 
9 
RESEARCH USE OF STORED HUMAN SAMPLES, SPECIMENS, OR DATA 41 
10 
DATA SHARING PLAN ....................................................................................... 42 
10.1 
Human data generated in this study will be shared for future research as follows:
 ............................................................................................................................ 42 
10.2 
Data will be shared through ................................................................................ 42 
10.3 
The data will be shared: ...................................................................................... 42 
11 
REMUNERATION PLAN ..................................................................................... 42 
12 
ASSESSMENT OF SAFETY ............................................................................... 43 
12.1 
Documenting, Recording, and Reporting AEs .................................................... 43 
12.2 
Definitions ........................................................................................................... 43 
12.3 
Investigator Assessment of AEs ......................................................................... 45 
12.3.1 
Severity ................................................................................................... 45 
12.3.2 
Causality .................................................................................................. 46 
12.4 
Investigator Reporting Responsibilities to the Sponsor ....................................... 47 
12.4.1 
AEs .......................................................................................................... 47 
12.4.2 
SAEs ....................................................................................................... 47 
12.4.3 
Unanticipated Problems .......................................................................... 47 
12.4.4 
Pregnancy ............................................................................................... 47 
12.5 
Investigator Reporting Responsibilities to the NIAID IRB ................................... 48 
12.5.1 
Special Reporting Situations ................................................................... 48 
12.5.2 
Expedited Reporting to the NIAID IRB .................................................... 48 
12.5.3 
Waiver of Reporting Anticipated Protocol Deviations, Expected 
UPnonAEs and Deaths to the NIAID IRB ................................................ 48 
12.5.4 
Annual Reporting to the NIAID IRB ......................................................... 48 
12.6 
Follow-Up of AEs and SAEs ............................................................................... 49 
12.7 
Sponsor’s Reporting Responsibilities .................................................................. 49 
12.8 
Pausing Rules for an Individual Subject .............................................................. 49 
12.8.1 
Reporting a Pause ................................................................................... 50 
12.8.2 
Resumption of a Paused Study ............................................................... 50 
12.9 
Halting Rules for the Protocol ............................................................................. 50 
12.9.1 
Reporting a Study Halt ............................................................................ 50 
12.9.2 
Resumption of a Halted Study ................................................................. 50 
12.10 Study Discontinuation ......................................................................................... 51 
12.11 Premature Withdrawal of a Participant ................................................................ 51 
12.12 Replacement of a Participant .............................................................................. 52 
12.13 Safety Oversight .................................................................................................. 52 
12.13.1 
Safety Review and Communications Plan (SRCP) ................................. 52 
12.13.2 
Sponsor Medical Monitor ......................................................................... 52 
12.13.3 
Data and Safety Monitoring Board .......................................................... 52 
13 
SITE MONITORING PLAN ................................................................................. 53 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 6 of 62 
14 
STATISTICAL CONSIDERATIONS .................................................................... 53 
14.1 
Study Hypothesis ................................................................................................ 53 
14.2 
Sample Size Justification and Analysis Plan ....................................................... 53 
14.3 
Analysis Plan ....................................................................................................... 55 
15 
ETHICS/PROTECTION OF HUMAN PARTICIPANTS ....................................... 55 
15.1 
Informed Consent Process .................................................................................. 55 
15.2 
Non–English-Speaking Participants .................................................................... 56 
15.3 
Participant Confidentiality .................................................................................... 56 
16 
DATA HANDLING AND RECORD KEEPING ..................................................... 56 
16.1 
Data Capture and Management .......................................................................... 56 
16.2 
Record Retention ................................................................................................ 57 
APPENDIX A: SCHEDULE OF PROCEDURES/EVALUATIONS .................................. 61 
APPENDIX B: BLOOD VOLUMES FOR SPECIMEN COLLECTION ............................. 62 
LIST OF TABLES 
Table 1. Participant remuneration ................................................................................... 42 
Table 2. Probability of observing at least one AE. .......................................................... 54 
Table 3. Difference in probabilities that can be detected with 80% power for sample 
sizes of 15 in each group or 15 in one group and 30 in the other ................................... 55 
  
List of Figures 
Figure 1. Graph representing the mosquito % survival post feeding .............................. 15 
Figure 2. Effect of immunization with salivary gland protein fractions on mosquito 
fecundity. (vehicle (control), <20 kDa, 20-40 kDa, 40-80 kDa and >80 kDa) 
fecundity. .......................................................................................................... 16 
Figure 3. IFN-g response in mice immunized with AGS-mix  (AGS-v) or vehicle. .......... 17 
Figure 4.Total Ig and IgG2a specific-AGS-mix (AGS-v) ................................................. 18 
Figure 5.Animals succumbing to infection ...................................................................... 19 
Figure 6. Study Schedule ................................................................................................ 31 
 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 7 of 62 
LIST OF ABBREVIATIONS 
ACT 
 
Artemisinin-based combination therapy 
AE 
 
Adverse Event 
AR 
 
Adverse Reaction 
BMI 
 
Body mass index  
CC 
 
Clinical Center 
CFR 
 
Code of Federal Regulations 
CRF 
 
Case Report Form 
CSO 
 
Clinical Safety Office 
CSU 
 
Clinical Studies Unit 
DCR 
 
Division of Clinical Research 
DLM 
Department of Laboratory Medicine 
DSMB 
Data Safety Monitoring Board  
ECG 
 
Electrocardiogram 
ELISA  
Enzyme-linked Immunosorbent Assay 
EP 
 
European Pharmacopoeia 
FDA 
 
Food and Drug Administration 
GCP 
 
Good Clinical Practice 
HIV 
 
Human Immunodeficiency Virus 
HRPP  
Human Research Protection Program 
ICH 
 
International Council on Harmonization 
IFA 
 
Incomplete Freund’s adjuvant 
Ig 
 
Immunoglobulin 
IND 
 
Investigational New Drug 
IP 
 
Investigational Product 
IRB 
 
Institutional Review Board 
IV 
 
Intravenous 
LID 
 
Laboratory of Infectious Disease 
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 
PCR   
Polymerase chain reaction 
PI 
 
Principal Investigator 
RT 
 
Room Temperature 
SAE 
 
Serious Adverse Event 
SAR 
 
Serious Adverse Reaction 
SCSU   
Special Clinical Studies Unit 
SERF  
Safety Expedited Report Form 
SRCP  
Safety Review and Communications Plan 
SUSAR 
Serious, Unexpected, Suspected Adverse Events 
Th1 
 
CD4+ T helper (Th1) cells 
UP 
 
Unanticipated Problem 
UPnonAE 
Unexpected Problem that is not an AE 
USP 
 
United States Pharmacopeia 
WFI 
 
Water for Injection 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 8 of 62 
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, a Universal Mosquito-Borne Disease and Mosquito Control 
Vaccine  
Short Title: 
Universal Mosquito Vaccine  
IND Sponsor:  
 
OCRPRO, DCR, NIAID, NIH/ NIAID LID CSU 
Conducted by: 
 
LID 
Principal Investigator: 
Matthew J. Memoli, MD, MS 
Sample Size: 
45 participants 
Accrual Ceiling: 
60 participants   
Study Population: 
 
Healthy volunteers 18 to 50 years of age 
Accrual Period: 
 
1 year  
Study Design: 
 
This is a randomized, double-blind, placebo-controlled, 
single-center, Phase 1 study of AGS-v administered as 
2 vaccinations prior to a non-infected Aedes aegypti 
mosquito feeding. 
Study Duration: 
The study will begin approximately in December 2016 and 
will require approximately 3 years to complete. The length 
of individual subject participation is about 1 year. 
Study Agents 
 
1. AGS-v 
 
 
2. AGS-v + adjuvant 
 
 
3. Placebo 
Intervention Description: 
Individuals will be vaccinated subcutaneously with vaccine 
or placebo.    
Primary Objective:  
The primary objectives of the study are: 
1. To determine the safety of AGS-v as measured by 
incidence and type of adverse events (AEs) observed 
2. To identify an immune response produced by AGS-v 
by an increase in one or both of the following immune 
markers: 
a. Total AGS-v specific immunoglobulin from 
participant serum 21 days after the first and/or 
second vaccination 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 9 of 62 
b. Th1 and Th2-associated cytokine release after 
incubation of peripheral blood mononuclear 
cells (PBMCs) collected 21 days after the first 
or second vaccination with AGS-v antigens  
 
 
 
Secondary Objectives: 
The secondary objectives of this study are to identify other 
measures of immunogenicity including:  
 
1. Measurement of immune response after clean Aedes 
aegypti mosquito feeding on participants by: 
a. measuring AGS-v specific total 
immunoglobulin from serum  
b. measuring Th1 and Th2-related cytokines 
after incubation of PBMCs with AGS-v 
antigens  
 
2. Measure AGS-v specific immunoglobulins, primarily 
IgG1, IgG3, and IgM both after vaccination and clean 
Aedes aegypti mosquito feeding 
3. Evaluate the effect of the AGS-v immune response on 
Aedes aegypti mosquito feeding and fecundity after 
feeding on a vaccinated individual or whole blood 
collected from those individuals in vitro.   
4. Evaluate the effect of incubating Zika virus coated in 
vector saliva with PBMCs and/or serum collected from 
vaccinated individuals. 
Endpoints: 
 
 
Primary:  
1. Incidence and severity (by grading) AEs in AGS-v vaccinated 
individuals 
2. Total AGS-v specific immunoglobulin measured in serum 21 
days after the first and/or second vaccination. 
3. Interferon-gamma and other cytokine markers of Th1 and Th2 
response measured in vitro from PBMCs incubated with AGS-
v antigens as indicators of the Th1 vs. Th2 response 
 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 10 of 62 
Secondary:  
1. Total AGS-v specific immunoglobulin in acute serum collected 
immediately after a clean Aedes aegypti mosquito feeding and 
convalescent serum collected on follow-up visits (60 days later 
+/- 14 days)               
2. Interferon-gamma and other cytokine markers of a Th1 or 
Th1/Th2 mixed response measured in vitro from acute PBMCs 
and serum collected immediately after a clean Aedes aegypti 
mosquito feeding incubated with AGS-v antigens and 
convalescent PBMCs and serum collected on follow-up visits 
(60 days later +/- 14 days) 
3. Measurement of changes to Aedes aegypti mosquito life cycle 
post feeding on blood from participants after vaccination both 
in vivo and in vitro, including survival, egg laying, and other 
measures of reproduction. 
4. Infectivity of Zika virus after incubation with serum and/or 
PBMCs from vaccinated participants 
 
 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 11 of 62 
Précis 
Mosquito-borne diseases continue to cause significant morbidity and mortality worldwide 
despite on-going control efforts.  In 2015, there were >200 million cases of malaria 
worldwide, causing nearly half a million deaths, with most of the deaths occurring among 
children under the age of 5 years 1.  Mosquitos also transmit arboviruses, including 
dengue, yellow fever, West Nile virus, chikungunya, Rift Valley fever, Japanese 
encephalitis, and Zika virus.  The current new outbreak of Zika virus 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. 
 
In this protocol we plan to perform a Phase I study of a novel 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 hope to demonstrate the safety of this vaccine similar to SEEK’s other 
peptide based vaccines Flu-v and HIV-v that have been found to have very good safety 
profiles in previous Phase I trials.  We also hope to demonstrate immunomodulation 
after a controlled clean Aedes aegypti mosquito feeding to demonstrate proof of concept 
efficacy of the vaccine. With the current rise of Zika in the Americas and the threat of 
local mosquito transmission in 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 and malaria that make dealing 
with this newly emerging epidemic a challenge. 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 12 of 62 
1 
Background Information and Scientific Rationale 
 
Mosquito-borne diseases continue to cause significant morbidity and mortality worldwide 
despite on-going vector control efforts.  In 2015, there were >200 million cases of 
malaria worldwide, causing nearly half a million deaths, with most of the deaths 
occurring among children under the age of 5 years 1.  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 current 
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. 
 
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-prong 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 the development of the disease in a healthy 
individual (prophylactic vaccine) and block the transmission of the disease 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, respectively 4. Even if the RTS,S vaccine is licensed, there will be a 
recognized need for the continued implementation of current malaria prevention 
programs 5. 
 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 13 of 62 
In this protocol we propose to perform a Phase I study of a 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 the prophylactic and the transmission blocking 
components.  SEEK’s approach in developing such a vaccine is defined by the following 
three principles: 
 
• 
Because the complexity and variability of the pathogens’ life cycle and antigenic 
make up, the prophylactic effect of the vaccine is not aimed at destroying the 
parasite itself, but rather to decrease the development of the essential 
physiologic environment that leads to a successful infection in humans. 
• 
Because the varied number of mosquito species capable of transmitting the 
pathogens, the transmission blocking effect of the vaccine is not aimed at killing 
the vector on contact, but rather to prevent the very process of blood feeding, 
which is essential for the successful procreation of the vector. 
• 
Because most nations where malaria and arboviral diseases are endemic do not 
have well developed healthcare systems and limited healthcare budgets, the 
vaccine must be economically viable as a large-scale product entirely 
manufactured synthetically from raw components, which are widely available, 
and must not require complex storage and delivery systems. 
 
Female mosquitoes have an absolute physiological need for blood-feeding in order 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 host 6. 
Mosquito saliva has been shown to inhibit T and B cell proliferation 7 and downregulate 
the expression of interferon gamma (IFN-g) 8, a soluble Th1 pro-inflammatory immune 
mediator known to have in vivo therapeutic and prophylactic effects against other 
arthropod transmitted diseases such as leishmaniasis 9. Interestingly, mosquito saliva 
also triggers a non-inflammatory Th2 immune response in the host that causes the 
characteristic irritation and allergic reaction associated with mosquito bites 10. 
 
With time and continuous exposure, individuals become desensitized to mosquito bites 
due to a reduction in Th2 cytokine mediators, thus allowing for the more effective 
development of pro-inflammatory Th1 responses 11. This process is thought to be 
associated with the gradual acquisition of partial immunity to infection observed in adults 
living in malaria-endemic regions. Studies in patients infected by Plasmodium falciparum 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 14 of 62 
presenting with uncomplicated malaria suggest that an increase of Th1 cytokines (e.g. 
IL-12 and IFN-g) in the acute phase provides protective immunity and may also be 
involved in preventing severe malaria and complications from malaria. 12 Recent studies 
support this conclusion by showing that 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 saliva13. 
  
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 minutes  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 macrophages 16-18. 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 virus 19, and 
arboviruses in general 20. 
 
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 kDa mosquito salivary gland protein significantly reduced the invasion by 
Plasmodium yoelii sporozoites of salivary glands in mosquitoes that fed on blood 
containing that antibody 21. 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 antibodies 22,23. Therefore, the antibodies against salivary 
antigens may 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 the 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 infection by the pathogen 
and (b) block transmission of the disease by preventing invasion of the arthropod 
vector’s salivary glands and/or reduce the survival and/or fecundity of the vector. 
 
As indicated earlier, SEEK aims at manufacturing 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 Fmoc 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 15 of 62 
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 the proteins of a 
salivary gland lysate from Anopheles gambiae 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 Fig. 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 the disease.  
 
 
Figure 1. Graph representing the mosquito % survival post feeding 
 
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. 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). 
 
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 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 16 of 62 
from eggs and mature adult offspring by the surviving mosquitoes (Fig. 2). 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 
transmission rate of the disease. 
  
Figure 2. Effect of immunization with salivary gland protein fractions on mosquito 
fecundity. (vehicle (control), <20 kDa, 20-40 kDa, 40-80 kDa and >80 kDa) fecundity. 
 
 
 
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 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 SwissProt). Any region showing significant sequence similarity was 
discarded in order 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 24 and HIV 
viruses. The identified sequences constitute two independent candidate vaccine 
preparations currently undergoing clinical trials in the UK. 
 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 17 of 62 
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 the saliva of the mosquito vector is associated 
with an increased 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) and identified as AGS-v (or AGS-mix) were used 
to immunize mice and to establish the type of induced immune response.  While there is 
no specific reason why four peptides are 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-g 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-g and IgG2a are considered primary mediators of a Th1 response and hence 
immunization with AGS-v can and does induce the desired Th1 response. 
 
Figure 3. IFN-g response in mice immunized with AGS-mix  (AGS-v) or vehicle. 
 
Splenocytes were isolated from mice spleens and incubated with media (blank), a non-
specific positive control (Concanavalin A), or different concentrations of the vaccine 
preparation (2-4uM AGS-mix). After 72h incubation, the levels of IFN-g (Th1 cytokine) 
were measured by ELISA. 
 
 
0
200
400
600
800
1000
1200
1400
blank
ConA
AGS-mix 2uM AGS-mix 4uM
control group
AGS-mix group
Splenocyte IFNg production
IU/ml 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 18 of 62 
Figure 4.Total Ig and IgG2a specific-AGS-mix (AGS-v)  
response in mouse sera as measured by ELISA. 
 
 
 
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 of the disease.  
 
Traditional experimental models of malaria infection rely on the intravenous injection of 
large numbers of parasites in order 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 infection 25. 
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 show that mice vaccinated with AGS-v are 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

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 19 of 62 
Figure 5.Animals succumbing to infection 
 
 
Survival of mice vaccinated with AGS-v vs. mice vaccinated with a control preparation 
(NRP or non-relevant product) upon challenge via malaria-infected mosquito bite. 
Groups of 5-6 mice were challenged by bite of infected mosquitoes (low to moderate 
parasite oocyst burden) on Day 21 post-vaccination (animals received two doses of 
either AGS-v or NRP-V on Days 1 and 15) 
 
Further experiments in C57BL6, BALBc, and CD1 mice have been performed to 
measure immunogenicity.  All had good responses to the vaccine after two weeks, and  
antibodies to AGS-v have been raised to the vaccine many months after vaccination and 
will likely last multiple years.  T-cell responses are seen two weeks after vaccination, but 
are harder to predict long-term. 
 
SEEK has performed clinical studies with two other vaccines using the same technology 
as AGS-v.  Based on their experience with FLU-v and HIV-v in clinical trials it is clear 
that a combination of four peptides allows the immune system to mount a response 
without peptides competing with each other.   These studies have also assisted in 
determination of the appropriate dose as 50 nmol of each peptide proved to be the best 
dose which replicated the ratio nmol/Kg in mouse studies, and therefore was the most 
immunogenic and safe in humans for these other vaccines.  In Phase 1 studies for both 
HIV-v in HIV-positive males and FLU-v in healthy adults, a dose of 250 ug versus 500 ug 
with and without adjuvant were given (with approximately 10 people in each arm, for a 
total of 40 volunteers per Phase 1 study).  In the HIV-v Phase 1 trial, either dose induced 
a significant and sustained antibody response in 60% of volunteers but only the higher 
500 ug 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-g response in more than 80% of volunteers in a dose-dependent manner.  A FLU-v 
Phase II study is evaluating the efficacy of a single dose of adjuvanted 500 ug of FLU-v 
(15 volunteers have received FLU-v vaccine and 12 have received control). 
 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 20 of 62 
A parenteral tolerance study in New Zealand White Rabbits was conducted to evaluate 
the local tolerance and irritation potential associated with a single subcutaneous injection 
of AGS-v. 
 
All animals survived test article or vehicle control formulation administration and 
appeared active and healthy during the study. Erythema scores were very slight and 
comparable between Test Formulation 1 (AGS-v in suspension – lyophilized AGS-v 
reconstituted in 0.5 mL of water for injection) and contralateral Vehicle Control 1 (water 
for injection). Erythema resolved by the end of the observation period. There was no 
edema observed at any test site of administration-related findings on macroscopic 
evaluation. 
 
In this tolerance study with New Zealand white rabbits, the severity of edema, very slight 
to slight, and erythema, slight to well-defined, scores was comparable between Test 
Formulation 2 (AGS-v in emulsion – lysophilized AGS-v reconstituted in 0.25 mL of 
water for injection and emulsified with 0.25 of Montanide ISA-51) and contralateral 
Vehicle Control 2 (0.25 mL water for injection and adjuvant, 0.25 mL Montanide ISA-51, 
emulsion); however a slightly lower incidence associated with evidence of resolution at 
the end of the observation period was observed for Test Formulation 2. These scores 
correlated with a lower incidence of macroscopic observations for Test Formulation 2. 
 
Under the conditions of this study, a single subcutaneous injection of AGS-v (Test 
Formulation 1) caused minimal irritation comparable to contralateral vehicle control with 
full clinical resolution when administered as a suspension in water and minimal to mild 
irritation when administered as an emulsion using Montanide ISA-51 (Test Formulation 
2) with a slightly lower irritation incidence when compared to contralateral vehicle 
control. 
 
In summary, the data presented here supports the validity of SEEK’s scientific 
hypothesis. This states that immunization with mosquito salivary antigens can induce a 
Th1 specific response that provides both prophylactic and transmission blocking 
immunity. Equally importantly, and in contrast to other groups that have shown the ability 
of vaccination with salivary proteins to reduce infection in diseases such as 
leishmaniasis 26,27.  SEEK’s vaccine is composed entirely of synthetic products that do 
not require complex heterologous expression systems for manufacture and hence can 
readily be 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 preparation is a vaccine candidate capable of delivering on both the 
prophylactic and transmission blocking areas of malaria and arbovirus infection control. 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 21 of 62 
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 host 20. This is particularly relevant 
since many hematophagous arthropods such as Aedes aegypti are responsible for the 
transmission of more than one pathogen (e.g. dengue, Zika and West Nile virus).   
 
In this protocol we plan to demonstrate the safety of this vaccine similar to SEEK’s other 
peptide based vaccines Flu-v and HIV-v that have been found to have very good safety 
profiles in Phase I trials.  We also hope to demonstrate immunomodulation after a 
controlled clean Aedes aegypti mosquito feeding as proof of concept efficacy of the 
vaccine.  With the current rise of Zika 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 and malaria that make dealing with this newly 
emerging epidemic a challenge. 
 
2 
Study Objectives 
2.1 
Primary Objective  
The primary objectives of the study are: 
1. To determine the safety of AGS-v as measured by incidence and type of AEs 
observed  
2. To identify an immune response produced by AGS-v by an increase in one or 
both of the following immune markers: 
a. Total AGS-v specific immunoglobulin from participant serum 21 days 
after the first and/or second vaccination 
b. Th1 and Th2-associated cytokine release after incubation of 
peripheral blood mononuclear cells (PBMCs) collected 21 days after 
the first or second vaccination with AGS-v antigens  
2.2 
Secondary Objectives 
The secondary objectives of this study are to identify other measures of immunogenicity 
including:  
 
1. Measurement of immune response after clean Aedes aegypti mosquito 
feeding on participants by: 
a. measuring total AGS-v specific immunoglobulin from serum  
b. measuring Th1 and Th2-related cytokines after incubation of PBMCs 
with AGS-v antigens  

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 22 of 62 
2. Measure AGS-v specific immunoglobulins, primarily IgG1, IgG3, and IgM 
both after vaccination and clean Aedes aegypti mosquito feeding 
3. Evaluate the effect of the AGS-v immune response on Aedes aegypti 
mosquito feeding and fecundity after feeding on a vaccinated individual or 
whole blood collected from those individuals in vitro.   
4. Evaluate the effect of incubating Zika virus coated in vector saliva with 
PBMCs and/or serum collected from vaccinated individuals. 
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 administered at Day 0 and Day 21 prior to Aedes aegypti mosquito challenge at 
Day 42. Subjects will be randomized 1:1:1 to: 
• 
Group 1: 825 µg (50 nmol each peptide) adjuvanted AGS-v vaccine 2 doses 
• 
Group 2: 825 µg (50 nmol each peptide) non-adjuvanted AGS-v vaccine 2 doses 
• 
Group 3: placebo 2 doses  
 
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.  
3.2 
Study Endpoints 
Primary:  
1. Incidence and severity (by grading) AEs in AGS-v vaccinated 
individuals 
2. Total AGS-v specific immunoglobulin measured in serum 21 days 
after the first and/or second vaccination. 
3. Interferon-gamma and other cytokine markers of Th1 response 
measured in vitro from PBMCs incubated with AGS-v antigens as 
indicators of the expected Th1 response 
 
Secondary:  

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 23 of 62 
 
1. Total AGS-v specific immunoglobulin in acute serum collected 
immediately after a clean Aedes aegypti mosquito feeding and 
convalescent serum collected on follow-up visits (60 days later +/- 14 
days)               
2. Interferon-gamma and other cytokine markers of a Th1 or Th1/Th2 
mixed response measured in vitro from acute PBMCs and serum 
collected immediately after a clean Aedes aegypti mosquito feeding 
incubated with AGS-v antigens and convalescent PBMCs and serum 
collected on follow-up visits (60 days later +/- 14 days) 
3. Measurement of changes to Aedes aegypti mosquito life cycle post 
feeding on blood from participants after vaccination both in vivo and in 
vitro, including survival, egg laying, and other measures of 
reproduction. 
4. Infectivity of Zika virus after incubation with serum and/or PBMCs 
from vaccinated participants 
 
4 
Study Population 
4.1 
Rationale for Participant Selection  
Participants will be carefully selected using the inclusion and exclusion criteria described 
here to select the optimum participants for completing the study objectives and minimize 
the risk of AEs. NIH employees and members of their immediate families may participate 
in this protocol. We will follow the Guidelines for the Inclusion of Employees in NIH 
Research Studies and will give each employee a copy of the “NIH information sheet on 
Employee Research Participation.”  
 
For NIH employees:  
• 
Neither participation nor refusal to participate will have an effect, either beneficial 
or adverse, on the participant’s employment or work situation.  
• 
The NIH information sheet regarding NIH employee research participation will be 
distributed to all potential subjects who are NIH employees.  
• 
The employee subject’s privacy and confidentiality will be preserved in 
accordance with NIH Clinical Center and NIAID policies, which define the scope 
and limitations of the protections.  
• 
For NIH employee subjects, consent will be obtained by an individual 
independent of the employee’s team. Those in supervisory position to any 
employee and co-workers of the employee will not obtain consent.  

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 24 of 62 
• 
The importance of maintaining confidentiality when obtaining potentially sensitive 
and private information from co-workers or subordinates will be reviewed with the 
study staff at least annually and more often if warranted.   
 
4.2 
Recruitment Plan 
Participants will be recruited through the screening study #11-I-0183 “Screening of 
Volunteers for Influenza Human Challenge and Vaccine Studies”. Participants will be 
carefully screened and evaluated, and those who meet the study eligibility criteria will be 
contacted and given the opportunity to be enrolled into the study. A staggered 
enrollment procedure will be followed with the initial enrollment phase to include 6 
patients in the first cohort.  These will be block randomized so that 2 receive placebo, 2 
vaccine, and 2 vaccine plus adjuvant.  If no pausing or halting criteria are met per the 
safety evaluations described in Section 12 Assessment of Safety for these initial 6 
patients one week after the second vaccine administration (~Day 28), the following 
cohort will include 9 patients.  Then, if no pausing or halting criteria or safety concerns 
arise by one week after the second vaccine administration (~Day 28),  the remaining 30 
patients will be enrolled in appropriately sized cohorts with 15 patients in each arm to 
complete the study efficiently. 
 
If a participant has completed the screening study #11-I-0183 more than 60 days prior to 
Day 0 of this study, they will be asked to come to the NIH Clinical Center for another 
screening visit under the screening study #11-I-0183 within 60 days prior to the date of 
vaccination to repeat HIV testing and complete any laboratory or other testing as 
deemed necessary by the investigator to ensure that it remains safe for the participant to 
take part in this study.  All eligible participants will be consented and enrolled for this 
study only after completion of all necessary screening studies under protocol #11-I-0183 
a maximum of 60 days prior to vaccine administration.  
 
The screening visit will take place prior to Day 0 (Day of vaccine/placebo administration).   
4.3 
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 subject 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: 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 25 of 62 
• 
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 prior to study initiation through 12 weeks after the 
second vaccine administration. Acceptable methods of contraception 
include 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 (including genetic research). 
6. Agrees to abstain from alcohol intake for 24 hours prior to each study visit. 
7. Agrees to not donate blood or blood products throughout the study.  
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.  
2. Individual with body mass index (BMI) £18 and ≥40.  
3. Participants who have a clinically significant (as determined by the PI) baseline 
Grade 1 or greater toxicity, or any Grade 3 or greater toxicity (regardless of clinical 
significance) by the toxicity table.   
4. Receipt of blood or blood products (including immunoglobulins) within 3 months 
prior to enrollment. 
5. Receipt of any unlicensed drug within 3 months or 5.5 half-lives (whichever is 
greater) prior to enrollment. 
6. Receipt of any unlicensed vaccine within 6 months prior to enrollment. 
7. Self-reported or known history of alcoholism or drug abuse within 6 months prior 
to enrollment, or positive urine/serum test for drugs of abuse at screening  
8. Self-reported or known history of psychiatric or psychological issues that require 
treatment and are deemed by the PI to be a contraindication to protocol 
participation.  
9. History of a previous severe allergic reaction with generalized urticaria, 
angioedema, anaphylaxis or anaphylactoid reaction. 
10. Any condition or event that, in the judgment of the PI, is a contraindication to 
protocol participation or impairs the volunteer’s ability to give informed consent. 
11. Has a known allergy to any of the components of the vaccine. 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 26 of 62 
12. Has a history of severe immunization reaction. 
13. Has a severe allergic reaction to mosquito bites 
 
Co-enrollment Guidelines: Co-enrollment in other trials is restricted, but may take 
place with the approval of the PI and after study staff notification. 
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 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 reproductive safety tests. 
 
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. 
5 
Study Agents/Interventions  
5.1 
Agent 1: AGS-v, AGS-v + Adjuvant, or Placebo 
5.1.1 Formulation, Packaging and Labeling 
AGS-v contains four drug substances: AGS-6, AGS-30, AGS-31, and AGS-35.  These 
are peptides consisting of 32 to 44 amino acids.  They are obtained by chemical 
synthesis and are available as acetate salts.  The peptides contain multiple T-cell 
reactive regions capable of inducing Th1 responses. 
 
A solution of the four drug substances (50 nmol net peptide/g of each drug substance) is 
prepared by completely dissolving the drug substances in water for injection (WFI).  The 
four solubilized peptides are mixed to make up AGS-v solution.  This mix is filter-
sterilized before filling glass vials automatically with the sterile AGS-v solution.  The 
pharmaceutical formulation solution is filled into glass vials by weight which results in 
0.824g of AGS-v solution per vial which is then lyophilized before closing with a rubber 
stopper and a flip-off cap.  The lyophilization removes both the water component; 
whereupon a relatively neutral pH, suitable for human injection, is achieved by 
reconstitution. 
 
Bottle labels will bear the appropriate label text 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. 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 27 of 62 
5.1.2 Study Agent Storage and Stability 
The vials of WFI and Montanide ISA-51 should be stored at room temperature.  Stability 
will be performed to cover the duration of the trial.  Stability studies of previous batches 
of another vaccine Flu-v indicate that it should remain stable for 2 years when stored at -
15 to -25°C which should be the case for the AGS-v vaccine also.  None of the materials 
can be dispensed beyond the expiration date.  
5.1.3 Adjuvant 
The adjuvant is Montanide ISA 51, also known as incomplete Freund’s adjuvant (IFA).  
This 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 surfactant.28    
Drakeol 6 VR is a mineral oil with 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 and fatty tissues in the form of 
phospholipids and fatty acids.28 
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 remains.28 
This version of IFA , Montanide ISA 51, is highly purified and avoids having impurities 
and high levels of fatty acids that have lead to toxicities with other versions of 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 
malaria.28 
 
5.1.4 Placebo 
The placebo contains water for injection, which is the final product used as placebo. 
 
5.1.5 Preparation 
• 
Please see Pharmacy Manual Section 2.3. 
• 
Two members of the study staff, Dr. Alison Han (associate investigator) and 
Lindsay Czajkowski (NP), will perform emulsification and blinding of all 
syringes and accountability for the AGS-v study agent.  Therefore, these two 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 28 of 62 
members of the study staff will be unblinded and will not participate in any 
other aspects of the study or patient care. 
• 
Once prepared and drawn into the syringe, it should be administered within 3 
hours. 
• 
Once emulsification of the adjuvant and vaccine occurs, it should be 
administered within 15 minutes. 
 
5.2 
Dosing and Administration  
Subjects will be randomized to one of the following vaccination regimens: 
 
• 
Group 1: AGS-v (unadjuvanted) as a suspension in WFI (0.5 mL) on Day 0 
and on Day 21 
• 
Group 2: ISA-51-adjuvanted AGS-v emulsified in WFI (0.5 mL) on Day 0 and 
on Day 21 
• 
Group 3: WFI (0.5 mL) on Day 0 and Day 21 
 
All doses administered by subcutaneous injection in the fatty tissue of the triceps. 
 
The person administering the vaccine will be blinded to the type of formulation 
(adjuvanted or not adjuvanted) because an opaque label will be placed over the syringe 
by the pharmacy.  The NIH Special Clinical Studies Unit (SCSU) or other appropriate 
clinical unit nurses will administer the vaccines.  No member of the study team will be 
administering the blinded study product. 
 
Administration of the vaccine will be performed following these instructions as detailed in 
the NIH Clinical Center subcutaneous vaccine administration guidelines29: 
 
To locate the injection site on the arms, fold one arm across the chest.  Place 
your hand on the shoulder and draw an imaginary line below your hand.  Place 
another hand on the elbow.  Draw an imaginary line down the outer side of the 
arm and down the center front of the arm, starting at the elbow.  The area inside 
these imaginary lines is where injections are given.  
 
Cleanse the area of the skin thoroughly before a needle is inserted.  Cleanse the 
skin with a back and forth motion using an alcohol swab.  This motion moves 
bacteria away from the injection site.  Allow the alcohol to dry completely by air.  
 
• 
Take the cover off the needle.  Be careful not to contaminate the 
needle.  Place the cover on its side. Hold the syringe in one hand like 
pencil or a dart.  Grasp the skin between the thumb and index finger 
with your other hand and pinch up.  
• 
Quickly thrust the needle all the way into the skin.  Do not “push” the 
needle into the skin slowly or thrust the needle into the skin with great 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 29 of 62 
force.  Do not press down on the top of the plunger while piercing the 
skin.  
• 
Insert the needle at a 90° (right) angle.  This angle is important to 
ensure that the medications will be injected into the fatty tissue.  
However, for persons with little subcutaneous fat on thin skin, you 
may be taught to use a 45° angle.  
• 
After the needle is completely inserted into the skin, release the skin 
that you are grasping.  Press down on the plunger to release 
medication into the subcutaneous layer in a slow, steady pace. Count 
10 seconds before removing the needle from the skin.  
• 
As the needle is pulled out of the skin, gently press a gauze pad onto 
the needle insertion site. Pressure over the site while removing the 
needle prevents skin from pulling back, which may be uncomfortable.  
The gauze also helps seal the punctured tissue and prevents leakage.  
 
It is not serious if you notice blood at the site after the needle is removed.  You 
may have nicked a surface blood vessel when you injected, and blood is 
following the needle track out to the surface.  Simply press the site with a 2 x 2 
gauze pad.  Also, a small amount of clear fluid may appear at the site. This may 
be medication that is following the needle track to the surface.  Again, apply 
pressure using a 2 x 2 gauze pad. 
 
5.3 
Disposal and Drug Accountability of AGS-v 
5.3.1 Product Accountability 
The Clinical Center pharmacy 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 eCRFs and in the 
Drug Administration Record form.  The latter includes the subject identification, quantity 
(volume) and date of administration.  The containers from which the vaccine was 
administered to the subjects 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 and that no study supplies remain in the 
Investigator’s possession. 
 
All unused vaccine supplies, partially used and empty containers will be returned to the 
drug supplier. 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 30 of 62 
5.3.2 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 investigational product (IP) accountability, reconciliation 
and record maintenance.  In accordance with all applicable regulatory requirements, the 
investigator or designated site staff must maintain investigational product accountability 
records throughout the course of the study.  The investigator or designated site staff will 
document the amount of investigational product received from SEEK and the amount 
administered to subjects. 
 
A Drug Dispensing Log must be kept current and will contain the following information: 
 
• 
The identification of the subject to whom the drug was dispensed. 
• 
The date(s) of the drug dispensed to the subject. 
 
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 or returned by the investigator or designee to SEEK.  
When requested in writing by the drug supplier, unused drug supplies may be destroyed 
by the investigator provided such disposition does not expose humans to risks from the 
drug.  Records will be maintained by the investigator of any such alternate disposition of 
the test drug.  These records must show the identification and quantity of each unit 
disposed of, 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. 
 
5.3.3 Retention of Samples 
It will be the responsibility of SEEK to ensure adequate samples of all study drug are 
retained in accordance with the regulatory guidelines. 
5.4 
Concomitant Medications and Procedures 
All concomitant prescription medications, over-the-counter medications, or herbal 
remedies taken during study participation must be approved by the PI and will be 
recorded in the participant’s source documents. For this protocol, a prescription 
medication is defined as a medication that can be prescribed only by a properly 
authorized/licensed clinician.  
6 
Study Schedule 
This study will take place at the NIH Clinical Center. All aspects of the protocol will be 
carried out in accordance with NIH guidelines, GCP and ICH involving human-participant 
research.  Please see Figure 6 below. 
 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 31 of 62 
Figure 6. Study Schedule 
 
6.1 
Randomization and Blinding 
Randomization will occur using computer generated randomization codes.  This code 
will be sent to the pharmacy where the unblinded pharmacist will have the key and 
prepare the appropriate agent for administration.  All vaccination vials will be labeled and 
look identical so the blinded staff cannot identify placebo v AGS-v vaccine doses.   
 
Under normal circumstances, the blind should not be broken until all participants have 
completed the study and the database is finalized. 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 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. 
  
In general, randomization codes will be disclosed fully only if the study is completed and 
the clinical database is closed. However, for the interim analysis planned for this study 
unblinding will occur when the interim database is locked.  This unblinding is limited to 
the study statistician and Data Safety Monitoring Board (DSMB).  
 
6.2 
Randomization and Vaccine or Placebo Administration (Day 0)  
The first vaccine or placebo administration will take place on Day 0. This visit will occur 
in the SCSU or other appropriate CC unit/clinic. The research team will thoroughly 
discuss the consent with the volunteer prior to 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 of AGS-v +adjuvant 0.5 mL dose, 
AGS-v 0.5 mL dose, or placebo.  All participants will be educated by study staff on 
prevention of mosquito bites while participating in study including, but not limited to, 
minimizing skin exposure with long sleeves/pants and the use of repellent.  
 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 32 of 62 
 The following procedures will be performed after informed consent is obtained:  
• 
Review of medical/medication history 
• 
Documentation of usual responses to mosquito bites as none (0), mild (1), 
moderate (2), or severe (3) 
• 
Physician exam/assessment  
• 
Vital signs including weight pre vaccination 
• 
Vital signs (+/- 15 minutes) post vaccination at 30 minutes, 60 minutes, and 
120 minutes 
• 
Blood collection for routine safety labs (as per Appendix A), total serum IgE,  
as well as serum/whole blood for research (as per Appendix B). 
• 
Routine urinalysis 
• 
Serum pregnancy test (females only) 
• 
Review of inclusion/exclusion criteria  
• 
Vaccine or placebo administration  
• 
Required 2 (two) hour observation time (post vaccine injection) at the clinical 
research site 
• 
Vaccine injection site and AE assessment pre-vaccination, 60 minutes and 
120 minutes post vaccination (+/- 15 minutes) 
• 
Any other clinical tests that are medically indicated or appropriate to ensure 
the safety of the individual participant as deemed by the PI 
• 
Diary card, thermometer, and ruler will be distributed prior to discharge home 
 
6.3 
Day 7 (+/- 2 days) 
• 
Clinic visit 
• 
Review of medical/medication history 
• 
Physician exam/assessment 
• 
Vital signs including weight 
• 
Blood collection for routine safety labs 
• 
Routine urinalysis 
• 
Serum pregnancy test (females only) 
• 
Vaccine injection site assessment  
• 
Review of diary card 
6.4 
Day 14 (+/- 2 days) 
• 
Clinic visit 
• 
Review of medical/medication history 
• 
Physician exam/assessment 
• 
Vital signs including weight 
• 
Blood collection for routine safety labs 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 33 of 62 
• 
Routine urinalysis 
• 
Serum pregnancy test (females only) 
• 
Vaccine injection site assessment  
6.5 
Day 21 (+/- 2 days) 
This visit will take place in the SCSU or other appropriate CC unit/clinic.  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 
• 
Physician exam/assessment 
• 
Vital signs and including weight 
• 
Vital signs (+/- 15 minutes) post vaccination at 30 minutes, 60 minutes, and 
120 minutes 
• 
Blood collection for routine safety labs as well as serum/whole blood for 
research  
• 
Routine urinalysis 
• 
Serum pregnancy test (females only) 
• 
Vaccine or placebo administration  
• 
Required 2 (two) hour observation time (post vaccine injection) at the clinical 
research site 
• 
Vaccine injection site and AE assessment pre-vaccination, 60 minutes and 
120 minutes post vaccination (+/- 15 minutes) 
• 
Any other clinical tests that are medically indicated or appropriate to ensure 
the safety of safety the individual participant as deemed by the PI 
• 
Diary card, thermometer, and ruler will be distributed prior to discharge home 
6.6 
Day 28 (+/- 2 days) 
• 
Clinic visit 
• 
Review of medical/medication history 
• 
Physician exam/assessment 
• 
Vital signs including weight 
• 
Blood collection for routine safety labs 
• 
Routine urinalysis 
• 
Serum pregnancy test (females only) 
• 
Vaccine injection site assessment  
• 
Review of diary card 
6.7 
Day 35 (+/- 2 Days) 
• 
Clinic visit 
• 
Review of medical/medication history 
• 
Physician exam/assessment 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 34 of 62 
• 
Vital signs including weight 
• 
Blood collection for routine safety labs 
• 
Routine urinalysis 
• 
Serum pregnancy test (females only) 
• 
Vaccine injection site assessment  
6.8 
Day 42 Mosquito Feeding (+14 days) 
This will take place in the SCSU or other appropriate CC unit.  The following 
procedures/assessments and testing will be performed: 
• 
Review of medical/medication history and addition of any new information 
• 
Physician assessment and physical exam 
• 
Vital signs including weight 
• 
Routine urinalysis 
• 
Placement of IV catheter in arm 
• 
Serum pregnancy test (females only) 
• 
Blood collection for routine safety labs and serum/whole blood for research 
prior to mosquito feeding. 
• 
Per Appendix B: serum 8 ml tube x 1, whole blood 8.5 ml tube x 10, and 
Paxgene 2.5 ml x1 
• 
Mosquito feeding: 
• 
Starved female Aedes aegypti mosquitos will be selected from a mosquito 
colony approved for human feeding studies in Laboratory of Malaria and 
Vector Research (LMVR), NIAID.  5 -10 female mosquitoes will be 
secured in the feeding device prior to feeding and brought to the CC from 
the LMVR Insectary.  
• 
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 20 minutes.  The 
mosquitos will feed through a disposable mesh on the bottom of the 
feeding device..  In the unlikely event of no feeding or poor feeding (only 
0-2 mosquitoes fed or probed as noted by trained staff), the volunteer 
may undergo a repeat feed with 5-10 fresh mosquitoes ONCE.   
• 
Participants will be asked not to apply cream, perfume or deodorant the 
day of mosquito exposure. Once the mosquitos have fed they will remain 
in the same device and be brought back to the lab for further study. 
• 
The skin will be inspected pre-feeding.  Redness, swelling and number of 
visible bites will be recorded pre-feeding, immediately post feeding and 30 
minutes post feeding (+/- 15 minutes) 
• 
Research blood collection (+/- 15 minutes) after mosquito feeding will be 
collected at: 
• 
30 minutes: whole blood (8.5 mL tubes x 5), serum (8 ml tube x 1), and 
2.5 mL in Paxgene tube 
• 
60 minutes: 2.5 mL for Paxgene tube 
• 
120 minutes: 2.5 mL Paxgene tube 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 35 of 62 
• 
180 minutes: whole blood (8.5 mL tubes x 5), serum (8 ml tube x 1), and 
2.5 mL in Paxgene tube 
• 
Any other clinical tests that are medically indicated or appropriate to assure 
the safety of the individual participant as deemed by the PI. 
• 
AE assessment  
6.9 
Day 44 Telephone Call (+/- 1 day) 
A brief telephone call will be made on Study Day 44 to elicit general state of health and 
any adverse events. The volunteer will report the size of redness and swelling on their 
arm at 48 hours. 
 
6.10 
Follow-up 
All participants will be followed for a minimum of 10 months after completing Day 42 of 
the study. 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 in the clinic approximately every 60 days (±14 days) for 
the next 5 months or more often if deemed medically necessary; the 
procedures/evaluations to be conducted at these visits are shown in Appendix A. The 
10-month post Day 42 visit will be the final study visit unless the participant requires 
additional follow-up of study-related complications.  
 
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. 
7.1.1 Laboratory Testing of Collected Samples for Primary and Secondary 
Endpoints 
• 
Primary Endpoints: 
o AGS-v specific immunoglobulin assay will be performed using enzyme-
linked immunosorbent assay (ELISA). 
o Th1 and Th2 response assays: PBMCs collected from participants will be 
incubated with AGS-v and measurements of various Th1 and Th2 
cytokines will be performed using Luminex and/or ELISA 
• 
Secondary Endpoints: 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 36 of 62 
o AGS-v specific immunoglobulin and interferon-gamma assay will be 
performed as stated above on samples collected after Aedes aegypti 
mosquito feeding occurs.   
o Specific IgG subclasses will be measured using ELISA 
o Mosquito Studies:  
§ 
Feeding on human volunteers: 24 hour after emergence from pupae, 
male and female Aedes aegypti mosquitoes will be allowed to mate 
during 24-48 hour. After this period, female mosquitoes will be kept 
separated until human exposure. 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 42 (approximately 21 days after they have 
completed the vaccination schedule).  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 but on non-mammalian 
blood to minimize the presence of blood borne pathogens in them. 
o Zika virus killing, in vitro toxicity assay:  
§ 
Blood samples will be obtained from all volunteers 21 days after 
vaccination is completed. PBMCs+sera containing immune cells and 
antibodies generated against AGS-v will be tested for their ability to 
kill Zika virus mixed with salivary gland proteins from Aedes aegypti. 
This test will be carried out in 96 well plates.   
§ 
Salivary gland proteins will be obtained after dissecting the salivary 
glands from female Aedes aegypti, sonicating them and centrifuging 
the sonicate to obtain the supernatant containing all the mosquito 
salivary proteins.   
§ 
Zika virus will be obtained from a commercial supplier (ATCC® VR-
1838). This strain can be propagated in vitro by infecting VERO cells. 
Cells are plated 18-24 hours prior to infection and infected when 
cultures are 75-80% confluent. The medium is removed, cell 
monolayer is washed with PBS or serum free medium prior to 
inoculation with a small volume of inoculum (e.g. 1 mL per 25 cm2) 
diluted to provide an optimal MOI (e.g. 0.1-0.01). The inoculum is left 
to adsorb for 1-2 hours at 37°C in a humidified 5% CO2 atmosphere. 
End adsorption by adding virus growth medium. Cells are incubated 
for 2-4 days at 37°C in a humidified 5% CO2 atmosphere, until 
cytopathic effect is progressed through 80-90% of the monolayer. At 
this point the propagated virus is collected.   
§ 
In order to assess that the AGS-v specific immune responses have 
killed the Zika virus, the PBMC+Zika+salivary protein cultures will be 
spun to remove any cells. Any viable Zika virus will be found in the 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 37 of 62 
supernatant which will be used to attempt re-infection of VERO cells 
and/or PCR. If infection is not successful and/or PCR is negative it will 
be an indication that the virus was destroyed. 
 
7.2 
Reactogenicity 
On a daily basis during the 7-day period after each injection subjects will evaluate and 
record temperature, any local (redness, swelling, and pain) or general symptoms (e.g., 
fever, chills, headache, myalgia, arthralgia, sweats, fatigue, nausea, vomiting) they 
experience on the diary card.  The diary card will be brought to each clinic visit.  The 
diary cards will be reviewed and evaluated for AEs.   
 
The diary card will be completed by the subject in the evening.  Subjects will be 
instructed on the use and bring it to every clinic visit.  The diary card will be reviewed by 
the study team during the post injection follow up visits.  Diary cards will be collected 
from subjects, and retained as source documentation.  If diary cards are not completed 
or returned, the study team will review the AEs that occurred during that timeframe to the 
best of the subject’s recollection.   
 
8 
Potential Risks and Benefits 
8.1 
Risks of Placebo administration 
Subcutaneous injection of water for injection (WFI) poses minimal risk.  The primary 
risks of the placebo vaccinations are a local vaccine reaction or infection at the site of 
injection.  Signs of a local vaccine 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 drainage at the site. 
8.2 
Risks of AGS-v ± 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.  These 
reactions can include severe anaphylactic reactions and seizure.  Primarily topical 
symptomatic therapy will be administered if necessary to treat vaccine reactions 
including cold compresses, topical antipuriritics and pain relievers.  Additional standard 
of care therapy for pain or anaphylaxis will be administered if the reaction becomes 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 38 of 62 
severe or life threatening.  This therapy includes but is not limited to analgesics, 
epinephrine, diphenhydramine, hydroxyzine, and systemic steroids.  
 
SEEK has completed clinical trials in the EU for the FLU-v and HIV-v vaccines.  The 
technology utilized for peptide identification and vaccine development for the Flu-v and 
HIV-v vaccines is the same technology utilized for peptide identification and vaccine 
development for the AGS-v vaccine.  FLU-v, HIV-v and AGS-v all utilize the same 
adjuvant, Montanide ISA-51.  Therefore, we expect similar safety findings for AGS-v as 
was observed with FLU-v and HIV-v.  Minor vaccine reactions have been observed, but 
no severe vaccine reactions have been observed in any participants receiving either of 
these vaccines.  Safety results from the FLU-v and HIV-v studies are presented below 
and no safety concerns were identified during those Phase I trials. 
 
In the Phase 1a Study Flu-v-001, no deaths occurred and only 1 SAE was reported with 
this being considered unrelated to FLU-v (appendicitis).  There was no apparent 
increase in AEs with a change in dose from the 250 μg or 500 μg (500 µg of FLU-v is the 
equivalent of 825 µg of AGS-v as both contain 50 nmol of each peptide), but an 
approximate 2-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; however, the forearm site and 1 mL volume is suspected to be the cause 
rather than the adjuvant itself.  Montanide ISA-51 has been used in cancer vaccines and 
local reactogenicity has not been documented to be greater than moderate or Grade 2.  
Case reports of erythema nodosum and sterile abscesses have also been documented 
in vaccines adjuvanted with Montanide ISA-5130-34, but these were not observed in the 
FLU-v trials of healthy volunteers. 
 
In the Phase 1b Study Flu-v-002, all 32 subjects 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 Montanide ISA-51 
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, ECG). 
 
In the Phase 1b Study HIV-v-001 performed in HIV positive patients not healthy 
volunteers, all 59 subjects 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 subjects with 3 SAEs occurring in the 1 subject (lower abdominal pain, 
shigella infection, and gastroenteritis) 37 days prior to vaccination, 31 and 128 days after 
vaccination respectively.  The other 2 SAEs (perianal abscess with streptoccacl infection 
and Kaposi’s sarcoma) occurred at 101 and 56 days after vaccination.  None of the 
SAEs were considered to be related to the study drug. No subjects died and no subject 
discontinued the study due to an AE.   

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 39 of 62 
8.3 
Risks of adjuvant 
Given the use of Montanide ISA-51 in our trial and its theoretical potential for induction of 
autoimmune disease, we will monitor for the following list of potentially immune-
mediated medical conditions for 12 months after vaccination.   
 
We will monitor for the occurence of an unexpected event of a vaccine-related immune-
mediated condition by performing interim history at each interval follow-up visit, thorough 
review of systems of new symptoms, physical exams, and blood work to include 
comprehensive metabolic panel and complete blood count with differential.  If necessary 
as based on history or exam, we may refer subjects for evaluation or send additional 
tests such as but not limited to c-reactive protein, thyroid stimulating hormone, 
hemoglobin A1c, cryoglobulins, ANCA, and rheumatoid factor.  Below is the provided list 
of immune-mediated conditions that will be monitored. 
 
Gastrointestinal disorders 
- 
Celiac disease 
- 
Crohn’s idseas 
- 
Ulcerative colitis and proctitis 
 
Liver disorders 
- 
Autoimmune cholangitis 
- 
Autoimmune hepatitis 
- 
Primary biliary cirrhosis  
- 
Primary sclerosing cholangitis 
 
Metabolic diseases 
- 
Addison’s disease 
- 
Autoimmune thyroiditis 
- 
Diabetes mellitus type 1 
- 
Grave’s disease 
 
Musculoskeletal disorders  
- 
Antisynthetase syndrome 
- 
Dermatomyositis 
- 
Juvenile chronic arthritis 
- 
MCTD 
- 
Polymyalgia rheumatica 
- 
Polymyositis 
- 
Psoriatic arthropathy 
- 
Relapsing polychondritis 
- 
Rheumatoid arthritis 
- 
Sclerderma (including CREST And systemic diffuse form) 
- 
Spondyloarthritis 
- 
Systemic lupus erthematosus 
- 
Systemic sclerosis 
 
Neuroinflammatory disorders 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 40 of 62 
- 
Acute disseminated encephalomyelitis 
- 
Cranial nerve disorders 
- 
Guillain Barré syndrome 
- 
Immune-mediated peripheral neuropathies and plexopathies 
- 
Multiple sclerosis 
- 
Narcolepsy 
- 
Optic neuritis 
- 
Transverse myelitis 
- 
Myasthenia gravis 
 
Skin disorders 
- 
Alopecia areata 
- 
Autoimmune bullous skin disease such as pemphigus, pemphigoid, and 
dermatitis herpetiformis 
- 
Cutaneous lupus erythematosus 
- 
Erythema nodosum 
- 
Morphoea 
- 
Lichen planus 
- 
Psoriasis 
- 
Sweet’s syndrome 
- 
Vitiligo 
 
Vasculitides 
- 
Large vessel vasculitides such as giant cell arteritis and Takayasu’s arteritis and 
temporal arteritis 
- 
Medium and small sized vessels: polyarteritis nodosa, Kawasaki’s, miscrosocpic 
polyangiitis, Wegener’s granulomatosis, Churg-Staruss, Buerger’s, Henoch 
Schonlein purpeura and Behcet’s 
 
Others 
- 
Antiphospholipid syndrome 
- 
Autoimmune hemolytic anemia 
- 
Autoimmune glomerular nephritis 
- 
Autoimmune myocarditis/cardiomyopathy 
- 
Autoimmune thrombocytopenia 
- 
Goodpasture syndrome 
- 
Idiopathic pulmonary fibrosis 
- 
Pernicious anemia  
- 
Raynaud’s phenomenon 
- 
Sarcoidosis 
- 
Sjogrens’ syndrome 
- 
Steven-Johnson syndrome 
- 
Uveitis 
 
 
8.4 
Risks of blood draw 
Risks of blood draw include pain, bruising, bleeding, and rarely fainting or infection.  The 
amount of blood drawn will be within the limits allowed for adult subjects by the NIH 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 41 of 62 
Clinical Center (Medical Administrative Policy 95-9: Guidelines for Limits of Blood Drawn 
for Research Purposes in the Clinical Center:  
http://cc-internal.cc.nih.gov/policies/PDF/M95-9.pdf).  Exceptions to this limit will be 
submitted to the Institutional Review Board (IRB) for prior approval. 
8.5 
Risks of Aedes aegypti 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 
Potential Benefits 
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 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. Only investigators 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 (both at NIH and outside) may wish to study 
these samples and/or data. In that case, IRB approval must be sought prior to 
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 and data will either 
be destroyed, or after IRB approval, transferred to another existing protocol. 
Reporting the 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 collected for the study will be 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 42 of 62 
reported to the IRB. The PI will also notify the IRB if the decision is made to 
destroy the samples. 
• 
Additionally, subjects may decide at any point not to have their samples 
stored. In this case, the principal investigator 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 NIH. 
10 
Data Sharing Plan 
10.1 
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 BTRIS. 
• 
De-identified or identified data with approved outside collaborators under 
appropriate agreements. 
10.2 
Data will be shared through: 
• 
An NIH-funded or approved public repository. Insert name(s): 
ClinicalTrials.gov____ 
• 
BTRIS. 
• 
Approved outside collaborators under appropriate individual agreements. 
• 
Publication and/or public presentations. 
10.3 
The data will be shared: 
• 
At the time of publication or shortly thereafter. 
 
11 
Remuneration Plan  
Participants will be compensated according to Table 1. Participant remuneration 
.   
 
Table 1. Participant remuneration 
 
Day 0  
$200 
Day 7 (± 2 days) 
$70 
Day 14 (± 2 days) 
$70 
Day 21 (± 2 days) 
$200 
Day 28 (± 2 days) 
$80 
Day 35 (± 2 days) 
$80 
Day 42 (+ 14 days) 
$400   
 
 
Follow up visits ~60 days (± 14 days) X5  
$60 x 5=$300 
 
 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 43 of 62 
Expected total for completion of ALL 
study visits 
$1,400  
 
 
Study-requested interim visits: 
$75 
 
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 CRIMSON 
• 
Reported as outlined below (e.g., IND Sponsor, IRB, FDA). 
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 principal 
investigator 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  
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.  
 
Adverse Reaction (AR) 
An AE that is caused by an investigational agent (drug or biologic). 
 
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. An SAR implies a lesser degree of certainty 
about causality than AR, which implies a high degree of certainty. 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 44 of 62 
 
Serious Adverse Event (SAE) 
An SAE is an AE that results in 1 or more of the following outcomes: 
• 
Death 
• 
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: 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 an 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.) 
 
UP that is not an AE (UPnonAE)  
A 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 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 45 of 62 
non-serious UP. For example, breaches of confidentiality, accidental destruction of study 
records, or unaccounted-for study agent will be reported. 
 
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 the FDA “Toxicity 
Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive 
Vaccine Clinical Trials” September 2007, which can be found at: 
http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/
Guidances/Vaccines/ucm074775.htm   
 
Microscopic hematuria will be graded using the above toxicity table except for the Grade 
1 values, which are being adjusted to be consistent with the CC Department of 
Laboratory Medicine normal laboratory values.  Grade 1 will be >3-10 RBC/HPF instead 
of 1-10 RBC/HPF listed in the FDA toxicity table. 
 
All lab and vital sign abnormalities found prior to 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 
• 
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 
 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 46 of 62 
*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. 
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. 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 47 of 62 
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. 
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) and sent to the Sponsor Clinical 
Safety Office (CSO) by fax or e-mail attachment. 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. 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. 
• 
Unblind the subject. 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 48 of 62 
• 
Report to the DSMB and the IRB. 
• 
Advise the research subject to notify the obstetrician of study participation 
and study agent exposure. 
12.5 
Investigator Reporting Responsibilities to the NIAID IRB 
12.5.1 Special Reporting Situations 
Safety events of interest that may require expedited reporting 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 subject/patient exposure 
to the study drug, e.g., name confusion) 
 
Special reporting situations should be recorded. Any special reporting situation that 
meets the criteria of a serious adverse event should be recorded as a serious adverse 
event and reported as described in Section 12.4.2. 
12.5.2 Expedited Reporting to the NIAID IRB 
Serious and non-serious UPs, deaths, serious deviations, and serious or continuing 
non-compliance will be reported within 7 calendar days of investigator awareness. SAEs 
that are possibly, probably, or definitely related to the research will be reported to the 
NIAID IRB within 7 days of site awareness, regardless of expectedness. 
12.5.3 Waiver of Reporting Anticipated Protocol Deviations, Expected UPnonAEs 
and Deaths to the NIAID 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. 
12.5.4 Annual Reporting to the NIAID IRB 
The following items will be reported to the NIAID IRB in summary at the time of 
Continuing Review: 
• 
Serious and non-serious UPs  
• 
Expected SAEs that are possibly, probably, or definitely related to the 
research 
• 
SAEs that are not related to the research 
• 
All AEs, except expected AEs granted a waiver of reporting 
• 
Serious and non-serious protocol deviations  
• 
Serious, continuing, and minor non-compliance 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 49 of 62 
• 
Any trends or events which in the opinion of the investigator should be 
reported 
• 
A summary of accumulated safety data  
12.6 
Follow-Up of AEs and SAEs 
AEs that occur following enrollment of the participant (by signing the informed consent) 
will be followed until the final outcome is known or until the end of the 10-month 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), the reason a final outcome could not be obtained will be 
recorded by the investigator in CRIMSON and on the SERF.  
 
SAEs that occur after the 10-month study follow-up period 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). 
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 Subject 
Pausing is the suspension of administration of study agent to a single subject until a 
decision is made whether or not to resume administration of the study agent. 
 
The pausing criteria for a single subject in this study include any of the following: 
• 
A subject experiences an SAE that is possibly, probably, or definitely related 
to a study agent; 
• 
A subject 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 subject. 
 
The CSO, in collaboration with the principal investigator, may also pause for an entire 
group if a safety concern is identified. 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 50 of 62 
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 principal investigator within 1 business day 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 principal investigator and/or the relevant DSMB, will 
determine whether or not it is safe to resume administration of the study agent to the 
subject. The principal investigator will notify the IRB of the decision on resumption of the 
study agent. 
 
A subject who does not resume study agent will continue to be followed for safety. 
 
12.9 
Halting Rules for the Protocol 
Halting the study requires immediate discontinuation of study agent administered for all 
subjects 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 subjects 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 subjects that are grade 3 or 
above and are possibly, probably, or definitely related to the study agent; 
OR 
• 
any safety issue that the principal investigator and/or the CSO determines should 
halt the study. 
 
The principal investigator 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 
principal investigator within 1 business day to the 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 principal investigator and the DSMB will 
determine if it is safe to resume the study. The principal investigator will notify the IRB of 
the decision on resumption of the study. 
 
Subjects who do not resume study agent will continue to be followed for safety. 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 51 of 62 
 
12.10 Study Discontinuation 
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 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 prior to 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 prematurally 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 subject will be withdrawn for any of the following: 
• 
An individual subject’s decision. (The investigator should attempt to determine 
the reason for the subject’s decision.) 
• 
Non-compliance with study procedures to the extent that it is potentially harmful 
to the subject or to the integrity of the study data. 
• 
A change in the subject’s baseline condition after enrollment so that the subject 
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 subject. 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 52 of 62 
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.  If a participant does not receive 
2 vaccinations, but does not withdraw from the study, that participant’s data may be 
used in the analysis, but an additional participant may be enrolled who qualifies and 
consents to the study to receive 2 vaccinations.  This may require the overall sample 
size to be larger than 45 participants, but the number to have received two vaccinations 
will be 45 participants.  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.  
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 DSMB includes independent experts that do not have direct 
involvement in the conduct of the study and have no significant conflicts of interests as 
defined by NIAID policy. The DSMB will review the study prior to initiation, after the first 
group of participants has been vaccinated, and at least twice a year thereafter. The 
Board may convene additional reviews as necessary. The board will review the study 
data to evaluate the safety, study progress, and conduct of the study. All UPs and all 
IND Safety Reports will be reported by the PI to the DSMB at the same time they are 
submitted to the IRB or IND Sponsor. All SAEs that are possibly, probably or definitely 
related to the study agent will be reported to the DSMB within 1 business day after the 
site becomes aware of the event. The PI will notify the DSMB of any cases of intentional 
or unintentional unblinding as soon as possible. The PI will notify the board at the time 
pausing or halting criteria are met and obtain a recommendation concerning 
continuation, modification, or termination of the study. The PI will submit the written 
DSMB summary reports with recommendations to the IRB.  

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 53 of 62 
13 
Site Monitoring Plan 
According to the International Council on Harmonization (ICH) Good Clinical Practice 
(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 “NIAID Intramural Clinical Monitoring Guidelines.” Monitors under contract to the 
NIAID/OCRPRO 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 and 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. 
 
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 principal investigator and 
study staff prior to 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 specific total immunoglobulin will increase after 
vaccination with AGS-v or adjuvanted AGS-v and/or PBMCs collected after vaccination 
incubated with AGS-v antigens will show a greater Th1 response as measured by 
interferon-gamma as compared to placebo vaccination. 
14.2 
Sample Size Justification and Analysis Plan 
The sample size for this study is based on the primary comparison of total 
immunoglobulin and interferon-gamma release with each of the experimental vaccine 
arms to placebo vaccine.  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 subjects than necessary to an experimental vaccine but we also don’t 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 54 of 62 
wish to miss an active vaccine we will design the study to have 90% power with a 1-
sided type I error rate of .1. We also will not adjust for the 3 multiple comparisons  (all 3 
arms being compared).  
 
With 15 subjects per arm, there will be 90% power to detect a true difference in the 
primary efficacy endpoint of .95 standard deviations using t-test with significance level of 
.1 (one sided). 
 
For the safety endpoint we are interested in identifying AEs that may occur with the 
vaccines. With 15 patients in each group we would have a probability of .9 of observing 
at least one AE if the true underlying rate for that AE in a vaccinated arm is 0.14. If we 
collapse across the vaccinated arms we would have 30 patients. With 30 patients we 
would have a probability of .9 of observing at least one AE if the true rate is 0.075. With 
this sample size we will have a high probability of beginning to see an adverse safety 
signal if one exists. Table 2 shows the probability of observing at least 1 AE for various 
true AE rates with samples sizes of 15 and 30. 
 
Table 2. Probability of observing at least one AE. 
True rate of adverse 
events 
Probability of observing at least one AE 
 
N=15 
N=30 
0.01 
0.14 
0.26 
0.02 
0.261 
0.455 
0.03 
0.367 
0.599 
0.04 
0.458 
0.706 
0.05 
0.537 
0.785 
0.06 
0.605 
0.844 
0.07 
0.663 
0.887 
0.08 
0.714 
0.918 
0.09 
0.757 
0.941 
0.1 
0.794 
0.958 
.14 
0.900 
0.989 
 
With 15 patients per arm, there is low probability of being able to detect differences in 
adverse events rates between groups unless the differences are very large. But these 
comparisons will be made between the 3 arms of the study. In order to gain some 
power, the 2 experimental arms will also be collapsed and the AE event rates will be 
compared to the placebo arm. In order to be conservative and not miss a difference in 
significant safety issues, we will perform tests at the 1-sided .05 level without controlling 
for multiple comparisons. A Fishers exact test will be used to compare proportions of 
specific adverse events between groups. The following table shows the probability of 
observing at least one event for various true underlying rates for 15 (single arm) or 30 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 55 of 62 
patients (collapse across experimental arms). Table 3 gives the detectable differences 
with 80% power. 
 
Table 3. Difference in probabilities that can be detected with 80% power for 
sample sizes of 15 in each group or 15 in one group and 30 in the other 
15 per arm 
15 in arm 1 combined arms so n=30 
Arm 1 rate 
Arm 2 rate 
Arm 1 
combined 
0.01 
0.36 
0.01 
0.31 
0.025 
0.40 
.025 
0.34 
0.05 
0.47 
0.05 
0.38 
 
14.3 
Analysis Plan 
T-tests will be used to compare efficacy endpoint measurements for the primary 
endpoint and other continuous endpoints. For binary endpoints Fishers exact test will be 
used. AEs will be tabulated by treatment arm. 
 
For randomization, the first 6 patients will be block randomized.  Once an interim safety 
analysis is done on the initial enrollment, the remaining 39 patients will be randomized 
and enrolled in appropriately sized cohorts until there are 15 patients per arm. 
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 prior to 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.      
 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 56 of 62 
15.2 
Non–English-Speaking Participants 
If a non–English-speaking participant is unexpectedly eligible for enrollment, the 
participant will be provided with the CC Short Written Consent Form for Non–English-
Speaking Research Participants in the participant’s native language and a verbal 
explanation of the purpose, procedures and risks of the study as described in MAS 
Policy M77-2, NIH HRPP SOP 12, and 45 CFR 46.117(b)(2). The IRB-approved English 
consent form will serve as basis for the verbal explanation of the study. The investigator 
will obtain an interpreter unless the investigator is fluent in the prospective participant’s 
language. Preferably, the interpreter will be someone who is independent of the 
participant (i.e., not a family member). Interpreters provided by the CC will be used 
whenever possible. The interpreters will translate the IRB-approved English consent 
form verbatim and facilitate discussion between the participant and investigator. 
 
The IRB-approved English consent form will be signed by the investigator obtaining 
consent and a witness to the oral presentation. The CC Short Written Consent Form will 
be signed by the participant and a witness who observed the presentation of information. 
The interpreter may sign the consent document as the witness and, in this case, will note 
“Interpreter” under the signature line. A copy of both signed forms will be provided to the 
participant to take home. 
 
The investigator obtaining consent will document the consent process in the participant’s 
medical record (CRIMSON), including the name of the interpreter. Further, all instances 
of use of the CC Short Written Consent Form will be reported to the IRB at the time of 
annual review. If the CC Short Written Consent Form is used three times or more for the 
same language within an IRB approval period, this will be reported to the IRB 
immediately. 
15.3 
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. 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 CRIMSON and CRIS 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 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 57 of 62 
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 Guideline. Study records will be maintained by the PI for a minimum of 3 years and 
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. 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 58 of 62 
SCIENTIFIC REFERENCES 
 
1. 
World Malaria Report 2015. World Health Organization;2015. 
2. 
Bhattarai A, Ali AS, Kachur SP, et al. Impact of artemisinin-based 
combination therapy and insecticide-treated nets on malaria burden in 
Zanzibar. PLoS medicine. 2007;4(11):e309. 
3. 
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 phase 3 randomized, controlled trial in 
children and young infants at 11 African sites. PLoS medicine. 
2014;11(7):e1001685. 
5. 
Greenwood B, Targett G. Do we still need a malaria vaccine? Parasite 
immunology. 2009;31(9):582-586. 
6. 
Ribeiro JM. Role of saliva in blood-feeding by arthropods. Annual review 
of entomology. 1987;32:463-478. 
7. 
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. 
8. 
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. 
9. 
Silvestre R, Cordeiro-Da-Silva A, Santarem N, Vergnes B, Sereno D, 
Ouaissi A. SIR2-deficient Leishmania infantum induces a defined IFN-
gamma/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, & Immunology. 1996;77(3):238-244. 
11. 
Peng Z, Simons FE. Mosquito allergy: immune mechanisms and 
recombinant salivary allergens. International archives of allergy and 
immunology. 2004;133(2):198-209. 
12. 
Torre D, Speranza F, Giola M, Matteelli A, Tambini R, Biondi G. Role of 
Th1 and Th2 cytokines in immune response to uncomplicated 
Plasmodium falciparum malaria. Clinical and diagnostic laboratory 
immunology. 2002;9(2):348-351. 
13. 
Donovan MJ, Messmore AS, Scrafford DA, Sacks DL, Kamhawi S, 
McDowell MA. Uninfected mosquito bites confer protection against 
infection with malaria parasites. Infection and immunity. 2007;75(5):2523-
2530. 
14. 
Sidjanski S, Vanderberg JP. Delayed migration of Plasmodium 
sporozoites from the mosquito bite site to the blood. The American journal 
of tropical medicine and hygiene. 1997;57(4):426-429. 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 59 of 62 
15. 
Ponnudurai T, Lensen AH, van Gemert GJ, Bolmer MG, Meuwissen JH. 
Feeding behaviour and sporozoite ejection by infected Anopheles 
stephensi. Transactions of the Royal Society of Tropical Medicine and 
Hygiene. 1991;85(2):175-180. 
16. 
Vanderberg JP, Stewart MJ. Plasmodium sporozoite-host cell interactions 
during sporozoite invasion. Bulletin of the World Health Organization. 
1990;68 Suppl:74-79. 
17. 
Vanderberg JP, Chew S, Stewart MJ. Plasmodium sporozoite interactions 
with macrophages in vitro: a videomicroscopic analysis. The Journal of 
protozoology. 1990;37(6):528-536. 
18. 
Krettli AU, Dantas LA. Which routes do Plasmodium sporozoites use for 
successful infections of vertebrates? Infection and immunity. 
2000;68(5):3064-3065. 
19. 
Thangamani S, Higgs S, Ziegler S, Vanlandingham D, Tesh R, Wikel S. 
Host immune response to mosquito-transmitted chikungunya virus differs 
from that elicited by needle inoculated virus. PloS one. 2010;5(8):e12137. 
20. 
Schneider BS, Higgs S. The enhancement of arbovirus transmission and 
disease by mosquito saliva is associated with modulation of the host 
immune response. Transactions of the Royal Society of Tropical Medicine 
and Hygiene. 2008;102(5):400-408. 
21. 
Brennan JD, Kent M, Dhar R, Fujioka H, Kumar N. Anopheles gambiae 
salivary gland proteins as putative targets for blocking transmission of 
malaria parasites. Proceedings of the National Academy of Sciences of 
the United States of America. 2000;97(25):13859-13864. 
22. 
Suneja A, Gulia M, Gakhar SK. Blocking of malaria parasite development 
in mosquito and fecundity reduction by midgut antibodies in Anopheles 
stephensi (Diptera: Culicidae). Archives of insect biochemistry and 
physiology. 2003;52(2):63-70. 
23. 
Gulia M, Suneja A, Gakhar SK. Effect of anti-mosquito hemolymph 
antibodies on fecundity and on the infectivity of malarial parasite 
Plasmodium vivax to Anopheles stephensi (Diptera:Insecta). Japanese 
journal of infectious diseases. 2002;55(3):78-82. 
24. 
Stoloff GA, Caparros-Wanderley W. Synthetic multi-epitope peptides 
identified in silico induce protective immunity against multiple influenza 
serotypes. European journal of immunology. 2007;37(9):2441-2449. 
25. 
Vaughan JA, Scheller LF, Wirtz RA, Azad AF. Infectivity of Plasmodium 
berghei sporozoites delivered by intravenous inoculation versus mosquito 
bite: implications for sporozoite vaccine trials. Infection and immunity. 
1999;67(8):4285-4289. 
26. 
Valenzuela JG, Belkaid Y, Garfield MK, et al. Toward a defined anti-
Leishmania vaccine targeting vector antigens: characterization of a 
protective salivary protein. The Journal of experimental medicine. 
2001;194(3):331-342. 
27. 
Belkaid Y, Kamhawi S, Modi G, et al. Development of a natural model of 
cutaneous leishmaniasis: powerful effects of vector saliva and saliva 
preexposure on the long-term outcome of Leishmania major infection in 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 60 of 62 
the mouse ear dermis. The Journal of experimental medicine. 
1998;188(10):1941-1953. 
28. 
Wu Y, Ellis RD, Shaffer D, et al. Phase 1 trial of malaria transmission 
blocking vaccine candidates Pfs25 and Pvs25 formulated with montanide 
ISA 51. PloS one. 2008;3(7):e2636. 
29. 
National Institutes of Health: Giving a subcutaneous injection 2015; 
http://www.cc.nih.gov/ccc/patient_education/pepubs/subq.pdf, 2016. 
30. 
Bartlett JA, Wasserman SS, Hicks CB, et al. Safety and immunogenicity of 
an HLA-based HIV envelope polyvalent synthetic peptide immunogen. 
DATRI 010 Study Group. Division of AIDS Treatment Research Initiative. 
AIDS. 1998;12(11):1291-1300. 
31. 
Graham BS, McElrath MJ, Keefer MC, et al. Immunization with cocktail of 
HIV-derived peptides in montanide ISA-51 is immunogenic, but causes 
sterile abscesses and unacceptable reactogenicity. PloS one. 
2010;5(8):e11995. 
32. 
Pinto LA, Berzofsky JA, Fowke KR, et al. HIV-specific immunity following 
immunization with HIV synthetic envelope peptides in asymptomatic HIV-
infected patients. AIDS. 1999;13(15):2003-2012. 
33. 
Trauger RJ, Daigle AE, Giermakowska W, Moss RB, Jensen F, Carlo DJ. 
Safety and immunogenicity of a gp120-depleted, inactivated HIV-1 
immunogen: results of a double-blind, adjuvant controlled trial. J Acquir 
Immune Defic Syndr Hum Retrovirol. 1995;10 Suppl 2:S74-82. 
34. 
Trauger RJ, Ferre F, Daigle AE, et al. Effect of immunization with 
inactivated gp120-depleted human immunodeficiency virus type 1 (HIV-1) 
immunogen on HIV-1 immunity, viral DNA, and percentage of CD4 cells. 
The Journal of infectious diseases. 1994;169(6):1256-1264. 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 61 of 62 
Appendix A: Schedule of Procedures/Evaluations 
Study Phase > 
Screen 
Injection 
1 
 
 
Injection 
2 
 
 
Feeding 
Telephone 
Call 
Follow Up 
Study Day > 
~ DAY -60 
D0 
D7 ± 2 
days 
D14 ± 2 
days 
D21 
D28 ± 2 
days 
D35 ± 2 
days 
D42 +14 
days 
 
1 visit 
every 60 
days (± 14 
days) 
Under 
Protocol 
11-I-0183 
Day 44  
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
Outpatient Visit 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
Written Consent 
 
X 
 
 
 
 
 
 
 
 
Medical/Medication History 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
Assessment and PE 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
Review of I/E criteria and 
review of consent 
 
X 
 
 
 
 
 
 
 
 
Vital signs † 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
Review of Diary Card 
 
 
X 
 
 
X 
 
 
 
 
Telephone Call to Assess 
Symptoms 
 
 
 
 
 
 
 
 
X 
 
Pregnancy test‡ 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
Urinalysis 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
Vaccine administration 
 
X 
 
 
X 
 
 
 
 
 
Randomization 
 
X 
 
 
 
 
 
 
 
 
Mosquito Feeding 
 
 
 
 
 
 
 
X 
 
 
CBC + diff 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
Acute Care, Mineral, and 
Hepatic Panels 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
LDH, Uric Acid, Creatine 
Kinase, and Total Protein 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
Serum/Whole Blood 
Collection 
 
X 
 
 
X 
 
 
X 
 
X 
IgE 
 
X 
 
 
 
 
 
 
 
 
 
†Vital Signs: participants must be sitting for a minimum of 5 minutes prior to these procedures being performed; vital signs include blood pressure, mean 
arterial pressure, heart rate, respiratory rate, temperature, weight, pulse oximetry; height will be taken at the screening visit only.   
‡Serum pregnancy testing at the screening and all other visits. On Days 0, 21, 42, study vaccination or mosquito feeding will not be initiated until results 
are available. Females only. 
 

Universal Mosquito Vaccine 
Protocol Version 5.0 
09 Feb, 2018 
 
Page 62 of 62 
Appendix B: Blood Volumes for Specimen Collection 
 
 
 
Study 
Schedule/Procedures 
 
 
 
 
 
 
 
 
 
 
Day 0 
Day 7 
(+/- 2 
days) 
Day 14 
(+/- 2 days) 
Day 21 
(+/- 2 
days) 
Day 28 
(+/- 2 days) 
Day 35 
(+/- 2 days) 
Day 42 
(+ 14 days) 
1 visit every 60 
days (+/- 14 days) 
b-hCG, Pregnancy 
4mL per blood 
draw 
4 mL 
4 mL 
4 mL 
4 mL  
4 mL 
4 mL 
4 mL 
4 mL 
CBC + Diff  
3mL per blood 
draw 
3 mL 
3 mL 
3 mL 
3 mL 
3 mL 
3 mL 
3 mL 
3 mL 
Acute Care, Mineral, 
and Hepatic Panels,  
4mL per blood 
draw 
4 mL 
4 mL 
4 mL 
4 mL 
4 mL 
4 mL 
4 mL 
4 mL   
LDH, Uric Acid, 
Creatine Kinase, 
and Total Protein 
Included 
with the 
above 
X 
X 
X 
X 
X 
X 
X 
X 
IgE, Total 
4mL per blood 
draw 
4 mL 
 
 
 
 
 
 
 
Serum/Whole Blood 
Collection 
8 mL X 1 for 
serum, 8.5 mL 
X 5 for whole 
blood, and 2.5 
mL for RNA 
per blood draw 
 
53 mL + 8.5 mL 
x 5 more for 
whole blood 
 
 
53 mL 
 
 
95.5 mL prior 
to feeding, 
111 ml post 
feeding  
 
53 mL     
Daily Volume (mL) 
110.5 mL 
11 mL 
11 mL 
64 mL 
11 mL 
11 mL 
217.5 mL 
64 mL 
Cumulative Volume 
110.5 mL 
121.5 mL 132.5 mL 196.5 mL 
207.5 mL 
218.5 mL 
436 mL 
500 mL at first 
follow up,1152mL 
over the course of 
the whole study